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Absence of p53 allows direct immortalization of hematopoietic cells by the myc and raf oncogenes
Abstract
The p53 tumor suppressor is implicated here as a crucial barrier to unlimited cell proliferation. Its role in transformation of hematopoietic cells was studied by infecting fetal liver cells from wild-type or p53-/- mice with oncogenic retroviruses. Transformed colonies arose with a raf and a myc-raf virus. Absence of p53 did not affect their frequency but proved critical for their continued propagation. Colonies of p53-/- cells bearing both myc and raf readily yielded continuous cell lines without apparent requirement for genetic alteration. The lines, mainly of erythroid or myelomonocytic origin, were diploid but highly tumorigenic from their inception. These findings imply that p53 loss contributes directly to immortalization and tumorigenesis, probably by abrogating an intrinsic senescence program.
... brains, the neurons and other brain cells that remain appear to show multiple signs of at least partial immortalization: for example, AD patients' medial temporal lobe neurons and their NFT-131 bearing neocortical neurons were found to not have p53 proteins [80,82,908], the central G1 DNA damage checkpoint apoptosis-inducing tumor suppressor. Levels of oxidized, nitrated, and glutathionylated p53 proteins were found to be significantly increased in AD patients' IPL, the latter of which modification appeared to correlate with p53 monomers and dimers aggregating into fully-active tetramers less frequently [642,647]. ...
... p53-dependent forms of neuron death likely kill a considerable number of medial temporal lobe neurons especially [71,80,82,986]. AD patients' remaining medial temporal lobe neurons and NFT-bearing neocortical neurons are p53 deficient [80,82], resulting in their at least partial immortalization and enabling them to evade p53-dependent forms of cell death and survive chronic oxidative stress and DNA damage [71,80,82,115,908,986]. This pro-survival p53 loss might occur via several mechanisms, such as HIPK2 dysregulation [628], BMI1 transcriptional upregulation selectively in the medial temporal lobe but not the frontal lobe [21,136,[637][638][639][640], and NOS-promoted p53 nitration and exosomal secretion [82,184,546,[641][642][643][644][645]647,797,799]. ...
Abstract. Consistent with the numerous Aβ-targeting clinical trials that failed to meet their primary endpoints, Aβ does not correlate spatiotemporally with oxidative stress (OS), oxygen/glucose hypometabolism, or other AD hallmarks in sporadic Alzheimer’s disease (AD) patients, suggesting it is a compensatory response and an amplifying signal in sporadic AD, not the distal or primary cause of sporadic AD as some still believe. Diverse bacteria, fungi, and DNA viruses accumulate in AD patients’ brains. OS occurs in the preclinical AD (PCAD) medial temporal lobe (MTL), but not the PCAD neocortex. However, oxidatively damaged DNA (oxoDNA) accumulates in the MTLs and neocortices of PCAD, mild cognitive impairment (MCI), and AD patients. DNA-oxidizing iron and aluminum accumulate in AD patients’ hippocampal neuron chromatin. OS, pathogens, and cytokines drive Aβ42 deposition. At the intersection of OS and poly-microbial infections in AD, we hypothesize oxoDNA drives multiple AD hallmarks at all stages of AD pathogenesis. OxoDNA appears to drive Aβ42 deposition via <cGAS/cGAMP/STING/IFN/AIM2>, <MYD88→NFκB→BACE1>, <MAP4K4→JNK→AP1>, and <CK2→pSer529-p65-NFκB→BACE1>. OxoDNA appears to spread OS via Nrf2 mislocalization. OxoDNA appears to drive tau hyperphosphorylation via mitochondrial OS induction. OxoDNA appears to drive oxygen hypo-metabolism, ATP depletion, and mitochondrial OS via <PARP1→PAR→PARG→ADPR→TPRM2→AMP→ANT→ADP→ATP synthase inhibition>. OxoDNA appears to drive neurotoxicity via <PARP1→PAR→mPTP→AIF→parthanatos>, <ROS,ADPR,Ca2+→TRPM2→Ca2+,Zn2+→(Zn2+/LKB1/pThr172-AMPKα2)→p-FOXO3→Bim→oxytosis/ferroptosis>, <CK2→pSer529-p65→Noxa+Bim>, <ATM→p53→caspase9+BAX+APAF1→apoptosis>, <AIM2→caspase1→gasderminD→pyroptosis>. OxoDNA appears to drive synaptoxicity via PARP1, <ADPR→TRPM2→Ca2+>, p53, NFκB, IL-6, <AIM2→caspase1→IL-1β>. OxoDNA appears to drive immunosuppression via IFNs+IL-6→SOCS1/3, and <AIM2+(Ca2+/CaMKKβ/pThr183-AMPKα1)+EB1+LC3→IL-1β>. Via <ROS,ADPR,Ca2+→TRPM2→Ca2+,Zn2+→ZnT6→S-SMase→ceramide> and possibly <AIM2+(Ca2+/CaMKKβ/pThr183-AMPKα1)+EB1→LC3>, oxygen-hypometabolic and partially immortalized medial temporal lobe pyramidal neurons excite each other, neocortical neurons with trans-synaptic extracellular vesicles bearing Aβ42, Zn2+, p-tau, ceramides, IL-1β, RNA, and oxoDNA, promoting synaptotoxicity. The multi-dsDNA repair factor depletion/suppression in AD neurons appears to have been selected for to evade viral integration, e.g. HHV-6A telomere integration. The ATM and BRCA1 loss in AD neurons appears to have been selected for to evade chromosomal fusions at telomeres deprotected by Ku80 downregulation and triaging to dsDNA breaks. BRCA1 and sporadic-AD-specific BMI1 loss derepress heterochromatin. Tau-associated derepressed endogenous retroviruses induce immunodeficiency. In the hippocampus, oxoDNA/IFN/IL-6-induced SOCS1 and oxoDNA/IFN- plus oxoDNA/IL-6-induced SOCS3 promote JAK/STAT suppression. oxoDNA/IFNs may drive IL-1 resistance. Hippocampal <LPS→TLR4→NFκB→pro-IL-1β> + <oxoDNA→cGAS→IFNs→AIM2> + <AIM2+(Ca2+/CaMKKβ/pThr183-AMPKα1)+EB1+LC3>-mediated IL-1β secretion suppresses neocortex IFN immunity, enabling opportunistic herpesvirus-6A and herpesvirus-7, and drives glucose hypo-metabolism, evading parthanatos and sepsis. Stress-associated glucocorticoids also drive immunodeficiency. AD is an inflammatory/autoimmune/immunosuppressive/pseudo-cancerous brain acquired immunodeficiency meta-syndrome (AIDS) driven by chronic oxidative DNA damage, dsDNA repair factor depletion/suppression, endogenous retrovirus derepression, cytokines, and poly-pathogen dysbiosis.
... The extension of cellular lifespan has been associated with loss of the wild-type p53 allele (Rogan et al., 1995) and can be induced by expression of a dominant negative p53 mutant (Bond et al., 1994;Gollohan and Shay, 1996). Absence of p53 favors the immortalization of murine erythroid cells transformed by myc and raf (Metz et al., 1995). ...
... 1994b) bar (Miyashita et al. 1994a;Miyashita and Reed 1995), and Gadd4.5 Chin et al. 1997). p53 also plays a part in cellular senescence and differentiation (Metz et al. 1995;Bond et al. 1996;Eizenberg et al. 1996), and possibly other processes including monitoring recombination intermediates in spermatocytes (Montes de Oca Luna et al., 1995) and T cell receptors (Guidos et al., 1996) as well as in response to other cellular distress such as h-ypoxia (Graeber et al., 1996), low levels of nucleoside triphosphate pools ( Linke et al., 1996), and teratogenic agents (Nicol et al., 1995). The results presented here recapitulate in humans what has previously been observed in p5bdeficient mice, which suggest that perhaps these functions are not essential in human embryogenesis. ...
... The properties and functions of p53, a tumor suppressor and cellular transcription factor in cellular immortalization, have been well documented (Blattner et al., 1999;Harvey and Levine, 1991;Levine, 1997;Metz et al., 1995). The involvement of p53 in the process of cell differentiation is still discussed controversially. ...
... Molchadsky et al. noticed that many authors, who described differing results with respect to the regulative properties of p53 during cell differentiation, used different cell types in their studies (Metz et al., 1995;Molchadsky et al., 2008). Armesilla-Diaz et al. used MSCs and proposed negative regulation of p53 in the process of cell differentiation, and Qin et al. claimed that p53 plays a positive role in the regulation of cell differentiation when using hESCs (Armesilla-Diaz, et al. 2009;Qin et al. 2007). ...
Abstract Various diseases, injuries, and congenital abnormalities may result in degeneration and loss of organs and tissues. Recently, tissue engineering has offered new treatment options for these common, severe, and costly problems in human health care. Its application is often based on the usage of differentiated stem cells. However, despite intensive research and growing knowledge, many questions remain unresolved in the process of cell differentiation. The aim of this study was to find standardized cell models for analyzing molecular mechanisms of cell differentiation. We investigated the multipotency of three standardized murine embryonic fibroblast cell cultures using histological staining, western blotting, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Our results demonstrated that NIH-3T3 and mouse embryonic fibroblast (MEF) cells were able to differentiate into adipogenic, chondrogenic, and osteogenic lineages expressing typical differentiation markers. Interestingly, Flp-In-3T3 cells did not differentiate into any of the three mesenchymal lineages, although this cell line is genetically closely related to NIH-3T3. The results were confirmed by histological staining. Flp-In-3T3, NIH-3T3, and MEF cells have usually been used for DNA transfections, recombinant protein expression, and as "feeder cells." Unlike mesenchymal stem cells (MSCs) and mesenchymal progenitor cells (MPCs), they are easy to obtain and to expand and are less prone to change their structure and morphology, even at higher passages. Our results suggest that Flp-In-3T3, MEF, and NIH-3T3 cells are highly suitable to be used as models to analyze molecular mechanisms of cell differentiation.
... Although some genes such as p53 and PTEN are commonly mutated in many different types of cancer, most cancer genes are more lineage-specific. It is well established that human cells are harder to transform than rodent cells [5][6][7][8][9][10][11], which can be transformed using only MYC and RAS oncogenes [12][13][14]. Seminal experiments by Hahn and Weinberg established already 20 years ago that different human cell types can be transformed using a set of oncogenes that includes the powerful viral large-T and small-T oncoproteins from the SV40 virus [15]. ...
Cancer is the most complex genetic disease known, with mutations implicated in more than 250 genes. However, it is still elusive which specific mutations found in human patients lead to tumorigenesis. Here we show that a combination of oncogenes that is characteristic of liver cancer (CTNNB1, TERT, MYC) induces senescence in human fibroblasts and primary hepatocytes. However, reprogramming fibroblasts to a liver progenitor fate, induced hepatocytes (iHeps), makes them sensitive to transformation by the same oncogenes. The transformed iHeps are highly proliferative, tumorigenic in nude mice, and bear gene expression signatures of liver cancer. These results show that tumorigenesis is triggered by a combination of three elements: the set of driver mutations, the cellular lineage, and the state of differentiation of the cells along the lineage. Our results provide direct support for the role of cell identity as a key determinant in transformation and establish a paradigm for studying the dynamic role of oncogenic drivers in human tumorigenesis.
... The drugs and small molecules that targets TP53 or TP53 signaling pathway including the most widely understood were PRIMA-1 [2,2-bis(hydroxymethyl)quinuclidin-3-one or APR-017] and APR-246 [2-(hydroxymethyl)-2-(methoxymethyl)] that induced the cancer cell apoptosis (16). The TP53 may be the important regulator of apoptosis, senescence, and metabolism for cancer (6,17,18). From these, the TP53 may sever as a key role for WT as well. ...
Background:
Differential expression of tumor protein 53 (TP53, or p53) has been observed in multiple cancers. However, the expression levels and prognostic role of TP53 signaling pathway genes in Wilms' tumor (WT) have yet to be fully explored.
Methods:
The expression levels of TP53 signaling pathway genes including TP53, mouse double minute 2 (MDM2), mouse double minute 4 (MDM4), cyclin-dependent kinase 2A (CDKN2A), cyclin-dependent kinase 2B (CDKN2B), and tumor suppressor p53-binding protein 1 (TP53BP1) in WT were analyzed using the Oncomine database. Aberration types, co-mutations, mutation locations, signaling pathways, and the prognostic role of TP53 in WT were investigated using cBioPortal. MicroRNA (miRNA) and transcription factor (TF) targets were identified with miRTarBase, miWalk, and ChIP-X Enrichment Analysis 3 (CheA3), respectively. A protein-protein network was constructed using GeneMANIA. The expression of TP53 signaling genes were confirmed in WT samples and normal kidney tissues using the Human Protein Atlas (HPA). Cancer Therapeutics Response Portal (CTRP) was used to analyze the small molecules potentially targeting TP53.
Results:
TP53 was significantly expressed in the Cutcliffe Renal (P=0.010), but not in the Yusenko Renal (P=0.094). Meanwhile, MDM2 was significantly overexpressed in the Yusenko Renal (P=0.058), but not in the Cutcliffe Renal (P=0.058). The expression levels of MDM4 no significant difference between the tumor and normal tissue samples. The most common TP53 alteration was missense and the proportion of TP53 pathway-related mutations was 2.3%. Co-expressed genes included ZNF609 (zinc finger protein 609), WRAP53 (WD40-encoding RNA antisense to p53), CNOT2 (CC chemokine receptor 4-negative regulator of transcription 2), and CDH13 (cadherin 13). TP53 alterations indicated poor prognosis of WT (P=1.051e-4). The regulators of the TP53 pathway included miR-485-5p and TFs NR2F2 and KDM5B. The functions of TP53 signaling pathway were signal transduction in response to DNA damage and regulate the cell cycle. The small molecules targeting TP53 included PRIMA-1, RITA, SJ-172550, and SCH-529074.
Conclusions:
TP53 was found to be differentially expressed in WT tissues. TP53 mutations indicated poor outcomes of WT. Therefore, pifithrin-mu, PRIMA-1, RITA, SJ-172550, and SCH-529074 could be used in combination with traditional chemotherapy to treat WT.
... Immortalized cells are defined as cells whose proliferative capacity has been enhanced using different methods [30] . There are a variety of established approaches to immortalize cell lines including the introduction of oncogenes [31][32][33] , viral transformation [34,35] , the inactivation of tumor suppressor genes [36,37] or the inactivation of telomere-controlled senescence [38] . The establishment of immortalized cell lines has helped the scientific community to study different biological and molecular events [26] , although, this approach has been questioned since these immortalized cells differ significantly from cells with an intact cell cycle control and they are more similar to malignant cells in many respects. ...
The genetic cardiomyopathies are a group of disorders related by abnormal myocardial structure and function. Although individually rare, these diseases collectively represent a significant health burden since they usually develop early in life and are a major cause of morbidity and mortality amongst affected children. The heterogeneity and rarity of these disorders requires the use of an appropriate model system in order to characterize the mechanism of disease and develop useful therapeutics since standard drug trials are infeasible. A common approach to study human disease involves the use of animal models, especially rodents, but due to important biological and physiological differences, this model system may not recapitulate human disease. An alternative approach for studying the metabolic cardiomyopathies relies on the use of cellular models which have most frequently been immortalized cell lines or patient-derived fibroblasts. However, the recent introduction of induced pluripotent stem cells (iPSCs), which have the ability to differentiate into any cell type in the body, is of great interest and has the potential to revolutionize the study of rare diseases. In this paper we review the advantages and disadvantages of each model system by comparing their utility for the study of mitochondrial cardiomyopathy with a particular focus on the use of iPSCs in cardiovascular biology for the modeling of rare genetic or metabolic diseases.
Keywords: Cardiomyopathy, Mitochondria, Induced pluripotent stem cells, Fibroblasts, Cellular models
Core tip: Several experimental model systems exist for the modeling of cardiomyopathies, including those caused by rare metabolic or mitochondrial diseases. We compare and contrast the cellular models that have been used to date to model several different mitochondrial disorders with a particular focus on the advantages and disadvantages of induced pluripotent stem cells.
... For the generation of immortalized myoblast cell lines according to [16], homozygous R349P desmin knockin mice [14] were cross-bred with homozygous p53 knock-out mice [15] to generate offspring doubleheterozygous for the R349P desmin knock-in and p53 knock-out alleles. These mice were further mated to obtain offspring with the following genotypes for the isolation of skeletal muscle myoblasts, homozygous R349P desmin knock-in and homozygous p53 knock-out, heterozygous R349P desmin knock-in and homozygous p53 knock-out, wild-type desmin and homozygous p53 knock-out alleles. ...
... However, the fact that many apparently oncogenic mutations fail to transform fibroblastic cells in culture suggests that different cell types are susceptible to transformation by different sets of oncogenes. It is well established that human cells are harder to transform than rodent cells (5)(6)(7)(8)(9)(10)(11), which can be transformed using only MYC and RAS oncogenes (12)(13)(14). Many different human cell types can be transformed using a set of oncogenes that includes the powerful viral large-T and small-T oncoproteins from the SV40 virus (15). ...
Cancer is the most complex genetic disease known, with mutations in more than 250 genes contributing to different forms of the disease. Most mutations are specific to particular types of cancer, suggesting that cancer genes interact with cell lineage-determining factors to drive the transformation process. To identify the factors necessary and sufficient to define a lineage-specific cancer type, we have reprogrammed and transformed normal human fibroblasts to liver cancer cells. We show that reprogramming human fibroblasts to induced hepatocytes (iHeps) makes the cells sensitive to transformation by a combination of oncogenes that is characteristic of liver cancer (CTNNB1, TERT and MYC). The transformed iHeps are highly proliferative, tumorigenic in nude mice, and bear gene expression signatures of liver cancer. Our results show that lineage-determining factors collaborate with oncogenes to drive tumorigenesis and establish a paradigm for defining the molecular states of distinct types of human cancer.
... With these approaches, we modeled diverse hemopoietic malignancies. Salient findings were that N-Myc, like c-Myc, retards B cell development and that the two genes can cross-regulate each other's expression (Rosenbaum et al. 1989); that the expression threshold for c-Myc-induced tumorigenesis varies with hemopoietic lineage (Smith et al. , 2006; that v-Abl plus deregulated Myc preferentially transforms plasma cells (Rosenbaum et al. 1990); that a mutated N-Ras gene is highly tumorigenic for T cells and macrophages but not, by itself, for B cells (Harris et al. 1988a, Haupt et al. 1992; that the Cyclin D1 gene (CCCND1), implicated as an oncogene by the 11;14 chromosomal translocation in mantle cell lymphoma, synergizes with Myc (Bodrug et al. 1994); and that p53 loss coupled with expression of Raf or Myc plus Raf oncogenes fully transforms hemopoietic cells (Metz et al. 1995). ...
The war on cancer that began some 40 years ago with the discovery of oncogenes is starting to be won. We feel fortunate to have contributed to several advances. Here we recall how molecular biology became our scientific passion, how we met from opposite ends of the earth, and how our 50-year odyssey has taken us from gene expression through immunogenetics to exploring the molecular basis of cancer and cell death. We describe the scientific discoveries that motivated us and remarkable scientists who influenced us. We sketch our studies that clarified the role of chromosome translocations in cancer and established the value of genetically engineered mouse models of tumorigenesis. Finally, we outline how our findings with many talented close colleagues on cell death regulation stimulated the development of remarkable new anticancer agents called BH3 mimetics, which are encouraging hope that many more malignancies will become controllable and even curable. Expected final online publication date for the Annual Review of Cancer Biology Volume 3 is March 4, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... For the generation of immortalized myoblast cell lines according to [16], homozygous R349P desmin knockin mice [14] were cross-bred with homozygous p53 knock-out mice [15] to generate offspring doubleheterozygous for the R349P desmin knock-in and p53 knock-out alleles. These mice were further mated to obtain offspring with the following genotypes for the isolation of skeletal muscle myoblasts, homozygous R349P desmin knock-in and homozygous p53 knock-out, heterozygous R349P desmin knock-in and homozygous p53 knock-out, wild-type desmin and homozygous p53 knock-out alleles. ...
Aims
We investigated newly generated immortalized heterozygous and homozygous R349P desmin knock‐in myoblasts in conjunction with the corresponding desminopathy mice as models for desminopathies to analyse major protein quality control processes in response to the presence of R349P mutant desmin.
Methods
We used hetero‐ and homozygous R349P desmin knock‐in mice for analyses and for crossbreeding with p53 knock‐out mice to generate immortalized R349P desmin knock‐in skeletal muscle myoblasts and myotubes. Skeletal muscle sections and cultured muscle cells were investigated by indirect immunofluorescence microscopy, proteasomal activity measurements, and immunoblotting addressing autophagy rate, chaperone‐assisted selective autophagy, and heat shock protein levels. Muscle sections were further analysed by transmission and immunogold electron microscopy.
Results
We demonstrate that mutant desmin i) increases proteasomal activity, ii) stimulates macroautophagy, iii) dysregulates the chaperone assisted selective autophagy, and iv) elevates the protein levels of αB‐crystallin and Hsp27. Both αB‐crystallin and Hsp27 as well as Hsp90 displayed translocation patterns from Z‐discs as well as Z‐I junctions, respectively, to the level of sarcomeric I‐bands in dominant and recessive desminopathies.
Conclusions
Our findings demonstrate that the presence of R349P mutant desmin causes a general imbalance in skeletal muscle protein homeostasis via aberrant activity of all major protein quality control systems. The augmented activity of these systems and the subcellular shift of essential heat shock proteins may deleteriously contribute to the previously observed increased turnover of desmin itself and desmin‐binding partners, which triggers progressive dysfunction of the extrasarcomeric cytoskeleton and the myofibrillar apparatus in the course of the development of desminopathies.
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... To generate immortalized fibroblast cell lines according to (Metz et al., 1995), homozygous N471D strumpellin knock-in mice were crossbred with homozygous p53 knock-out mice (Jacks et al., 1994). This resulted in offspring double-heterozygous for the N471D strumpellin knock-in and p53 knock-out alleles, which were further mated to obtain mice containing the homozygous p53 knock-out and either the heterozygous or the homozygous N471D strumpellin knock-in or the wild-type strumpellin alleles. ...
Hereditary spastic paraplegias (HSPs) are genetically diverse and clinically characterised by lower limb weakness and spasticity. The N471D and several other point mutations of human strumpellin (Str; also known as WASHC5), a member of the Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) complex, have been shown to cause a form of HSP known as spastic paraplegia 8 (SPG8). To investigate the molecular functions of wild-type (WT) and N417D Str, we generated Dictyostelium Str⁻ cells and ectopically expressed StrWT-GFP or StrN471D-GFP in Str⁻ and WT cells. Overexpression of both proteins apparently caused a defect in cell division, as we observed a clear increase in multinucleate cells. Real-time PCR analyses revealed no transcriptional changes in WASH complex subunits in Str⁻ cells, but western blots showed a twofold decrease in the SWIP subunit. GFP-trap experiments in conjunction with mass-spectrometric analysis revealed many previously known, as well as new, Str-interacting proteins, and also proteins that no longer bind to StrN471D. At the cellular level, Str⁻ cells displayed defects in cell growth, phagocytosis, macropinocytosis, exocytosis and lysosomal function. Expression of StrWT-GFP in Str⁻ cells rescued all observed defects. In contrast, expression of StrN471D-GFP could not rescue lysosome morphology and exocytosis of indigestible material. Our results underscore a key role for the WASH complex and its core subunit, Str, in the endolysosomal system, and highlight the fundamental importance of the Str N471 residue for maintaining lysosome morphology and dynamics. Our data indicate that the SPG8-causing N471D mutation leads to a partial loss of Str function in the endolysosomal system.
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... Induction of cell senescence was first shown to play a critical role in TP53-mediated tumor suppression in a mouse model of erythroleukemia (Metz et al. 1995). Moreover, restoration of TP53 function in established solid-organ tumors (driven by loss of TP53) in vivo leads to the induction of cellular senescence in association with tumor regression (Ventura et al. 2007;Xue et al. 2007). ...
The fundamental biological importance of the Tp53 gene family is highlighted by its evolutionary conservation for more than one billion years dating back to the earliest multicellular organisms. The TP53 protein provides essential functions in the cellular response to diverse stresses and safeguards maintenance of genomic integrity, and this is manifest in its critical role in tumor suppression. The importance of Tp53 in tumor prevention is exemplified in human cancer where it is the most frequently detected genetic alteration. This is confirmed in animal models, in which a defective Tp53 gene leads inexorably to cancer development, whereas reinstatement of TP53 function results in regression of established tumors that had been initiated by loss of TP53. Remarkably, despite extensive investigation, the specific mechanisms by which TP53 acts as a tumor suppressor are yet to be fully defined. We review the history and current standing of efforts to understand these mechanisms and how they complement each other in tumor suppression.
... Cell Culture-Cells were grown in Dulbecco's modified Eagle's medium, 5% fetal calf serum (FCS). Differentiation of J2E (2), clone 11, and clone 24 (30) as well as ME17 (31) cell lines was initiated with epo (5 units/ml) or sodium butyrate (0.5 mM), whereas murine erythroleukemia (MEL) cells were stimulated with Me 2 SO (1.5%). Viability was determined by eosin dye exclusion (4) and hemoglobin synthesis by benzidine staining (32). ...
Erythropoietin (EPO) stimulates the immature erythroid J2E cell line to terminally di.fferentiate, proliferate and maintains their viability in the absence of serum. In contrast, a mutant J2E clone (J2E-NR) fails to mature in response to the hormone which we have shown is due to a very low expression level of the Lyn tyrosine kinase. Co-immunoprecipitation and yeast two-hybrid analysis indicates that Lyn directly associates with the EPO-receptor complex. Using the yeast two hybrid system we have identified LckBP-1/HS1 and several novel molecules as Lyn interactors. LckBP-1/HS1 has been shown to bind to the SH3 domain of Lck and contains four tandem helix-turn-helix motifs, a proline rich region, a proline and glutamine rich segment, and an SH3 domain. The importance of this interaction in EPO-induced signalling through Lyn is currently be!ng investigated. Three novel molecules were also identified in the two-hybrid screen as specifically interacting with Lyn. One of these has an ankyrin repeat most closely related to a K+ channel. Another novel protein bound specifically to a kinase inactive mutant of Lyn where tyrosine 397 had been mutated to phenyalanine. Full-length clones of these novel molecules are currently being isolated and their involvement in erythroid development will be analysed.
... Such cell lines have successfully been derived after crossing the corresponding knockout mouse line into a p53 À/À background. Absence of p53 facilitates immortalization of cells that otherwise are difficult to immortalize (Metz, Harris, & Adams, 1995). So far, the following plectin-deficient (total, i.e., all isoforms) cell lines have been established: basal keratinocytes (Andrä et al., 2003), dermal fibroblasts (Osmanagic-Myers & , and skeletal muscle myoblasts (Winter et al., 2014). ...
Plectin is a large cytoskeletal linker protein with a multitude of functions affecting various cellular processes. It is expressed as several different isoforms from a highly complex gene. Both, this transcript diversity (mainly caused by short 5′-sequences contained in alternative first exons) and the size (> 500 kDa) of the resulting proteins, present considerable challenges to plectin researchers. In this chapter, we will consider these problems and offer advice on how to tackle them best. As plectin has been studied most extensively in skin and muscle, we will focus on these types of tissues and describe some selected methods in detail. Foremost, however, we aim to give the readers some good pointers to available tools and into the existing literature.
... So wurde kürzlich für Mäuse gezeigt, dass eine signifikante Telomerverkürzung die Aktivierung der Expression von p53 sowie einen Wachstumsstillstand und eine Apoptose auslöst (Chin et al. 1999). Im Gegensatz dazu führt die Zerstörung des p53-Gens zur Immortalisierung von Zellen und zum Tumorwachstum (Metz et al. 1995). ...
Jeanne L. Calment, geboren am 21. Februar 1875, gestorben am 4. August 1997 im Alter von 122 Jahren in einem Alterspflegeheim in Arles, Frankreich, war der bisher älteste Mensch, dessen Lebensalter bestätigt ist. In den westlichen Industriestaaten steigt der Anteil der älteren Bevölkerung stetig an. Dies wird dazu führen, dass bis zum Jahr 2050 etwa ein Viertel der Bevölkerung über 65 Jahre alt sein wird. Noch zu Beginn des letzten Jahrhunderts lag die durchschnittliche Lebenserwartung des Menschen in den entwickelten Ländern bei etwa 37 Jahren. Heute beträgt diese etwa 73 Jahre, Tendenz steigend. Den steilsten Anstieg in der Lebenserwartung erlebt seit 1950 Japan, wo das durchschnittliche Lebensalter sogar 87 Jahre bei Frauen und 78 Jahre bei Männern beträgt (Wemmer 1993). Die Gründe für diese immense Verlängerung der Lebenserwartung seit dem letzten Jahrhundert sind vielfältig und werden unter anderem in der verbesserten Hygiene, der veränderten Ernährung und nicht zuletzt in den wesentlich weiterentwickelten Behandlungsmethoden der Medizin gesehen.
... So wurde kürzlich für Mäuse gezeigt, dass eine signifikante Telomerverkürzung die Aktivierung der Expression von p53 sowie einen Wachstumsstillstand und eine Apoptose auslöst (Chin et al. 1999). Im Gegensatz dazu führt die Zerstörung des p53-Gens zur Immortalisierung von Zellen und zum Tumorwachstum (Metz et al. 1995). ...
Altern ist ein grundlegender physiologischer Prozess, der ausnahmslos in allen arbeitsteiligen multizellulären Organismen auftritt. In dieser Arbeit werden wir uns beim Begriff „Spezies“ besonders auf Mammalia beziehen—auf Abweichungen wird hingewiesen. Aus medizinisch-biologischer Sicht umfasst der Begriff „Altern“ die regressive Phase des stetigen Wandels von Struktur und Funktion, den Bürger (1947) als Biomorphose bezeichnete (Abb. 1.2.1).
... Plectin-deficient myoblasts differentiate and form contractile multinucleated myotubes in vitro. The strategy pursued in this study for isolating immortalized myoblasts was based on mouse lines (9) that had been crossed into a p53 -/background (11). Cultures of immortalized plectin-deficient (Plec -/-) and plectin-positive (Plec +/+ ) myoblasts were isolated and purified from fibroblasts to a homogeneity of >90% of desmin-positive cells (L. ...
The ubiquitously expressed multifunctional cytolinker protein plectin is essential for muscle fiber integrity and myofiber cytoarchitecture. Patients suffering from plectinopathy-associated epidermolysis bullosa simplex with muscular dystrophy (EBS-MD) and mice lacking plectin in skeletal muscle display pathological desmin-positive protein aggregation and misalignment of Z-disks, which are hallmarks of myofibrillar myopathies (MFMs). Here, we developed immortalized murine myoblast cell lines to examine the pathogenesis of plectinopathies at the molecular and single cell level. Plectin-deficient myotubes, derived from myoblasts, were fully functional and mirrored the pathological features of EBS-MD myofibers, including the presence of desmin-positive protein aggregates and a concurrent disarrangement of the myofibrillar apparatus. Using this cell model, we demonstrated that plectin deficiency leads to increased intermediate filament network and sarcomere dynamics, marked upregulation of HSPs, and reduced myotube resilience following mechanical stretch. Currently, no specific therapy or treatment is available to improve plectin-related or other forms of MFMs; therefore, we assessed the therapeutic potential of chemical chaperones to relieve plectinopathies. Treatment with 4-phenylbutyrate resulted in remarkable amelioration of the pathological phenotypes in plectin-deficient myotubes as well as in plectin-deficient mice. Together, these data demonstrate the biological relevance of the MFM cell model and suggest that this model has potential use for the development of therapeutic approaches for EBS-MD.
... These extensive studies have shown that immortalization of erythroblasts can be achieved by manipulating the expression of transcription factors, tumor suppressors, and nuclear receptors. In addition to the factors mentioned above, v-Erb-a (THRA), v-Erb-b, and Raf, as well as several other genes, have been shown to have the potential to contribute to immortalization of erythroid precursors [32][33][34][35][36]. Some of the cell lines created with these factors have retained the capacity to differentiate and enucleate. ...
In the Western world, the volunteer-based collection system covers most transfusion needs, but transient shortages regularly develop and blood supplies are vulnerable to potentially major disruptions. The production of cultured red blood cells from stem cells is slowly emerging as a potential alternative. The various cell sources, the niche applications most likely to reach the clinic first, and some of the remaining technical issues are reviewed here. STEM CELLS TRANSLATIONAL MEDICINE 2012;1:927-933
... However, in addition, multiple posttranslational modifications, including phosphorylation, acetylation, and neddylation, also affect P53 stability, its binding to target genes, and/or its transcriptional activity (Vousden and Lane 2007). Upon activation, P53 binds as a homotetramer to specific sequences within the regulatory regions of a broad range of target genes (Riley et al. 2008) and thereby triggers a multitude of effector pathways, including cell cycle arrest (Livingstone et al. 1992), cellular senescence (Metz et al. 1995), coordination of DNA repair, and apoptosis (Vousden and Lane 2007) (Fig. 2). In addition, P53 triggers a process that regulates its own activity that operates mainly through direct transcriptional activation of MDM2 (Barak et al. 1993;Wu et al. 1993). ...
Multicellular organisms have evolved processes to prevent abnormal proliferation or inappropriate tissue infiltration of cells, and these tumor suppressive mechanisms serve to prevent tissue hyperplasia, tumor development, and metastatic spread of tumors. These include potentially reversible processes such as cell cycle arrest and cellular senescence, as well as apoptotic cell death, which in contrast eliminates dangerous cells that may initiate tumor development. Tumor suppressive processes are organized as complex, extensive signaling networks, controlled by central "nodes." These "nodes" are prominent tumor suppressors, such as P53 or PTEN, whose loss is responsible for the development of the majority of human cancers. In this review we discuss the processes by which some of these prominent tumor suppressors trigger apoptotic cell death and how this process protects us from cancer development.
... To establish immortalized plectin-deficient cell lines, we have intercrossed plectin (þ/À) and p53 (À/À) mice to obtain plectin (À/À)/p53 (À/À) double knockout mice (Andrä et al., 2003). This method generates a defined mutation (absence of p53) that facilitates immortalization of cells that otherwise are diYcult to immortalize (Metz et al., 1995). ...
This chapter provides an overview of the historical perspective of plectin and discusses its structural and molecular properties, gene organization and isoform diversity, the molecular interactions of plectin, and plectin's relation to human diseases. The important role of plectin for the mechanical stability of living cells can be understood by defining the molecular interactions of plectin with a number of cytoskeletal proteins. The most detailed analysis of a plectin gene locus, however, has been carried out in mouse. At the cellular level, plectin codistributes with different types of IFs and is located at plasma membrane attachment sites of intermediate filaments (Ifs) and microfilaments such as hemidesmosomes, desmosomes, Z-line structures and dense plaques of striated and smooth muscle, intercalated disks of cardiac muscle, and focal contacts. Plectin's widespread distribution, strategic localization, and multitude of known binding partners make it ideally fit to act as a scaffolding platform, bringing together the right molecules at the right time and location to perform their tasks. This role is further supported by an elaborate fine-tuning mechanism of plectin provided by the alternative splicing of its gene, leading to subtle, yet functionally significant, variations in the expressed isoforms. The future focus of plectin research will have to be put on the understanding of the roles of the individual isoforms. Pinpointing specific isoform functions might also be helpful to tailor practicable gene therapy approaches for patients with plectin gene mutations.
... While Ras-transformed cells are exquisitely sensitive to Cdc42 loss when compared to non-transformed control cells, whether Cdc42 is also important for transformation mediated by oncogenes of distinct pathways has not been addressed. c-Myc is another potent and widely studied oncogene which, like Ras, has been shown to cooperate with p53 loss to promote cellular trans-formation [27]. Cells immortalized by dominant negative p53 were transformed through overexpression of c-Myc (Figure 5a). ...
The ras proto-oncogene is one of the most frequently mutated genes in human cancer. However, given the prevalence of activating mutations in Ras and its association with aggressive forms of cancer, attempts to therapeutically target aberrant Ras signaling have been largely disappointing. This lack of progress highlights the deficiency in our understanding of cellular pathways required for Ras-mediated tumorigenesis and suggests the importance of identifying new molecular pathways associated with Ras-driven malignancies. Cdc42 is a Ras-related small GTPase that is known to play roles in oncogenic processes such as cell growth, survival, invasion, and migration. A pan-dominant negative mutant overexpression approach to suppress Cdc42 and related pathways has previously shown a requirement for Cdc42 in Ras-induced anchorage-independent cell growth, however the lack of specificity of such approaches make it difficult to determine if effects are directly related to changes in Cdc42 activity or other Rho family members. Therefore, in order to directly and unambiguously address the role of Cdc42 in Ras-mediated transformation, tumor formation and maintenance, we have developed a model of conditional cdc42 gene in Ras-transformed cells. Loss of Cdc42 drastically alters the cell morphology and inhibits proliferation, cell cycle progression and tumorigenicity of Ras-transformed cells, while non-transformed cells or c-Myc transformed cells are largely unaffected. The loss of Cdc42 in Ras-transformed cells results in reduced Akt signaling, restoration of which could partially rescues the proliferation defects associated with Cdc42 loss. Moreover, disruption of Cdc42 function in established tumors inhibited continued tumor growth. These studies implicate Cdc42 in Ras-driven tumor growth and suggest that targeting Cdc42 is beneficial in Ras-mediated malignancies.
... Our strategy to isolate immortalized neural precursor cells was to transfer the gene encoding a MycER regulatable oncoprotein into a primary culture of p53deficient neural precursor cells given a continuous supply of bFGF. This approach is basically the same as the method for immortalizing hematopoietic progenitor cells from p53 knockout mice using the myc and raf oncogenes (Metz et al., 1995), as the raf-signal could be activated by bFGF in our system. This triple signal modulation (p53 null mutation, myc, and raf) would promote the proliferation of both hematopoietic and neural precursor cells. ...
In the mammalian central nervous system, a complexcircuit of neurons contributes to higher behaviors.Each region of the brain has a unique function derivedfrom various types of neurons. Several neuralprecursor cell lines have been established from basalganglia of fetal brain. In this study, hippocampalneural precursor cell lines were established from thehippocampus of p53-/- embryos. By means ofintegration of a MycER regulatable oncoprotein intop53-/- neural precursor cells, several immortallines were established from embryonic hippocampalprimordium, with bFGF and estrogen continuouslysupplied for activation of the MycER protein. A dualluciferase study demonstrated that the MycER proteinblocked the expression of a glial cell marker protein,GFAP, probably contributing to the persistent celldivision of the immortalized neural precursor cells.These cell lines differentiate into neuronal and glialcell types after withdrawal of bFGF. The phenotype ofthe hippocampal cell lines differed from that of thebasal ganglia cell lines as observed in a clonaldensity culture. This result implies that each regionof the brain has a unique developmental program, thatmay be imprinted in each of the neural precursor cells.
... In this regard, loss of p53 function enables oncogenic proteins such as Myc, RAF, BCR/ABL, and RAS to exert their transformation potential (15,26,27). In contrast, ectopic expression of oncogenic RAS in primary cells is associated with a transient stimulation of cell proliferation, followed by a permanent G1 arrest and features of early senescence (28). ...
Distinct genetic abnormalities (loss-of-function mutations of APC and p53 and oncogenic activation of Ki-ras) are associated with specific stages of the sporadic, most common types of colorectal tumors. However, the inability to maintain primary colon epithelial cells in culture has hindered the analysis of the pathogenetic role of these abnormalities in colorectal tumorigenesis. We have now established primary cultures of epithelial cells from the colon crypts of p53-deficient mice; these cells are nontumorigenic as indicated by their failure to form colonies in soft agar and to grow as tumors in immunodeficient SCID mice and in immunocompetent syngeneic hosts. Upon ectopic expression of an activated Ki-ras gene, p53-deficient colon epithelial cells form colonies in soft agar and highly invasive subcutaneous tumors in both immunodeficient and immunocompetent mice. Ectopic expression of wild-type p53, but not of a DNA-binding-deficient mutant, markedly suppressed the colony-forming ability of the Ki-ras-transformed p53-deficient epithelial cells. Together, these findings establish a functional synergism in colorectal tumorigenesis dependent on the effects of an oncogenic Ki-ras in a p53-deficient background. This model of tumorigenic conversion of colon epithelial cells might be useful to identify genetic changes associated with disease progression and to evaluate the therapeutic response to conventional and novel anticancer drugs.
Background
The expression of aquaporin 4 (AQP4) and intermediate filament (IF) proteins is altered in malignant glioblastoma (GBM), yet the expression of the major IF-based cytolinker, plectin (PLEC), and its contribution to GBM migration and invasiveness, are unknown. Here, we assessed the contribution of plectin in affecting the distribution of plasmalemmal AQP4 aggregates, migratory properties, and regulation of cell volume in astrocytes.
Methods
In human GBM, the expression of glial fibrillary acidic protein ( GFAP) , AQP4 and PLEC transcripts was analyzed using publicly available datasets, and the colocalization of PLEC with AQP4 and with GFAP was determined by immunohistochemistry. We performed experiments on wild-type and plectin-deficient primary and immortalized mouse astrocytes, human astrocytes and permanent cell lines (U-251 MG and T98G) derived from a human malignant GBM. The expression of plectin isoforms in mouse astrocytes was assessed by quantitative real-time PCR. Transfection, immunolabeling and confocal microscopy were used to assess plectin-induced alterations in the distribution of the cytoskeleton, the influence of plectin and its isoforms on the abundance and size of plasmalemmal AQP4 aggregates, and the presence of plectin at the plasma membrane. The release of plectin from cells was measured by ELISA. The migration and dynamics of cell volume regulation of immortalized astrocytes were assessed by the wound-healing assay and calcein labeling, respectively.
Results
A positive correlation was found between plectin and AQP4 at the level of gene expression and protein localization in tumorous brain samples. Deficiency of plectin led to a decrease in the abundance and size of plasmalemmal AQP4 aggregates and altered distribution and bundling of the cytoskeleton. Astrocytes predominantly expressed P1c, P1e, and P1g plectin isoforms. The predominant plectin isoform associated with plasmalemmal AQP4 aggregates was P1c, which also affected the mobility of astrocytes most prominently. In the absence of plectin, the collective migration of astrocytes was impaired and the dynamics of cytoplasmic volume changes in peripheral cell regions decreased. Plectin’s abundance on the plasma membrane surface and its release from cells were increased in the GBM cell lines.
Conclusions
Plectin affects cellular properties that contribute to the pathology of GBM. The observed increase in both cell surface and released plectin levels represents a potential biomarker and therapeutic target in the diagnostics and treatment of GBMs.
The zinc-finger transcription factor GATA-2 plays a critical role in maintaining the pool of early hematopoietic cells. To define its specific functions in the proliferation, survival, and differentiation of hematopoietic cells, we analyzed the hematopoietic potential of GATA-2−/− cells in in vitro culture systems for proliferation and maintenance of uncommitted progenitors or differentiation of specific lineages. From a two-step in vitro differentiation assay of embryonic stem cells and in vitro culture of yolk sac cells, we demonstrate that GATA-2 is required for the expansion of multipotential hematopoietic progenitors and the formation of mast cells, but dispensable for the terminal differentiation of erythroid cells and macrophages. The rare GATA-2−/− multipotential progenitors that survive proliferate poorly and generate small colonies with extensive cell death, implying that GATA-2 may play a role in both the proliferation and survival of early hematopoietic cells. To explore possible mechanisms resulting in the hematopoietic defects of GATA-2−/− cells, we interbred mutant mouse strains to assess the effects of p53 loss on the behavior of GATA-2−/− hematopoietic cells. Analysis of GATA-2−/−/p53−/− compound-mutant embryos shows that the absence of p53 partially restores the number of total GATA-2−/− hematopoietic cells, and therefore suggests a potential link between GATA-2 and p53 pathways.
Purpose:
The tumor suppressor p53 is a master regulator of apoptosis and also plays a key role in cell cycle checking. In our previous studies, we demonstrated that p53 directly regulates Bak in mouse JB6 cells and that p53-Bak signaling axis plays an important role in mediating EGCG-induced apoptosis. Furthermore, we have recently demonstrated that the same p53-Bak apoptotic signaling axis executes an essential role in regulating lens cell differentiation. In addition, we have also shown that p53 controls both transcription factors, C-Maf and Prox-1 as well as lens crystallin genes, αA, β- and γ-crystallins. Here, we have examined whether p53 also regulates other known target genes during its modulation of lens differentiation. The human and mouse lens epithelial cells, FHL124 and αTN4-1 were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin.
Methods:
Mice used in this study were handled in compliance with the "Protocol for the Care and Use of Laboratory Animals" (Sun Yat-sen University). Adult mice were used for the collection of lens cells. These samples were used for extraction of total proteins. A total of 32 embryonic mice {8 at 14.5 ED, 8 at 17.5 ED and 8 newborns for wild type} were used for immunohistochemistry, which were used for co-localization study. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J.
Results:
Immunohistochemistry revealed that both the cell cycle checking genes, p21 and Gadd45α and the apoptotic genes, Bcl-2 and PUMA, display developmental changes associated with p53 during mouse lens development. Knockdown of p53 in the mouse lens epithelial cells caused inhibition of lens differentiation. Associated with this inhibition, the cell cycle genes displayed significant downreglation, the apoptotic genes was also attenuated but to a much less degree. In addition, we found that bFGF can induce dose-dependent upregulation of the upstream kinases, CHK1/2 and ERK1/2, both known to phosphorylate p53 and activate the later. Furthermore, We showed that in both developing lens and human lens epithelial cells, p53 can be co-localized with the catalytic subunit of the protein phoshphatase-1 (PP-1), suggesting that PP-1 regulates p53 phosphorylation status both in vivo and in vitro.
Conclusion:
Taken together, our results suggest that during mouse lens development, p53 activity is regulated by ERK and CHK kinases-mediated activation, and by PP-1-mediated inactivation. p53 can regulate multiple groups of genes to mediate lens differentiation.
The tumor suppressor p53 is a master regulator of apoptosis and also plays a key role in cell cycle progress and cell differentiation. It mainly acts as a transcription factor. In addition, it can also directly interact with apoptosis regulators in mitochondria to control apoptosis. Recent studies from our laboratory and others have shown that p53 plays an active role in regulating lens differentiation. It does so by modulating developmental apoptosis and also controlling expression of lens differentiation-specific genes. In this chapter, we summary the current progresses in this field.
Neuronal and glial cells making the central nervous system are generated from central nervous system (CNS) stem cells during the development of CNS. CNS stem cells are potentially applicable for regeneration of damaged brain, such as in Parkinson’s Disease, or Alzheimer’s Disease. Recent studies have shown that mitogen and/or oncogene keep CNS stem cells proliferating. However, the long-term expansion of CNS stem cells needs a period of crisis, when almost all cells stop dividing and presumably wait for another intrinsic genomic change. The p53 tumor suppressor gene encodes a protein that is important in the regulation of cell proliferation and apoptosis. In this study, we used p53 knock-out embryos to analyze the effect of the p53 gene on apoptosis of the CNS stem cells. In a primary culture assay, p53 knock-out CNS stem cells are continuously growing in the presence of basic Fibroblast Growth Factor (bFGF), whereas expansion of wild-type CNS stem cells stopped shortly after cultivation due to substantial apoptosis. This indicates the critical role of the p53 locus on apoptosis of CNS stem cells. In this study, we have established immortalized CNS stem cells from p53 knock-out forebrain, MSP-1. To inhibit spontaneous differentiation during cell exparsion, MSP-1 had been introduced by c-Myc-Estrogen Receptor (ER) fusion gene, in which c-Myc can be conditionally activated by adding beta-estradiol. The integration of introducing c-Myc-ER has been confirmed in MSP-1 cells by Southern blot, Northern blot, and Western blot. The MSP-1 precursor cells express nestin and proliferate exponentially under the growing media, supplemented with bFGF, beta-estradiol and 10% FCS. The vigorous growth of MSP-1 may be supported both by c-Myc and the P53 null mutation. MSP-1 cells differentiate into neuronal and glial cell types after switching to the differentiating media (N2 serum-free media without growth factor) for 6 days. MSP-1 can be expanded from just one cell. Clonally expanded MSP-1 cells differentiated into neuronal and glial cell types after withdrawal of growth factors. The availability of immortal stem cells capable of neuronal differentiation offers an unusually powerful way of identifying the molecular basis of neuronal commitment.
Seit der zweiten Hälfte der 70er Jahre wird Krebs überwiegend als Ergebnis von genetischen Veränderungen angesehen, die in dominanter Art eine normale Zelle entgleisen lassen. Die daran beteiligten, zellulären Gene (Proto-Onkogene) sind mutiert, in Teilen deletiert, amplifiziert, rearrangiert oder sonstwie gegenüber dem Wildtyp verändert. Der große Gewinn dieses Ansatzes lag in den umfassenden neuen Erkenntnissen über die Wachstumsregulation von Zellen durch Rezeptor/Ligandensysteme und den daran beteiligten Signalübertragungsmechanismen.
Cell cultures are used extensively in many areas of biological research. Whereas the hazards to personnel associated with the use of infected tissues are well documented and appropriate safety measures have been introduced to minimize these risks, very little is known about the potential risks associated with the experimental manipulation of primary cultures or established cell lines. In particular, little information is available on which to base a reliable risk assessment of, for example, the experimental introduction of activated oncogenes into cell cultures or the targeted disruption of endogenous tumour suppressor genes [1]. This chapter seeks to address relevant safety aspects of experimental manipulations which are applied to cells in vitro: it is not intended as an exhaustive survey of the experimental procedures involved, but rather to provide an insight into some of the general principles that may aid in determining a level of risk.
Physiological cell death, known as apoptosis, is an evolutionarily conserved process that is required for normal development and function of multicellular organisms. Abnormalities in cell death control are implicated as a cause or contributing factor in a range of diseases, including cancer, autoimmunity, and degenerative disorders. Importantly, the propensity of a cell to undergo apoptosis is one of the determinants of the sensitivity of tumor cells to antineoplastic therapy. Apoptosis can be triggered by stress-induced signals that arise from within the doomed cell or by signals that are elicited by binding of extracellular "death ligands" to their "death receptors." Cysteine proteases have been recognized as essential effecters of all pathways to apoptosis. Experiments with transgenic mice and gene knockout mice have shown that different caspases and their adaptor molecules are needed for "death receptor" signaling and apoptotic pathways elicited by cytokine withdrawal, DNA damage, or corticosteroids. These differences allow the pathways to be regulated by distinct inhibitors. It has been published that chemotherapeutic drugs and gamma-radiation induce apoptosis by "death ligand"-mediated activation of "death receptors," but this model has been challenged. Our review discusses this controversy in the light of current knowledge of the molecular control of apoptosis.
It is well established that the tumor suppressor p53 plays major roles in regulating apoptosis and cell cycle progression. In addition, recent studies have demonstrated that p53 is actively involved in regulating cell differentiation in muscle, the circulatory system and various carcinoma tissues. We have recently shown that p53 also controls lens differentiation. Regarding the mechanism, we reveal that p53 directly regulates c-Maf and Prox1, two important transcription factors to control cell differentiation in the ocular lens. In the present study, we present further evidence to show that p53 can regulate lens differentiation by controlling expression of the differentiation genes coding for the lens crystallins. First, the alpha A and beta A3/A1 gene promoters or introns all contain putative p53 binding sites. Second, gel mobility shifting assays revealed that the p53 protein in nuclear extracts from lens epithelial cells directly binds to the p53 binding sites found in these crystallin gene promoters or introns. Third, exogenous wild type p53 induces dose-dependent expression of the luciferase reporter gene driven by different crystallin gene promoters and the exogenous dominant negative mutant p53 causes dose-dependent inhibition of the same crystallin genes. Fourth, ChIP assays revealed that p53 binds to crystallin gene promoters in vivo. Finally, in the p53 knockout mouse lenses, expression levels of various crystallins were found down-regulated in comparison with those from the wild type mouse lenses. Together, our results reveal that p53 directly regulates expression of different sets of genes to control lens differentiation.
In the past few years, substantial evidence has been put forth that suggests increasing complexity of the mechanism of action of cytotoxic, mutagenic or carcinogenic agents, commonly known as stress-inducers. The developing theme in a stress-induced cellular response is stimulation of a signal transduction pathway regulated by either cell growth and proliferation-related (survival) factors or programmed cell death-related (apoptotic) factors. The balance between the survival and apoptotic signals depends on the cell type, and dictates the fate of the cell. This review summarises current knowledge of the intracellular signal transduction responses elicited by ionising radiation in rodent and mammalian cells, and focuses on the involvement of Raf-1 protein serine-threonine kinase in the signalling pathways initiated by diverse stress-inducers including ionising radiation, ultraviolet radiation, growth factor-deprivation, and cytokines.
The Greek word apoptosis was used first by Hippocrates as a synonyme of dislocations of the bones, structural changes related to tissue, by Marcus Aurelius in political and social context as failure and decline. The physician Galen extended the medical meaning of apoptosis to wound healing and inflammation. Apoptosis, or cell suicide is an integral part of life cycle of plants and animals indicated by the loss of 140-190g (50-70 billion) cells each day in the human adult, amounting to the body weight in one year. The growing interest in apoptosis is indicated by the number of scientific publications since the 1990s which is now more than 140,000 and will exceed 160,000 by the end of 2008. The unique feature of this book is the use of synhronized and reversibly permeabilized cells allowing to visualize the dynamic nature of chromatin condensation through transitory chromatin and chromosomal forms including changes upon genotoxic treatment, which were not seen earlier. The chromatin condensation process is illustrated from string (DNA) to rope (chromosomes) in more than 160 figures. The interdisciplinary nature of studies summarized in the book facilitate the global view of readers interested in the higher order structure of nucleic acids. The wealth of additional information will attract a wide population of readers. The natural audience engaged in DNA research such as genetics, cell biology, biochemistry, molecular biology will find that it contains essential material.
The tumor suppressor, p53 regulates a large number of target genes to control cell proliferation and apoptosis. In addition, it is also implicated in the regulation of cell differentiation in muscle, the circulatory system and various carcinoma tissues. We have recently shown that p53 also controls lens differentiation. Regarding the mechanism, we reveal that p53 directly regulates several genes including c-Maf and Prox1, two important transcription factors for lens differentiation, and αA and βA3/A1, the lens differentiation markers. In the present study, we present evidence to show that the γA-crystallin gene distal promoter and the first intron also contain p53 binding sites and are capable of mediating p53 control during mouse lens development. First, gel mobility shifting assays revealed that the p53 protein in nuclear extracts from human lens epithelial cells (HLE) directly binds to the p53 binding sites present in the γA-crystallin gene. Second, the exogenous wild type p53 induces the dose-dependent expression of the luciferase reporter gene driven by the basic promoter containing the γA-crystallin gene p53 binding site. In contrast, the exogenous dominant negative mutant p53 causes a dose-dependent inhibition of the same promoter. Third, ChIP assays revealed that p53 binds to the γA-crystallin gene promoter in vivo. Finally, in the p53 knockout mouse lenses, the expression level of the γAcrystallin gene was found attenuated in comparison with that in the wild type mouse lenses. Together, our results reveal that p53 regulates γA-crystallin gene expression during mouse lens development. Thus, p53 directly regulates all 3 types of crystallin genes to control lens differentiation.
The human body consists of a huge number of cells. At the base of all these cells is one
fertilized oocyte. By cell divisions the number of cells is increased during development,
and cells specialize into various types of cells, i.e. muscle, brain and blood cells. Groups of
specialized cells form the various tissues. DNA contains the blueprint of all cells. By each
cell division the DNA is duplicated and equally distributed over the two daughter cells.
Hence, practically all the cells contain identical genetic information. The DNA in each cell
contains about 25.000 genes. Each gene contains information necessary to produce a particular
protein. These proteins are involved in many processes such as energy metabolism,
cell composition and gene regulation.
Although all cells contain the same genetic information, they specialize into various cell
types. For this, the genes are specifically activated or repressed. A tight regulation of the
genes makes it possible to specialize into a particular type of cell. Transcription factors play
an important role in this tight regulation of activating or repressing genes. EKLF is such a
transcription factor. EKLF is mainly expressed in red blood cells. These cells give blood
its red color. They carry oxygen from lungs to tissues in the body that require the oxygen
for energy metabolism. Red blood cells are filled with hemoglobin that actually binds the
oxygen. Each hemoglobin particle consists of two a-globin, two ß-globin and four heme
molecules. The genetic blueprint for the ß-globin gene is called the ß-globin locus. In the
mouse, this locus contains four globin genes. Two of them, the embryonic ß-globin genes,
are active during embryonic development of the mice and the other two, the adult ß-globin
genes are activated later in development and stay active during the rest of the lifespan of the
mouse. Apart from the four ß-globin genes, the locus contains regulatory elements. These
include the Locus Control Region (LCR), and a number of other hypersensitive sites. The
LCR is required for a proper regulation of the globin genes; in absence of the LCR the genes
are not activated, and mutations in the LCR can lead to impaired activation. The LCR is
located relatively far from the genes it controls. However, when a globin gene is active in
red blood cells, the LCR is physically in close proximity to this gene. In fact, the globin
locus forms a spatial organization that consists not only of the active gene and the LCR,
but also of the other hypersensitive sites that are located even further away on both sides
of the genes. This structure is called the Active Chromatin Hub (ACH). Prior to the stage
in which the genes are active, a substructure of this ACH is formed. Unlike the complete
ACH, this substructure does not contain the globin genes and part of the LCR. In Chapter
4 we show that a similar substructure is found in red blood cells that are EKLF deficient.
We demonstrated that EKLF is necessary for the completion of the ACH, a requirement for
activating the genes.
The transcription factor EKLF is necessary for the proper development of red blood cells;
apart from the regulation of the ß-globin genes it regulates also other genes. However, it
was not known which other genes. To study the genes EKLF activates (or represses), we
used a culture method with erythroid progenitor cells. These progenitor cells are restricted
to the red blood cell lineage, but have not yet developed to fully differentiated, hemoglobin-
containing red blood cells. The culture method allows us to culture the cells as progenitors
or induce them to start their terminal differentiation to red blood cells.
Telomerase activity constitutes an important mechanism in stabilizing the structure of telomeres and the genome. Activation of telomerase requires de novo transcription of telomerase reverse transcriptase (TERT) gene, appropriate splicing of TERT mRNA, and post-translational modification and incorporation of full-length TERT protein into the holoenzyme. Regulation thus can occur at multiple levels, sites and stages, each involving complex interactions and cooperation. The result is the precise temporal and spatial pattern of telomerase activity throughout cell proliferative cycles during development and oncogenesis. The regulation of TERT gene expression is likely to be primarily at the promoter level; oncogenes and tumor suppressor genes including the c-Myc/Max/Mad system regulate the human telomerase reverse transcriptase (hTERT) gene expression. Post-translation, hTERT is imported into the nucleus and interacts with 14-3-3 proteins thus avoiding being exported by chromosome region maintenance 1 (CRM1). In the presence of heat shock proteins, telomerase undergoes appropriate assembly and activation with serine/threonine phosphorylation by protein kinase C (PKC) and protein kinase B (PKB), Telomerase activity is inhibited by hTERT gene down-regulation, intra-molecular peptide interactions, the tumor suppressor p53, specific dephosphorylation by protein phosphatase 2A (PP2A), and tyrosine phosphorylation by tyrosine kinase c-Abl.
Oncogenic ras can transform most immortal rodent cells to a tumorigenic state. However, transformation of primary cells by ras requires either a cooperating oncogene or the inactivation of tumor suppressors such as p53 or p16. Here we show that expression of oncogenic ras in primary human or rodent cells results in a permanent G1 arrest. The arrest induced by ras is accompanied by accumulation of p53 and p16, and is phenotypically indistinguishable from cellular senescence. Inactivation of either p53 or p16 prevents ras-induced arrest in rodent cells, and E1A achieves a similar effect in human cells. These observations suggest that the onset of cellular senescence does not simply reflect the accumulation of cell divisions, but can be prematurely activated in response to an oncogenic stimulus. Negation of ras-induced senescence may be relevant during multistep tumorigenesis.
It is well established that the tumor suppressor p53 plays major roles in regulating apoptosis and cell cycle progression. In addition, recent studies have demonstrated that p53 is actively involved in regulating cell differentiation in muscle, the circulatory system and various carcinoma tissues. We have recently shown that p53 also controls lens differentiation. Regarding the mechanism, we reveal that p53 directly regulates c-Maf and Prox1, two important transcription factors to control cell differentiation in the ocular lens. In the present study, we present further evidence to show that p53 can regulate lens differentiation by controlling expression of the differentiation genes coding for the lens crystallins. First, the aA and bA3/A1 gene promoters or introns all contain putative p53 binding sites. Second, gel mobility shifting assays revealed that the p53 protein in nuclear extracts from lens epithelial cells directly binds to the p53 binding sites found in these crystallin gene promoters or introns. Third, exogenous wild type p53 induces dose-dependent expression of the luciferase reporter gene driven by different crystallin gene promoters and the exogenous dominant negative mutant p53 causes dose-dependent inhibition of the same crystallin genes. Fourth, ChIP assays revealed that p53 binds to crystallin gene promoters in vivo. Finally, in the p53 knockout mouse lenses, expression levels of various crystallins were found down-regulated in comparison with those from the wild type mouse lenses. Together, our results reveal that p53 directly regulates expression of different sets of genes to control lens differentiation.
We have examined the effects of inactivation of the p53 tumor suppressor gene on the incidence of apoptotic cell death in two stages of the adenoma-to-carcinoma progression in the
intestine: in early adenomas where p53 mutations are rare and in highly dysplastic adenomas where loss of p53 occurs frequently. Homozygosity for an inactivating germ-line mutation of p53 had no effect on the incidence or the rate of progression of ApcMin/+-induced adenomas in mice and also did not affect the frequency of apoptosis in the cells of these adenomas. To examine the
effect of p53 loss on apoptosis in late-stage adenomas, we compared the incidence of apoptotic cell death before and after the appearance
of highly dysplastic cells in human colonic adenomas. The appearance of highly dysplastic cells, which usually coincides during
colon tumor progression with loss of heterozygosity at the p53 locus, did not correlate with a reduction in the incidence of apoptosis. These studies suggest that p53 is only one of the genes that determine the incidence of apoptotic in colon carcinomas and that wild-type p53 retards the progression of many benign colonic adenoma to malignant carcinomas by mechanism(s) other than the promotion of
apoptosis.
The tumor suppressor p53 plays a key role in regulating apoptosis and cell cycle progression. In addition, p53 is implicated in control of cell differentiation in muscle, the circulatory system, ocular lens and various carcinoma tissues. However, the mechanisms by which p53 controls cell differentiation are not fully understood. Here we present evidence that p53 directly regulates c-Maf and Prox1, two important transcription factors controlling differentiation in the ocular lens. First, human and murine c-Maf and Prox1 gene promoters contain authentic p53 DNA binding sites. Second, p53 directly binds to the p53 binding sites found in the promoter regions. Third, exogenous p53 induces dose-dependent expression of the luciferase report gene driven by both c-Maf and Prox1 promoters, and p53 binds to both promoters in the ChIP assays. Fourth, in the in vitro differentiation model, knockdown of p53 significantly inhibits lens differentiation which is associated with downregulated expression of c-Maf and Prox1. Finally, in p53 knockout mice, the expression of c-Maf and Prox1 are significantly altered. Together, our results reveal that p53 regulates lens differentiation through modulation of two important transcription factors, c-Maf and Prox1, and through them p53 thus controls expression of various differentiation-related downstream crystallin genes.
In vitro cell transformation is a valuable approach for studying the mechanisms of multistep carcinogenesis of human cells. Since immortalization is an essential step for in vitro neoplastic transformation of human cells, this study addresses the question of whether mutant p53 contributes to the immortalization process of human cells. The mutant p53 gene (mp53: codon273Arg-His) was introduced into normal human fibroblasts (OUMS-24 line) and a G418-resistant clone, OUMS-24/P6 line, was obtained. This clone showed an extended life span and chromosome abnormalities, but senesced at the 79th population doubling level (PDL). When these cells were subjected to intermittent X-ray treatment, they became an immortalized cell line (OUMS-24/P6X). Although these immortalized cells showed chromosome abnormalities, they were not tumorigenic. On the other hand, normal OUMS-24 cells into which mp53 had not been introduced were not immortalized by the same X-ray treatment. These results indicate that introduction and expression of mp53 alone were not sufficient for immortalization of human cells, and that mutations of the remaining wild-type p53 or other genes may have been necessary for immortalization. In fact, no expression of the wild-type p53 was detected in the immortalized cells by RT-PCR. Expression of p21, which is located downstream of p53, was remarkably reduced in the immortalized cells, resulting in an increase in cdk2 and cdc2 kinase activity. These findings indicate that the p53-p21 cascade may play some role in the immortalization of human cells. On the other hand, there was no significant difference in expression of proteins such as Rb, p16, cdk4, cdk6, cyclin A and cyclin D1 between the normal and immortalized human fibroblasts. © 1997 Wiley-Liss, Inc.
BACKGROUND
Alterations of the p53 gene and of MDM2, a gene coding for a p53 binding protein, have been implicated in the pathogenesis of osteosarcoma (OS).METHODS
To determine the frequency of alterations of the p53/MDM2 pathway in OS and their possible correlation with clinicopathologic features, MDM2 copy number and p53 protein levels were determined in a series of 83 samples of OS by quantitative Southern blot analysis and immunohistochemistry, respectively.RESULTSPositivity for p53 was found in 26.5% and MDM2 amplification in 6.6% of the samples analyzed in a mutually exclusive fashion with one exception. Overall, alterations of the p53/MDM2 pathway occurred in 34% of cases; p53 accumulation was not associated with a higher proliferative rate. The mean age of patients with p53 positive OS (40 years) was older than that of the p53 negative group (28 years) (P < 0.04). Furthermore, three of the four cases of OS arising in Paget's disease showed p53 accumulation.CONCLUSIONS
Alterations of the p53/MDM2 pathway are frequent in OS and usually represent mutually exclusive tumorigenic events. p53 does not appear to be a major determinant of proliferative rate in OS. Cancer 1997; 79:1541-7. © 1997 American Cancer Society.
The incidence of postmenopausal osteoporosis is increasing as the population ages. Even though estrogen replacement therapy has proven beneficial in reducing the number of skeletal fractures, the known risks and associated side-effects of estrogen replacement therapy make compliance poor. Recent research has focused on the development of tissue specific estrogen agonist/anatagonists such as droloxifene which can prevent estrogen deficiency-induced bone loss without causing uterine hypertrophy. Furthermore, droloxifene acts as a full estrogen antagonist on breast tissue and is being evaluated for treatment of advanced breast cancer. In this report we propose a common mechanism of action for droloxifene that underlies its estrogen agonist and antagonist effects in different tissues. Droloxifene and estrogen, which have identical effects on bone in vivo, both induced p53 expression and apoptosis in cells of in vitro rat bone marrow cultures resulting in a decrease in the number of bone-resorbing osteoclasts. Droloxifene is growth inhibitory in MCF-7 human breast cancer cells and therefore acts as an antagonist, whereas estrogen is mitogenic to these cells and acts as an agonist. Droloxifene, but not estrogen, induced p53 expression and apoptosis in MCF-7 cells. These results indicate that the induction of apoptosis by droloxifene may be the common mechanism for both its estrogen agonist effects in bone and its antagonist effects in breast tissue. J. Cell. Biochem. 65:159–171. © 1997 Wiley-Liss, Inc.
The tumor suppressor p53 is a master regulator of apoptosis and also plays a key role in cell cycle checking. In our previous studies, we demonstrated that p53 directly regulates Bak in mouse JB6 cells (Qin et al. 2008. Cancer Research. 68(11):4150) and that p53-Bak signaling axis plays an important role in mediating EGCG-induced apoptosis. Here, we demonstrate that the same p53-Bak apoptotic signaling axis executes an essential role in regulating lens cell differentiation. First, during mouse lens development, p53 is expressed and differentially phosphorylated at different residues. Associated with p53 expression, Bak is also significantly expressed during mouse lens development. Second, human p53 directly regulates Bak promoter and Bak expression in p53 knockout mice (p53-/-) was significantly downregulated. Third, during in vitro bFGF-induced lens cell differentiation, knockdown of p53 or Bak leads to significant inhibition of lens cell differentiation. Fourth, besides the major distribution of Bak in cytoplasm, it is also localized in the nucleus in normal lens or bFGF-induced differentiating lens cells. Finally, p53 and Bak are co-localized in both cytoplasm and nucleus, and their interaction regulates the stability of p53. Together, these results demonstrate for the first time that the p53-Bak apoptotic signaling axis plays an essential role in regulating lens differentiation.
We are investigating cooperating genetic events in the genesis of breast cancer, using the mouse as a model system. We have shown cooperativity between a mutant allele of p53 (p53-172H) and overexpressed ErbB2 in mammary tumorigenesis in transgenic mice. We are now performing additional crosses to further examine oncogene cooperativity with ErbB2 and p53-172H. We attempted to test the dominant oncogenic potential of p53-172H in an in vivo setting by crossing the p53-172H transgene together with ErbB2 onto either a p53−/− or a p53+/− background. We show that the p53-172H allele and the heterozygous p53 genotype have an identical impact on the latency of ErbB2-induced mammary tumors; there was no evidence of additivity or synergy between p53-172H and the p53+/− genotype. On the p53−/− background, we obtained no mammary tumors due to the early onset of lymphomas and sarcomas, thus precluding assessment of the effect of the p53-172H transgene on mammary tumorigenesis in a p53-null background. Thus, in this in vivo model for breast cancer, we failed to find evidence that p53-172H can function as a dominant oncogenic allele, but rather found support for its being essentially equivalent to a null allele in its impact on ErbB2-induced mammary tumorigenesis. By comparative genome analysis, we showed that a common feature of tumors arising in ErbB2/mutant p53 mice (p53-null allele with or without p53-172H) is a loss of chromosome 4, a feature of many epithelial tumors in mice and one that is consistent with a role for loss of INK4a/ARF in such tumors. We also attempted to accelerate ErbB2-induced mammary tumorigenesis with mouse mammary tumor virus (MMTV) proviral tagging mutagenesis, but we were surprised to find that mice with MMTV alone had the same latency as mice with both MMTV and ErbB2, indicating no cooperativity between ErbB2 and MMTV. This may have been due to the mixed C3H/HeN × FVB strain background used in this cross.
The isolation and characterization of 25 strains of human diploid fibroblasts derived from fetuses are described. Routine tissue culture techniques were employed. Other than maintenance of the diploid karyotype, ten other criteria serve to distinguish these strains from heteroploid cell lines. These include retention of sex chromatin, histotypical differentiation, inadaptability to suspended culture, non-malignant characteristics in vivo, finite limit of cultivation, similar virus spectrum to primary tissue, similar cell morphology to primary tissue, increased acid production compared to cell lines, retention of Coxsackie A9 receptor substance, and ease with which strains can be developed. Survival of cell strains at - 70 °C with retention of all characteristics insures an almost unlimited supply of any strain regardless of the fact that they degenerate after about 50 subcultivations and one year in culture. A consideration of the cause of the eventual degeneration of these strains leads to the hypothesis that non-cumulative external factors are excluded and that the phenomenon is attributable to intrinsic factors which are expressed as senescence at the cellular level. With these characteristics and their extremely broad virus spectrum, the use of diploid human cell strains for human virus vaccine production is suggested. In view of these observations a number of terms used by cell culturists are redefined.
Clones of mortal chicken fibroblasts and erythroblasts transformed by temperature-sensitive v-src and v-erb B oncoproteins have been developed into immortal cell lines that retain the conditional transformed phenotype. The expressions of two tumor suppressor genes, the retinoblastoma (Rb) gene and the p53 gene, were investigated during senescence, crisis, and cell line establishment. In temperature-sensitive (ts)-v-erb B erythroblasts and ts-v-src fibroblasts (as well as in v-myc macrophages), loss of p53 mRNA or expression of a mutated p53 gene invariably occurred in the early phase of immortalization. In contrast, expression of the Rb gene was unchanged at all stages of immortalization. Inactivation of the original temperature-sensitive oncogene led to loss of the transformed phenotype in fibroblasts and to differentiation in erythroblasts, even in lines that were immortal and lacked p53. The results demonstrate that the process of immortalization is distinct from cell transformation, probably requiring different mutational events.
Murine bone marrow cells infected with replication-defective retroviruses containing v-raf alone or v-myc alone yielded transformed
pre-B cell lines, while a retroviral construct containing both v-raf and v-myc oncogenes produced clonally related populations
of mature B cells and mature macrophages. The genealogy of these transformants demonstrates that mature myeloid cells were
derived from cells with apparent B-lineage commitment and functional immunoglobulin rearrangements. This system should facilitate
studies of developmental relationships in hematopoietic differentiation and analysis of lineage determination.
Families of patients with the Li-Fraumeni cancer syndrome have an inherited pattern of sarcomas and various other types of cancers that follow a dominant mode of transmission, an early age of onset, and exhibit multiple primary tumors. As soft tissue sarcomas (including fibrosarcomas) are frequently observed with this syndrome, the in vitro growth characteristics of fibroblasts derived from skin biopsies of Li-Fraumeni syndrome patients were studied. Control fibroblasts maintained a normal morphology and eventually senesced in culture. Fibroblasts from seven of eight affected individuals developed changes in morphology, anchorage-independent growth, and chromosomal abnormalities. In a fashion similar to that of fibroblasts from normal donors they underwent a growth crisis during which their growth was slow, but they continued to grow past the point at which control samples had stopped dividing (35 population doublings). Fibroblasts from Li-Fraumeni cancer patients escape senescence, growing well beyond 35 population doublings with growth rates similar to early-passage cells. Patient fibroblasts maintain the morphology of a transformed cell but remain nontumorigenic in nude mice. These observations of the behavior of fibroblasts from patients with the Li-Fraumeni syndrome may have predictive value for the determination of gene carriers within these families who are at high risk of cancer.
In vitro infection of murine fetal liver cells with a retrovirus containing v-raf and v-myc oncogenes has produced continuous lines of immature erythroid cells that are leukemogenic. These cells synthesized a factor that stimulated their growth in vitro before autonomous variants emerged. Approximately 1000 high-affinity erythropoietin receptors could be detected per cell, and the hormone induced terminal differentiation in these cells. The lines were generated at an extremely low frequency (approximately 1 in 10(7) cells), suggesting that the combination of raf and myc is insufficient to develop erythroid cell lines and that additional events are necessary for transformation.
A comparison was made of the in vitro erythroid colony-forming abilities of v-raf-, v-myc-, and v-raf/v-myc-containing retroviruses. In methylcellulose, v-raf efficiently produced colonies of well-differentiated hemoglobin-synthesizing erythroid cells, whereas v-raf/v-myc-infected erythroid cells were inhibited from terminally differentiating but retained the ability to replicate extensively. In contrast, v-myc was unable to stimulate the formation of erythroid colonies.
A murine retrovirus (MRSV) containing the src gene of Rous sarcoma virus has been shown to cause an erythroproliferative disease in mice (S. M. Anderson and E. M. Scolnick, J. Virol. 46:594-605, 1983). We now demonstrate that this same virus can transform erythroid progenitor cells in vitro. Infection of fetal liver cells or spleen and bone marrow cells from phenylhydrazine-treated adult mice gave rise to colonies of erythroid cells which grew in methylcellulose under conditions not favorable for the growth of normal erythroid cells. The presence of pp60src in the transformed erythroid cells was demonstrated by an immune complex protein kinase assay. The time course of appearance and subsequent differentiation of erythroid colonies indicated that the target cell for MRSV was a 6- to 8-day burst-forming unit. Differentiation of the erythroid progenitors was not blocked by the presence of pp60src, and the cells retained sensitivity to the hormone erythropoietin. In fact, the transformed cells exhibited increased hormone sensitivity since the number, the size, and the extent of hemoglobinization of the colonies were all increased by the addition of small amounts of erythropoietin. MRSV was not susceptible to restriction by the Fv-2 locus, as MRSV could transform hematopoietic cells from C57BL/6 mice. These results indicate that (i) the erythroid proliferation observed in vivo is caused by a direct effect of MRSV on erythroid progenitors and (ii) the transformed erythroid precursors acquire a growth advantage over uninfected cells without losing the ability to differentiate and respond to physiologic regulators.
Synthesis of DNA at chromosome ends by telomerase may be necessary for indefinite proliferation of human cells. A highly sensitive
assay for measuring telomerase activity was developed. In cultured cells representing 18 different human tissues, 98 of 100
immortal and none of 22 mortal populations were positive for telomerase. Similarly, 90 of 101 biopsies representing 12 human
tumor types and none of 50 normal somatic tissues were positive. Normal ovaries and testes were positive, but benign tumors
such as fibroids were negative. Thus, telomerase appears to be stringently repressed in normal human somatic tissues but reactivated
in cancer, where immortal cells are likely required to maintain tumor growth.
In this review, we have reported on the distribution of genetic alterations detected in the various hematologic malignancies. In addition to the 235 specific point mutations scored, gross changes in the structure of p53 were also reported. Figure 3 represents a summary of the distribution of the 235 p53 mutations detected in a variety of hematologic fresh samples and established cell lines. This group includes the following malignancies: ALL, AML, ATCL, B-CLL, CML, lymphoma, MDS, and MM. Analysis of the various genetic events led us to the following generalizations. (1) The majority of point mutations map to conserves regions II-V (mainly to conserved region IV and V). The dinucleotide CpG, although underrepresented in DNA, is a hot spot and accounts for 34% (79/235) of events in hematologic malignancies. More than 86% of these were transitions from G to A or C to T transitions, respectively. These results are in agreement with those reported in the literature. CpG dinucleotides are preferentially involved in spontaneous mutations and result from methylation-induced deamination of 5 methyl cytosine. The remaining mutations are relatively evenly distributed between G:C (scored after subtraction of mutations occurring at CpG) and A:T, with a slight preference of G or C basepair in MDS and lymphoma patients. Of note are frequent G:C to A:T transitions, not localized in CpG in cases of MM patients (7/14 patients). These genetic changes were reported to be induced by DNA damaging agents. (2) The most common mutations are transitions (in which purine is substituted for a purine or a pyrimidine for a pyrimidine) followed by transversions (in which a purine is substituted for a pyrimidine or vise versa) and deletions/insertions. (3) Arginine is a preferential target of the mutations (76 times), mainly at the following codons: 248, 273, 175, 213, 282. Because arginine is frequently coded by CGN, it is not surprising that this amino acid is a preferential target of spontaneous mutations. Additional amino acids that are frequent targets for mutations are glycine, aspartic acid, aspargine, methionine, and cysteine, which are hit at the following codons, respectively: 245, 281, 239, 237, and 176. In conclusion although the number of cases reported in the literature cited in the present review are rather small, it is clear that the DNA lesions occurring in the p53 gene in hematologic malignancies seem to fall in a similar pattern as those observed in other malignancies. Both the types of mutations and their distribution in the p53 protein structure agree with the general trend of mutations observed in malignancies of other tissues. Because most mutations seem to span the central part of the p53 protein, which was suggested to be involved in DNA binding, it is tempting to speculate that the mechanism by which p53 is inactivated is connected with conformational protein modification that affects the interactions of the p53 protein and the specific DNA targets. Development of the p53 field may advance two major aspects of cancer therapy in general. The first is associated with early diagnosis and prognosis of cancer patients and the second is related to the development of potential gene therapy that will restore the inactivated p53 in cancer cells. Elucidation of the biochemical and biologic functions of p53, along with the discovery of alterations in human cancer in epidemiologic studies, led to the development of functional screening tests to identify carriers of p53 germline mutations. Hopefully, the analysis of spectrum of p53 mutations will provide us with hypothesis regarding the environmental factors that contribute to development of human leukemia and lymphoma. Finally, the clinicians have included p53 in the arsenal of the clinical tools that enable detection of residual disease, establishing more accurate prognosis and remission.
Wild-type p53 is a tumor-suppressor gene that can induce cell death by apoptosis when expressed in myeloid leukemic and some other types of tumor cells. However, the question remained as to what extent wild-type p53 is a mediator of apoptosis in normal cells. We have used mice deficient in wild-type p53 to determine whether induction of apoptosis in hematopoietic cells from these p53 deficient mice is defective. We show here that bone marrow myeloid progenitor cells from p53-deficient mice are more resistant to induction of apoptosis when there was only a low concentration of the viability factors granulocyte-macrophage colony-stimulating factor; interleukins-1 alpha, -3, and -6; or stem cell factor; or when apoptosis was induced in these cells by irradiation or heat shock. The loss of one allele of wild-type p53 was sufficient for increased resistance. The higher resistance to apoptosis in p53-deficient mice was also found in irradiated thymocytes, but not in thymocytes treated with dexamethasone or in mature peritoneal granulocytes. The degree of resistance in irradiated myeloid progenitors and thymocytes showed a dosage effect of the number of wild- type p53 genes. The results show that wild-type p53 is involved in the induction of apoptosis by some agents in normal hematopoietic cells. Loss of wild-type p53 can, therefore, contribute to tumor development by decreasing cell death at low concentrations of viability factors and after exposure to a DNA-damaging agent. The results also show that there are wild-type p53-dependent and -independent pathways of normal cell apoptosis.
Gene amplification occurs at high frequency in transformed cells (10(-3)-10(-5)), but is undetectable in normal diploid fibroblasts (less than 10(-9)). This study examines whether alterations of one or both p53 alleles were sufficient to allow gene amplification to occur. Cells retaining one wild-type p53 allele mimicked the behavior of primary diploid cells: they arrested growth in the presence of drug and failed to demonstrate amplification. Cells losing the second p53 allele failed to arrest when placed in drug and displayed the ability to amplify at a high frequency. Thus, loss of wild-type p53 may lead to amplification, possibly caused by changes in cell cycle progression. Other determinants can by-pass this p53 function, however, since tumor cells with wild-type p53 have the ability to amplify genes.
The hematopoietic target cells of the three prototype strains of replication defective avian leukemia viruses (DLVs) were studied, using a newly developed, quantitative in vitro transformation assay. Our results show that the target cells of avian erythroblastosis virus (AEV) belong to the erythroid lineage while those of myelocytomatosis virus 29 (MC29) and avian myeloblastosis virus (AMV) belong to the myeloid lineage. As judged from suicide experiments using BUdR incorporation and irradiation with visible light, a higher proportion of AEV- and AMV-target cells are in cycle than MC29-target cells. Using differentiation specific antisera directed against cell surface antigens, we could demonstrate that the target cells of AEV express erythroblast-specific antigen(s) and less intensively erythrocyte-specific antigen(s), while those of MC29 and AMV express myeloblast-specific antigen(s). In addition, MC29-target cells express macrophage-specific antigen(s). None of the AEV-target cells are adherent or phagocytic, while a small proportion of the AMV-target cells are adherent and about half of the MC29-target cells are both adherent and phagocytic. Our results support the concept that DLVs specifically transform certain types of committed erythroid and myeloid progenitor cells. The target cells of AEV and AMV appear to resemble the respective transformed cells in their state of differentiation, whereas those of MC29 appear to be more immature than the corresponding transformed cells.
A method for cloning of chicken embryo fibroblasts (CEF) was developed, yielding a cloning efficiency of up to 50% without use of feeder cells or conditioned medium. An analysis of the growth potential of over 200 randomly selected clones showed that only approx. 4% of the clones were capable of doubling more than 35 times before undergoing cellular senescence. A positive correlation between initial growth rate and in vitro lifespan was observed. This served as a basis for a simple selection procedure for fibroblast strains suitable for long-term culturing. None of over 200 clones thus isolated could be established into a line. Subclones from clonal CEF strains were more homogeneous than uncloned CEF cultures with respect to morphology and growth behaviour, but still heterogeneous in their in vitro life span. All fibroblast strains tested could be effectively infected and transformed by a variety of avian sarcoma and leukosis viruses.
We have asked whether p53 mutations are involved in the process of spontaneous immortalization of mouse embryo cells. Cells from Swiss mouse embryos were used to prepare 3T3 and 3T12 lines according to the protocol of Todaro & Green [(1963). J. Cell Biol., 17, 299-313]. After the cells emerged from crisis, p53 sequences were amplified by polymerase chain reaction (PCR) from both RNA and DNA. The sequence of the aggregated cDNA from each of six 3T3 lines showed no evidence of mutation. PCR-amplified p53 cDNA from two 3T3 lines was cloned, and individual clones in M13mp19 were partially sequenced. One cell ine showed a single, non-coding nucleotide change in 2/8 independent clones. Nine cDNA clones from the second 3T3 lines were sequenced, and no single nucleotide changes appeared more than once. The mutations which appeared only once were not detected in clones of genomic DNA. Since these apparent mutations are probably reverse transcriptase or Taq polymerase errors, we conclude that both the 3T3 lines contained only wild-type p53. In two out of three independent 3T12 lines however, missense mutations were readily observed in the aggregate cDNA sequence. Restriction fragment length polymorphism and Southern blot analyses of the genomic DNA indicated that these cells were homozygous for the mutations. The p53 protein molecules in four cell lines were analysed by immunoprecipitation: one 3T12 line showed the pattern of antibody reactivity characteristic of some p53 mutants, while the others displayed the wild-type pattern. We conclude that p53 mutations arise and are strongly selected for during immortalization according to the 3T12 but not the 3T3 protocol.
Gene amplification occurs at high frequency in transformed cells (10(-3)-10(-5)), but is undetectable in normal diploid fibroblasts (less than 10(-9)). This study examines whether alterations of one or both p53 alleles were sufficient to allow gene amplification to occur. Cells retaining one wild-type p53 allele mimicked the behavior of primary diploid cells: they arrested growth in the presence of drug and failed to demonstrate amplification. Cells losing the second p53 allele failed to arrest when placed in drug and displayed the ability to amplify at a high frequency. Thus, loss of wild-type p53 may lead to amplification, possibly caused by changes in cell cycle progression. Other determinants can by-pass this p53 function, however, since tumor cells with wild-type p53 have the ability to amplify genes.
Loss of cell cycle control and acquisition of chromosomal rearrangements such as gene amplification often occur during tumor progression, suggesting that they may be correlated. We show here that the wild-type p53 allele is lost when fibroblasts from patients with the Li-Fraumeni syndrome (LFS) are passaged in vitro. Normal and LFS cells containing wild-type p53 arrested in G1 when challenged with the uridine biosynthesis inhibitor PALA and did not undergo PALA-selected gene amplification. The converse occurred in cells lacking wild-type p53 expression. Expression of wild-type p53 in transformants of immortal and tumor cells containing mutant p53 alleles restored G1 control and reduced the frequency of gene amplification to undetectable levels. These studies reveal that p53 contributes to a metabolically regulated G1 check-point, and they provide a model for understanding how abnormal cell cycle progression leads to the genetic rearrangements involved in tumor progression.
It has been shown previously that mutant p53 can act as an immortalizing gene when cotransfected into primary rat embryo fibroblasts along with a selectable marker. To determine whether a mutation at the p53 locus is a common event in the pathways leading to spontaneous cellular immortalization, 11 clonally derived BALB/c murine embryo fibroblast lines were established by passage on a 3T3 schedule and examined for p53 alterations. By the following criteria, all 11 independently established lines contain at least one mutant allele of p53. Seven of these lines have a PAb240-reactive p53 species and exhibit an extended p53 half-life as determined by pulse-chase analysis. The p53 protein species in a subset of these lines is also capable of complex formation with the constitutive heat shock protein hsc70. p53 cytoplasmic DNAs (cDNAs) from several of these lines have been cloned by reverse transcription of cytoplasmic RNA followed by PCR amplification, and the mutations have been mapped by DNA sequence analysis. Point mutation in conserved domains of p53 appears to be a common alteration in these lines, although one established line carries a 24-bp in-frame deletion of p53. The remaining four cell lines do not express detectable p53 protein. For each line there is a different molecular event underlying the lack of p53 expression: (1) deletion of at least the first 6 exons of both p53 alleles; (2) expression of a single p53 mRNA encoding a stop codon at amino acid position 173; (3) no detectable p53 mRNA; and (4) greatly diminished expression of p53 mRNA. These findings indicate that p53 alteration commonly occurs in spontaneously immortalized BALB/c mouse embryo fibroblasts passaged on a 3T3 schedule and, therefore, may be an important event for the immortalization process.
The transforming potential of the c-myc gene is shown here, for the first time, to include murine erythroid cells. Continuously growing cell lines were reproducibly generated by infection of day 13 CBA fetal liver cells with novel recombinant c-myc retroviruses. By cytostaining, most cells resembled early erythroblasts, but certain lines also contained significant numbers of hemoglobinized cells. RNA analysis revealed substantial expression of the genes encoding beta-globin and the erythroid-specific transcription factor GF-1. Although apparently immortal, the lines were not initially transplantable. Thus, constitutive myc expression in early erythroid cells can enhance their self-renewal capacity but is insufficient to fully transform them. The cell lines proliferated without the addition of exogenous factors, but their clonogenicity in semisolid medium was enhanced in the presence of erythropoietin, interleukin 3, and/or leukemia-inhibitory factor. In combination with either interleukin 3 or erythropoietin, leukemia-inhibitory factor also facilitated differentiation of certain lines. These results suggest that leukemia-inhibitory factor may have a previously unsuspected role in the regulation of erythropoiesis and could be considered as a possible therapeutic agent for the clinical management of erythroleukemia.
Early death is the fate of most developing T lymphocytes. Because bcl-2 can promote cell survival, we tested its impact in mice expressing an E mu-bcl-2 transgene within the T lymphoid compartment. The T cells showed remarkably sustained viability and some spontaneous differentiation in vitro. They also resisted killing by lymphotoxic agents. Although total T cell numbers and the rate of thymic involution were unaltered, the response to immunization was enhanced, consistent with reduced death of activated T cells. No T cells reactive with self-superantigens appeared in the lymph nodes, but an excess was found in the thymus. These observations, together with previous findings on B cells, suggest that modulated bcl-2 expression is a determinant of life and death in normal lymphocytes.
Avian erythroblastosis virus (AEV) is a replication-defective retrovirus that transforms erythroid and fibroblast cells in vitro and in vivo. The transforming ability of AEV is due primarily to the oncogene v-erb-B. A recombinant murine retrovirus has been constructed by inserting a chimeric gag-v-erb-B gene into a Moloney murine leukemia virus based vector. This retrovirus was used to examine v-erb-B-induced transformation of murine hematopoietic cells. Infection of murine primary fetal liver, adult bone marrow or adult spleen cells with the recombinant virus generated large hemoglobinized erythroid colonies in the absence of exogenous growth factors. Generation of such colonies usually requires the presence of erythropoietin (Epo) and interleukin-3 (IL-3). These growth-factor independent colonies were shown to be derived from early (BFU-E) and not late (CFU-E) erythroid progenitor cells, and the effect was not attributable to growth factors elicited by the virus-producing cell lines. In order to confirm that the recombinant virus was responsible for this transformation of BFU-E to growth factor independence, bone marrow cells from post 5-fluorouracil treated mice were infected and used to repopulate lethally-irradiated mice. Growth factor-independent BFU-E were obtained in up to 30% of day-13 spleen colonies and it was shown by DNA analysis that cells from these colonies contained integrated provirus. Our results indicate that v-erb-B transforms early erythroid progenitors to growth factor independent growth and subsequent differentiation to erythrocytes -a process that normally requires Epo plus either IL-3 or granulocyte-macrophage colony stimulating factor (GM-CSF).
A general characteristic of tumour cells is their unstable karyotype. It is suggested here that maintenance of the normal diploid cell depends on the presence of two copies of specific genes; a change in gene dosage of one or more of these genes, by chromosome nondisjunction or rearrangement, may trigger a general loss of accuracy in chromosome segregation at mitosis.
Mouse cell lines of different lineages have been established which constitutively secrete large quantities of recombinant mouse interleukins (mIL2, mIL3, mIL4 or mIL5). An existing bovine papilloma virus-based expression vector, pBV-1MTHA, was modified to allow transformed X63Ag8-653 myeloma cells, NIH 3T3 fibroblasts and C127 mammary tumor cells to stably carry multiple copies of the vector, to express the inserted cDNA encoding a single interleukin constitutively, and to secrete the interleukin in high quantities. Cell lines transformed with mIL2 cDNA stably carried 30-100 copies of the plasmid per cell and constitutively secreted biologically active mIL2 in quantities similar to those produced by murine EL4 thymoma cells or rat spleen cells stimulated with mitogens. Deletion of the 3' untranslated region containing AT-rich sequences from the mIL2 cDNA resulted in a 100-fold increase in the constitutive production and secretion of mIL2 by the transformants. Addition of a heavy metal further increased the production 2 to 6-fold. Cells transformed with 3'-deleted mIL3 cDNA constitutively secreted 300-1000 times higher activities of mIL3 than the myelomonocytic leukemia line WEHI3. mIL4 produced by the similar transformants induced [3H]thymidine uptake of a T cell line, a mast cell line and B leukemia cells, and enhanced the production of IgG1 by B cells. IL4 titers were 150 times higher than those produced by the concanavalin A-stimulated T cell line 2.19. mIL5 was secreted by similar transformants at 10-fold higher titers than those produced by concanavalin A-stimulated 2.19 T cells, as judged by the proliferation and maturation of B cell leukemia BCL1. The expression vectors should be useful in establishing eukaryotic cell lines producing proteins from full length cDNA clones at higher rates. The established cell lines secreting IL2, 3, 4 or 5 at high rate should be useful sources for these interleukins in the investigation of their function in the immune system.
To assess the erythroproliferative effects of a variety of retroviruses in vitro, hemopoietic precursors were incubated with the viruses then plated in methylcellulose. Only replication defective transforming viruses were active in this assay and produced colonies of erythroid cells. The effective transforming viruses were: Friend virus and viruses containing the abl, fes, src, Ha-ras, Ki-ras, bas, raf, raf/myc, erb B and cbl oncogenes. Replication competent viruses and those bearing the myc, myb and fos oncogenes were unable to initiate colony formation. Significant differences were observed in the colonies induced by several of the transforming genes based on (i) colony size, (ii) morphology, (iii) time course of development and (iv) sensitivity to erythropoietin. The oncogene-expressing retroviruses appeared to have the same target cell, which may be less differentiated erythroid precursor than the target cell for Friend virus.
Intact and rearranged p53 genes from Friend virus-induced erythroleukemic cell lines which code for proteins of 53- (p53) and 44-kDa (p44), respectively, were cloned into pUC18 and tested for their ability to immortalize rat embryo fibroblasts. The functional p53 gene from normal Balb/c mouse liver was also tested for immortalizing activity. Immortal cells were obtained with the three genes although the efficiency of immortalization by p44 was lower than that by p53. Expression of murine p53 and p44 in the established rat cell lines was confirmed by metabolic labeling and Western Blotting. Our results demonstrate that elevated expression of the mouse p53 gene, driven by its natural promoter and in the absence of strong heterologous promoters and/or enhancers, can efficiently immortalize early-passage rat embryo fibroblasts. p53-immortalized cells but not p44-immortalized cells could be morphologically transformed by secondary transfection with activated Ha-ras. Thus 5'-coding sequences of the p53 gene appear necessary for ras complementation but not for immortalization.
Mouse erythroleukaemia cells (also called Friend cells) can be isolated from the spleen of certain strains of mice that have been infected with the Friend virus complex. The cells resemble proerythroblasts and, when exposed to dimethyl sulphoxide (DMSO) or a variety of other chemicals, can be induced to undergo a programme of differentiation which closely resembles the final stages of normal erythropoiesis. This includes the cessation of proliferation and large increases in the production of messenger RNA for both alpha- and beta-globin. In addition, DMSO induces a rapid (less than 2 h) decrease in c-myc mRNA levels. The c-myc oncogene is expressed in the majority of proliferating normal cells and altered expression of the gene has been implicated in the genesis of a wide variety of tumours. To study the influence of oncogene activation on differentiation, we have transfected viral-promoter-driven c-myc genes into mouse erythroleukaemia cells. Constitutive c-myc expression was found to block DMSO-induced differentiation.
Myeloid precursors can be grown in vitro in the presence of specific growth factors; however, their expansion is limited by a competing process of terminal differentiation. Proto-oncogenes seem to be involved in cellular proliferation and/or differentiation and may also play a role in the myelopoietic process. Murine myeloid precursors which are grown in vitro with growth factors respond with augmented self-renewal upon infection with recombinant retroviruses carrying the v-myc or v-src oncogenes, suggesting a synergism or complementation between some viral oncogenes (v-onc) and certain growth factors. We now show that the combination of two v-onc genes (raf and myc) induces the selective proliferation of monocytic cells from fresh murine bone marrow (BM) in the absence of a specific growth factor supplement. Depending on the culture conditions these cells can either differentiate and cease to proliferate or grow continuously, thus mimicking the alternative pathways that can be followed by committed BM stem cells in vivo.
A method is described for the colonial growth, in semi-solid medium, of erythropoietin-responsive erythroid cell precursors. The erythroid cell precursors were isolated by immune hemolysis from fetal mouse liver. Both the number of precursor cells triggered to proliferate and differentiate, and the size of the erythropoietic colonies formed, are directly dependent upon the concentration of erythropoietin included in the culture.
Malignant transformation of primary cells requires at least two distinct and characteristic alterations in cellular behaviour. The first, cellular immortality, can be induced by chemical carcinogens or by cloned oncogenes such as polyoma large T (ref. 4), adenovirus early region 1A (E1A) or the oncogene from avian (MC29) myelocytomatosis virus, v-myc. Cells whose in vitro life-span has been extended by these procedures can be fully transformed by transfection with oncogenes belonging to a different complementation group, including genes of the ras family, adenovirus E1b and polyoma virus middle T (refs 4, 5). The unstable cellular phosphoprotein p53 is frequently present at elevated levels in transformed cells and is stabilized by the formation of complexes with simian virus 40 (SV40) large T or adenovirus E1b 57K protein. Although several reports have associated p53 with cell proliferation, its role remains obscure. We have cloned complementary DNA sequences encoding murine p53 and report here that transfection of p53 expression constructs into cells of finite lifespan in vitro results in cellular immortality and susceptibility to transformation by a ras oncogene.
An examination of Abelson murine leukemia virus (A-MuL V)-hematopoietic cell interaction in cultures of fetal tissues reveals that A-MuLV can stimulate the formation of two different types of colonies. One type of colony is white and composed of A-MuLV-transformed lymphoid cells that can develop into established cell lines. These cells are indistinguishable in morphology from typical adult-derived lymphoid transformants. The second type of colony is pink or red and composed of erythroid cells in various stages of differentiation. Although A-MuLV is required to induce the erythroid colonies, and at least some cells in all of these colonies are infected with the virus, no permanently growing cell lines have been established from the cells in these colonies. The frequency of the two types of colonies varies depending upon the tissue and the gestational age of the embryo. Erythroid colonies are found following infection of early and mid gestation tissues while lymphoid colonies are found following infection of mid and late gestation tissues. Mixing experiments indicate that the two types of colonies arise from distinct target cells. Because A-MuLV mutants that are defective for lymphoid cell transformation are also defective for erythroid colony induction, expression of a functional Abelson protein is probably required for colony induction. Thus A-MuLV is capable of stimulating the cells of two distinct hematopoietic lineages. In one case, infection leads to transformation, while in the second, it leads to growth and differentiation. Both types of interaction are mediated, at least in part, by the same A-MuLV gene product, a molecule previously considered to induce transformation in all stably infected cells.
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We have molecularly cloned a unique acutely transforming replication-defective mouse type C virus (3611-MSV) and characterized its acquired oncogene. The viral genome closely resembles Moloney (M) murine leukemia virus (MuLV), except for a substitution in M-MuLV in the middle of p30 and the middle of the polymerase gene (pol). Heteroduplex analysis revealed that 2.4 kilobases of M-MuLV DNA were replaced by 1.2 kilobases of cellular DNA. The junctions between viral and cellular sequences were determined by DNA sequence analysis to be 517 nucleotides into the p30 sequence and 1,920 nucleotides into the polymerase sequence. Comparison of the transforming gene from 3611-MSV, designated v-raf, with previously isolated retrovirus oncogenes either by direct hybridization or by comparison of restriction fragments of their cellular homologs shows it to be unique. Transfection of NIH 3T3 cells with cloned 3611-MSV proviral DNA leads to highly efficient transformation and the recovered virus elicits tumors in mice typical of the 3611-MSV virus. Transfected NIH 3T3 cells express two 3611-MSV-specific polyproteins (P75 and P90), both of which contain NH2-terminal gag gene-encoded components linked to the acquired sequence (v-raf) translational product. The cellular homolog, c-raf, is present in one or two copies per haploid genome in mouse and human DNA.
We develop a murine retrovirus shuttle vector system for the efficient introduction of selectable and nonselectable DNA sequences into mammalian cells and recovery of the inserted sequences as molecular clones. Three protocols allow rapid recovery of vector DNA sequences from mammalian cells. Two of the methods rely on SV40 T-antigen-mediated replication of the vector sequences and yield thousands of bacterial transformants per 5 X 10(6) mammalian cells. The majority of plasmids recovered by all three protocols exhibited the proper structure and were as active as the parental vector in the generation of transmissible retrovirus genomes upon transfection of mammalian cells. One of the rescue methods, which relies on "onion skin" replication and excision of an integrated provirus from the host chromosome, enables facile recovery of the chromosomal site of proviral integration. The system was also used to generate, and then efficiently recover, a cDNA version of a genomic insert from the adenovirus E1A region.
The replicative life spans of mammalian fibroblasts in vitro were studied in a number of cell cultures representing eight species. Emphasis was placed on determining the population doubling level at which phase III (a period of decrease in the rate of proliferation) and chromosomal alterations occur. All the cell cultures studied went through a growth crisis, a period of apparent growth cessation lasting for at least 2 weeks. In most cultures, the crisis represented the end of their replicative capacities, but in some cultures cell proliferation was resumed after the crisis. A predominantly diploid chromosome constitution (more than 75%) was demonstrated prior to the growth crisis. In cultures in which cell proliferation was resumed after the crisis, a nondiploid constitution prevailed in all cases except the rat (with 90% or more diploid cells all the time). The growth crisis occurred at population doubling levels that were characteristic for the species and was shown to be related to the species' maximal life span by a strict power law, being proportional to the square root of the maximal life span. Based on data in the literature, the same relationship was also valid for the lifespans of circulating mammalian erythrocytes in vivo. These results may indicate the prevalence of a common functional basis regulating the life span of fibroblasts and erythrocytes and thus operating in replicative as well as postmitotic cells in vitro and in vivo.
Normal somatic cells are endowed with limited doubling potential in culture, and the process of immortalization is an inevitable step in neoplastic transformation of the cells. To examine the roles of p53 in this process, the cells of p53-deficient mice were examined for doubling potential. Fibroblast-like cells from a variety of tissues of these mice proliferated continuously without showing aging or crisis. The aneuploid cells overcome the population with passage, but cloning experiment indicated that chromosomal changes were not essential to this process. The enhanced proliferative potential in culture of cells from the p53-deficient mice was also observed in epithelial cells of lens, mammary glands and seminal vesicles and in neural precursor cells. Proliferation of bone marrow cells in response to stem cell factor was enhanced in long term culture, but not in in vitro colony assay; no permanent cell lines could be obtained. No effects of p53-deficiency were found in proliferation of cardiac muscle cells or hepatocytes.
Cell cycle checkpoints enhance genetic fidelity by causing arrest at specific stages of the cell cycle when previous events
have not been completed. The tumor suppressor p53 has been implicated in a G1 checkpoint. To investigate whether p53 also
participates in a mitotic checkpoint, cultured fibroblasts from p53-deficient mouse embryos were exposed to spindle inhibitors.
The fibroblasts underwent multiple rounds of DNA synthesis without completing chromosome segregation, thus forming tetraploid
and octaploid cells. Deficiency of p53 was also associated with the development of tetraploidy in vivo. These results suggest
that murine p53 is a component of a spindle checkpoint that ensures the maintenance of diploidy.
Hematopoietic growth factors control the growth and differentiation of hematopoietic progenitor cells and bind to specific receptors that are expressed on the surface of immature hematopoietic cells found in the bone marrow. Many studies have demonstrated that these growth factors stimulate cellular growth and division by receptor activation. More recently, it has become apparent that they also influence, either directly or indirectly, the process of cellular differentiation.
While the centrality of aberrant cell proliferation in cancer was widely acknowledged long ago, it is only recently that the control of cell death has been recognized as an important target in carcinogenesis. Various lines of evidence now suggest that p53 is a positive regulator of cell death, and particularly of apoptosis. Initial studies have shown that the forced overexpression of wild-type p53 can induce apoptosis in a number of cell types, mostly of hematopoietic origin. Subsequent work has confirmed that non-manipulated, endogenous wild-type p53 is required for the efficient induction of apoptotic death by a variety of signals. In particular, the lack of functional p53 interferes with the ability of ionizing radiation, and probably other types of DNA damage, to elicit apoptosis. In addition, p53 function appears to contribute to the dependence of certain cell types on survival factors, and to the induction of apoptosis by viral proteins. The decision whether the activation of wild-type p53 will lead to a growth arrest or to apoptosis, as well as the extent to which a cell is at all responsive to p53, depends on the intracellular context. DNA damage, as well as the constitutive activation of certain growth-promoting genes, are likely to be among the determinants of this context. Illegitimate cell survival may be an important consequence of the loss of p53 function, and may contribute to the carcinogenic effects of p53 inactivation.
Committed erythroid progenitors are the cellular targets of oncogene-carrying avian and murine retroviruses that induce acute erythroleukemia. Normally, these cells undergo a fixed program of 5-10 cell divisions while terminally differentiating into erythrocytes. The sustained self-renewal observed in the context of their retrovirus-mediated leukemic transformation has been viewed as an abnormal, oncogene-induced effect. Recently, however, it was found that certain combinations of growth/differentiation hormones (e.g. TGF-alpha and estradiol) can induce normal avian erythroid progenitors to undergo a similarly extensive self-renewal as caused by oncogenes. This leads to the hypothesis that leukemia-inducing oncogenes take advantage of the committed erythroid progenitors' capacity for self-renewal rather than reprogramming their targets to abnormal growth, as suspected previously. These findings and their implications will be discussed.
To determine the contribution of p53 loss to tumor progression, we have induced abnormal proliferation in the brain choroid plexus epithelium of transgenic mice using a SV40 T antigen fragment that perturbs pRB family function but does not affect p53 function. Tumors induced by this mutant develop slowly compared with those induced by wild-type T antigen. Suppressed tumor growth is directly attributable to p53 function, since rapid tumor development occurs when the T antigen fragment is expressed in p53-null mice. In p53-heterozygous mice, stochastic loss of the wild-type p53 allele results in the focal emergence of aggressive tumor nodules characteristic of tumor progression. In each case, aggressive tumor development in the absence of p53 function corresponds to a decrease in the level of apoptosis. These results provide in vivo evidence that p53-dependent apoptosis, occurring in response to oncogenic events, is a critical regulator of tumorigenesis.
Fibroblast cultures were derived from mouse embryos containing either one (p53+/-) or two (p53-/-) inactivated p53 alleles and compared to normal embryo fibroblasts for a number of growth parameters. Early passage p53-deficient embryo fibroblasts (p53-/-) divided faster than normal embryo fibroblasts, achieved higher confluent densities, and had a higher fraction of division-competent cells under conditions of low cell density. Flow cytometry studies of early passage embryo fibroblasts showed that the percent of p53-deficient cells in G0/G1 was lower than in normal cells, consistent with the argument that p53 mediates a G1 block. When p53-deficient and normal cells were passaged for long periods of time, the homozygote (p53-/-) fibroblasts grew at a high rate for over 50 passages and never entered a non-growing senescent phase characteristic of the heterozygote (p53+/-) and normal (p53+/+) cells. The p53-deficient fibroblasts were genetically unstable during passaging, with the p53-/- cells showing a high degree of aneuploidy and the p53+/- cells displaying a moderate level of chromosomal abnormalities by passage 25. Surprisingly, the heterozygote cells lost their single wild type allele very early during culturing and in spite of this loss most heterozygote lines entered into senescence. We conclude that the loss of p53 by itself is insufficient to confer immortality on a cell, but does confer a growth advantage. Taken together, the findings confirm that the absence of p53 promotes genomic instability, which in turn may result in genetic alterations which directly produce immortality.
We here describe a rapid and simple expression screen method that has allowed us to isolate cDNAs coding for inhibitors of DNA synthesis from senescent human diploid fibroblasts. The assay involved transient transcriptional overexpression of a gene product encoded by a cDNA in a proliferating cell, on the assumption that this would be sufficient to block DNA synthesis in a short-term assay using tritiated thymidine autoradiography. Three cDNAs, referred to as senescent cell-derived inhibitors (sdi), that exhibit DNA synthesis-inhibitory activity when introduced into young cycling cells, were successfully identified. Expression of one of the cDNAs, sdi1, increased 10- to 20-fold in senescent compared with young cells and the increase in RNA closely paralleled the onset of the senescent phenotype and loss of cell proliferation. sdi1 expression was also increased in young cells made nondividing (quiescent) by deprivation of growth factors or contact inhibition. Following serum stimulation, RNA levels of sdi1 in quiescent cells were initially increased, but then declined to low levels just prior to the entry of the cells into S phase. In contrast, RNA levels of sdi1 in senescent cells failed to decline, suggesting a role for this gene in maintaining the senescent phenotype. The sdi1 gene has been mapped to the p arm of chromosome 6.
Cellular senescence is thought to be a key restraint on the progression of human tumours, escape from which involves loss of function of tumour suppressor genes. The number and nature of the genes involved however is uncertain, in particular the role of p53 mutation, which is commonly correlated with tumour progression. To address this question, we used the novel approach of directly assessing the effect of mutant p53 on 'pre-aged' human diploid fibroblasts (HDF), thereby avoiding the uncertainty of additional cooperating events, inherent in transgenic models. HDF were passaged till near-senescent and then infected with an amphotropic retroviral vector encoding an ala143 human mutant p53. The results show conclusively that p53 mutation alone is sufficient to extend the proliferative lifespan of normal fibroblasts by approximately 17 population doublings, but has no phenotypic effect on 'young' fibroblasts. We conclude that a key tumour-limiting function of wild-type p53 is to mediate growth arrest after a given number of cell divisions, in agreement with data implicating a p53-regulated gene, WAF-1/sdi-1, in cellular senescence. This may be reconciled with its 'guardian of the genome' role, if telomere erosion, a key change in senescence, is perceived by the cell as a form of DNA 'damage'.
The ability of p53 to activate transcription from specific sequences suggests that genes induced by p53 may mediate its biological role as a tumor suppressor. Using a subtractive hybridization approach, we identified a gene, named WAF1, whose induction was associated with wild-type but not mutant p53 gene expression in a human brain tumor cell line. The WAF1 gene was localized to chromosome 6p21.2, and its sequence, structure, and activation by p53 was conserved in rodents. Introduction of WAF1 cDNA suppressed the growth of human brain, lung, and colon tumor cells in culture. Using a yeast enhancer trap, a p53-binding site was identified 2.4 kb upstream of WAF1 coding sequences. The WAF1 promoter, including this p53-binding site, conferred p53-dependent inducibility upon a heterologous reporter gene. These studies define a gene whose expression is directly induced by p53 and that could be an important mediator of p53-dependent tumor growth suppression.
The cyclin-dependent kinase Cdk2 associates with cyclins A, D, and E and has been implicated in the control of the G1 to S phase transition in mammals. To identify potential Cdk2 regulators, we have employed an improved two-hybrid system to isolate human genes encoding Cdk-interacting proteins (Cips). CIP1 encodes a novel 21 kd protein that is found in cyclin A, cyclin D1, cyclin E, and Cdk2 immunoprecipitates. p21CIP1 is a potent, tight-binding inhibitor of Cdks and can inhibit the phosphorylation of Rb by cyclin A-Cdk2, cyclin E-Cdk2, cyclin D1-Cdk4, and cyclin D2-Cdk4 complexes. Cotransfection experiments indicate that CIP1 and SV40 T antigen function in a mutually antagonistic manner to control cell cycle progression.