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Suppression of Human Colorectal Carcinoma Cell Growth by Wild-Type p53
Abstract
Mutations of the p53 gene occur commonly in colorectal carcinomas and the wild-type p53 allele is often concomitantly deleted.
These findings suggest that the wild-type gene may act as a suppressor of colorectal carcinoma cell growth. To test this hypothesis,
wild-type or mutant human p53 genes were transfected into human colorectal carcinoma cell lines. Cells transfected with the
wild-type gene formed colonies five- to tenfold less efficiently than those transfected with a mutant p53 gene. In those colonies
that did form after wild-type gene transfection, the p53 sequences were found to be deleted or rearranged, or both, and no
exogenous p53 messenger RNA expression was observed. In contrast, transfection with the wild-type gene had no apparent effect
on the growth of epithelial cells derived from a benign colorectal tumor that had only wild-type p53 alleles. Immunocytochemical
techniques demonstrated that carcinoma cells expressing the wild-type gene did not progress through the cell cycle, as evidenced
by their failure to incorporate thymidine into DNA. These studies show that the wild-type gene can specifically suppress the
growth of human colorectal carcinoma cells in vitro and that an in vivo-derived mutation resulting in a single conservative
amino acid substitution in the p53 gene product abrogates this suppressive ability.
... Saos-2 cells at 70% confluency were transfected with p53R273C [13], p53R175H [14], p53R248Q [13], p53WT [15] or empty vector. Transfections corresponding to p53 (R273C, R175H, or R248Q) and transient (p53WT) were performed using Lipofectamine 3000 Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. ...
... Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 July 2023 doi:10.20944/preprints202307.0842.v114 ...
Some p53 mutants lose their tumor suppressor activity and acquire new oncogenic functions, known as a gain of function. Recent studies have shown that p53 mutants can exert oncogenic effects through specific miRNAs. We identified the differentially expressed miRNA profiles of the three most frequent p53 mutants (p53R273C, p53R248Q, and p53R175H) in the Saos-2 cell line (null p53) transfected as compared with p53WT transfected cells. The association between these miRNAs and the signaling pathways in which they might participate was performed through the miRPath Software. QRT-PCR was employed to validate the miRNAs profiles. In addition, we explored if the induction of cell migration and invasion by the p53R48Q mutant was dependent on the miR-182-5p. We observed that p53 mutants have an overall negative effect on miRNA expression. Likewise, we found miRNAs differentially expressed associated with regulating Oncogenic signaling pathways. We found overexpression of miR-182-5p, which is associated with processes such as cell migration and invasion. Inhibition of mutant p53R248Q and miR-182-5p increased FOXF2-MTSS1 levels and decreased cell migration and invasion. Our results suggest that p53 mutants increase the expression of miR-182-5p, and this miRNA is also necessary to induce cell migration and invasion by the p53R248Q mutant in a cancer cell model.
... Remarkably, GEM sensitivity of AsPC-1 cells was counteracted when cultivated in CM derived by sip53-PaCa3 cells (Figure 2), indicating a role for p53-driven secretome in the response to GEM. These data functionally demonstrate that wild-type p53 can exert its oncosuppressor role in PDAC cells through the regulation of its secretome, in line with the tumor suppressor role of p53 [27,28]. ...
... functionally demonstrate that wild-type p53 can exert its oncosuppressor role in PDAC cells through the regulation of its secretome, in line with the tumor suppressor role of p53 [27,28]. ...
The study of the cancer secretome is gaining even more importance in cancers such as pancreatic ductal adenocarcinoma (PDAC), whose lack of recognizable symptoms and early detection assays make this type of cancer highly lethal. The wild-type p53 protein, frequently mutated in PDAC, prevents tumorigenesis by regulating a plethora of signaling pathways. The importance of the p53 tumor suppressive activity is not only primarily involved within cells to limit tumor cell proliferation but also in the extracellular space. Thus, loss of p53 has a profound impact on the secretome composition of cancer cells and marks the transition to invasiveness. Here, we demonstrate the tumor suppressive role of wild-type p53 on cancer cell secretome, showing the anti-proliferative, apoptotic and chemosensitivity effects of wild-type p53 driven conditioned medium. By using high-resolution SWATH-MS technology, we characterized the secretomes of p53-deficient and p53-expressing PDAC cells. We found a great number of secreted proteins that have known roles in cancer-related processes, 30 of which showed enhanced and 17 reduced secretion in response to p53 silencing. These results are important to advance our understanding on the link between wt-p53 and cancer microenvironment. In conclusion, this approach may detect a secreted signature specifically driven by wild-type p53 in PDAC.
... The luciferase reporter assay was performed as described recently [7]. In short, U-2 OS cells were co-transfected using FuGENE 6 (Promega, Madison, WI, USA) with a combination of reporter vector, encoding firefly luciferase under the control of tested promoter (wild type or mutant), and expression vector pC53-SN3, encoding wild-type p53 or pC53-SCX3 encoding Val143Ala p53 mutant (a gift from Dr. Bert Vogelstein and Dr. Kenneth W. Kinzler from Johns Hopkins University, Baltimore, MD, USA [17]). As a negative control, the p53 plasmid was replaced by empty vector. ...
The p53 tumor suppressor protein is an activator of transcription. Diverse stress factors lead to various sets of posttranslational modifications of p53 what results in different sets of upregulat-ed genes. We noticed that actinomycin D and nutlin-3a (A+N) synergize in inducing activating phosphorylations of p53 and upregulation of selected p53-target genes. Here we found that one of these genes is DUSP13, which codes for poorly-studied, dual-specificity phosphatase having at least two isoforms, one expressed in testis and the other in skeletal muscles. In cancer cells ex-posed to A+N, DUSP13 is expressed from an alternative promoter in intron, what results in ex-pression of isoform named TMDP-L1. The luciferase reporter tests demonstrated that this pro-moter is activated by both endogenous and ectopically expressed p53. We showed for the first time that mRNA expressed from this promoter actually produces the protein, which can be de-tected by Western blotting in all examined cancer cell lines with wild-type p53 exposed to A+N. In some cell lines it is also induced by clinically relevant camptothecin or by nutlin-3a acting alone. This isoform, fused with green fluorescent protein localizes in perinuclear region of cells. Thus, TMDP-L1 isoform may be an important element of p53-regulated stress response system.
... Thus, loss or inactivation of (wild-type) p53 gene permits tumorogenesis. [24] Inactivation of the tumor suppressing activity of the p53 gene can occur by many different point mutations. Some mutations are also able to activate the p53 gene to act as a dominant transforming oncogene. ...
... While carcinogenesis implies biallelic loss of functional tumor suppressor genes, the most typical TP53 mutation configuration is a single TP53 mutation with loss of the remaining TP53 allele through a large-scale deletion on chromosome 17p. 39,40 Additionally, mutant p53 can have a dominant negative effect over p53 WT and/or gain of function activity independently of the wild-type protein. 35 There is also evidence that single mutations of TP53 are associated with loss of a single allele ($ 2/3 of tumors) and a high distortion CNV, whereas tumors with more than two mutations usually retain both alleles (diploid in almost one-third of cases). ...
TP53, the Guardian of the Genome, is the most frequently mutated gene in human cancers and the functional characterization of its regulation is fundamental. To address this we employ two strategies: machine learning to predict the mutation status of TP53from transcriptomic data, and directed regulatory networks to reconstruct the effect of mutations on the transcipt levels of TP53 targets. Using data from established databases (Cancer Cell Line Encyclopedia, The Cancer Genome Atlas), machine learning could predict the mutation status, but not resolve different mutations. On the contrary, directed network optimization allowed to infer the TP53 regulatory profile across: (1) mutations, (2) irradiation in lung cancer, and (3) hypoxia in breast cancer, and we could observe differential regulatory profiles dictated by (1) mutation type, (2) deleterious consequences of the mutation, (3) known hotspots, (4) protein changes, (5) stress condition (irradiation/hypoxia). This is an important first step toward using regulatory networks for the characterization of the functional consequences of mutations, and could be extended to other perturbations, with implications for drug design and precision medicine.
... Saos-2 cells at 70% confluence were transfected with p53R273C [15], p53R175H [16], p53R248Q [15], p53WT [17], or empty vector. Transfections corresponding to p53 mutants (R273C, R175H, or R248Q) and wild type (p53WT) were performed using Lipofectamine 3000 Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. ...
The master-key TP53 gene is a tumor suppressor that is mutated in more than 50% of human cancers. Some p53 mutants lose their tumor suppressor activity and acquire new oncogenic functions, known as a gain of function (GOF). Recent studies have shown that p53 mutants can exert oncogenic effects through specific miRNAs. We identified the differentially expressed miRNA profiles of the three most frequent p53 mutants (p53R273C, p53R248Q, and p53R175H) after their transfection into the Saos-2 cell line (null p53) as compared with p53WT transfected cells. The associations between these miRNAs and the signaling pathways in which they might participate were identified with miRPath Software V3.0. QRT-PCR was employed to validate the miRNA profiles. We observed that p53 mutants have an overall negative effect on miRNA expression. In the global expression profile of the human miRNome regulated by the p53R273C mutant, 72 miRNAs were underexpressed and 35 overexpressed; in the p53R175H miRNAs profile, our results showed the downregulation of 93 and upregulation of 10 miRNAs; and in the miRNAs expression profile regulated by the p53R248Q mutant, we found 167 decreased and 6 increased miRNAs compared with p53WT. However, we found overexpression of some miRNAs, like miR-182-5p, in association with processes such as cell migration and invasion. In addition, we explored whether the induction of cell migration and invasion by the p53R48Q mutant was dependent on miR-182-5p because we found overexpression of miR-182-5p, which is associated with processes such as cell migration and invasion. Inhibition of mutant p53R248Q and miR-182-5p increased FOXF2-MTSS1 levels and decreased cell migration and invasion. In summary, our results suggest that p53 mutants increase the expression of miR-182-5p, and this miRNA is necessary for the p53R248Q mutant to induce cell migration and invasion in a cancer cell model.
... The luciferase reporter assay was performed as described recently [7]. In short, U-2 OS cells were co-transfected using FuGENE 6 (Promega, Madison, WI, USA) with a combination of reporter vector, encoding firefly luciferase under the control of tested promoter (wild type or mutant), and expression vector pC53-SN3, encoding wild-type p53 or pC53-SCX3 encoding Val143Ala p53 mutant (a gift from Dr. Bert Vogelstein and Dr. Kenneth W. Kinzler from Johns Hopkins University, Baltimore, MD, USA [17]). As a negative control, the p53 plasmid was replaced by empty vector. ...
The p53 tumor suppressor protein is an activator of transcription. Diverse stress factors lead to various sets of posttranslational modifications of p53 what results in different sets of upregulated genes. We noticed that actinomycin D and nutlin-3a (A+N) synergize in inducing activating phosphorylations of p53 and upregulation of selected p53-target genes. Here we found that one of these genes is DUSP13, which codes for poorly-studied, dual-specificity phosphatase having at least two isoforms, one expressed in testis and the other in skeletal muscles. We found that in cancer cells exposed to A+N, DUSP13 is expressed from an alternative promoter in intron 3, what results in expression of isoform named TMDP-L1. The luciferase reporter tests demonstrated that this promoter is activated by both endogenous and ectopically expressed p53. We showed for the first time that mRNA expressed from this promoter actually produces the protein, which can be detected by Western blotting in all examined cancer cell lines with wild-type p53 exposed to A+N. In some cell lines it is also induced by clinically relevant camptothecin. This isoform, fused with green fluorescent protein localizes in perinuclear region of cells. Thus, TMDP-L1 isoform may be an important element of p53-regulated stress response system.
... A truncated protein is formed as a result of APC mutations in a region of open reading frame called MCR (mutation cluster region).The changes in question at APC have been characterized by the inactivation of Axin binding sites (SAMP), C-terminal basic region, nuclear localization signals, and multiple β-catenin binding regions (20R)(Parker & Neufeld, 2020). The lack of β-catenin reaching the nucleus as a result of continuous elimination leads to suppression of Wnt target genes by DNA-bound TCF (T cell factor) /LEF (lymphoid enhancer factor) family proteins(MacDonald et al., 2009).The tumor suppressor p53 protein known to be mutated abundantly in CRC(Baker et al., 1990), is a TF that triggers apoptosis and affects the normal developmental process of the cell by stopping the cell cycle under cellular stress or by inducing aging(Li et al., 2015). There are several clinical trials and laboratory evidence supporting that restoration or reactivation of p53 by various small molecule inhibitors or medicines can induce apoptotic pathways such as death-receptorinduced or mitochondrial(Tan et al., 2005; Zhou et al., 2008). ...
... Transfection of expression plasmids, shRNA, and siRNA cDNA of GADD34 was obtained from GE Healthcare Life Sciences (Buckinghamshire, United Kingdom). The expression plasmids, pCMV-p53WT (22) and pCMV-MDM2, were provided by Dr. Bert Vogelstein (Howard Hughes Medical Institute). shRNA expression plasmids were purchased from Merck (MISSION ® shRNAs; Merck, Darmstadt, Germany). ...
We previously identified growth arrest and DNA-damage-inducible gene 34 (GADD34) as a marker of ischemic stroke. In the present study, serum levels of anti-GADD34 antibodies were found to be significantly higher in patients with acute ischemic stroke or chronic kidney disease compared to healthy donors. We then examined the biological function of GADD34 by transfection into U2OS human osteosarcoma and U87 human glioblastoma cells. Knockdown of GADD34 by siRNA resulted in enhanced cell proliferation, which was reversed by co-knockdown of MDM2. Luciferase reporter assays revealed that the transactivation ability of p53 enhanced by genotoxic anticancer drugs such as camptothecin and etoposide was further potentiated by enforced expression of GADD34 but attenuated by co-transfection with p53 shRNA expression plasmids. Western blotting demonstrated increased p53 protein levels after treatment with camptothecin, which was also potentiated by GADD34 but suppressed by GADD34 siRNA, ATM siRNA, and ATM inhibitor wortmannin. GADD34 levels also increased in response to treatment with camptothecin or adriamycin, and this increase was attenuated by MDM2 siRNA. Immunoprecipitation with anti-GADD34 antibody followed by Western blotting with anti-MDM2 antibodies indicated ubiquitination of GADD34 is mediated by MDM2. Accordingly, GADD34 may function as a ubiquitination decoy to reduce p53 ubiquitination and increase p53 protein levels. Increased neuronal cell death due to activation of p53 by GADD34 may account for the elevated serum levels of anti-GADD34 antibodies observed in patients with acute ischemic stroke.
... LN18 cells (2.5 × 10 5 cells/well) were co-transfected with the plasmid carrying the gene encoding a wild-type p53 under the CMV promoter (pC53-SN3, 0.1 µg) [45] or empty vector (pcDNA3.1, 0.3 µg) and the plasmid encoding EGFP protein (pEGFP-N1, 0.3 µg) using Lipofectamine 2000 (Invitrogen, USA). ...
Most of anti-tumour therapies eliminate neoplastic cells by introducing DNA damage which ultimately triggers cell death. These effects are counteracted by activated DNA repair pathways to sustain tumour proliferation capacity. RECQL helicases family, including BLM, participate in DNA damage and repair, and prevent the replication stress. Glioblastoma (GBM) is a common, malignant brain tumour that inevitably recurs despite surgical resection, radiotherapy, and chemotherapy with temozolomide (TMZ). Expression and functions of the BLM helicase in GBM therapy resistance have not been elucidated. We analysed expression and localisation of BLM in human gliomas and several glioma cell lines using TCGA datasets, immunostaining and Western blotting. BLM depleted human glioma cells were generated with CRISPR/Cas9 system. Effects of chemotherapeutics on cell proliferation, DNA damage and apoptosis were determined with flow cytometry, immunofluorescence, Western blotting and RNA sequencing. We found upregulated BLM mRNA levels in malignant gliomas, increased cytosolic localisation and poor survival of GBM patients with high BLM expression. BLM deficiency in LN18 and LN229 glioma cells resulted in profound transcriptomic alterations, reduced cell proliferation, and altered cell responses to chemotherapeutics. BLM-deficient glioma cells were resistant to the TMZ and PARP inhibitor treatment and underwent polyploidy or senescence depending on the TP53 activity. Our findings of high BLM expression in GBMs and its roles in responses to chemotherapeutics provide a rationale for targeting BLM helicase in brain tumours. BLM deficiency affects responses of glioma cells to chemotherapeutics targeting PARP1 dependent pathways.
... The second crucial genetic stage in colorectal cancer is the TP53 mutation that results in the inactivation of the p53 pathway ( Fig. 2) [62 -64]. The two TP53 alleles are typically inactivated in malignancies by a combination of a 17p chromosomal deletion and a missense mutation that inhibits the transcriptional activity of p53 and removes the second TP53 allele [78][79]. Wild-type p53 mediates cell-cycle arrest and a cell-death checkpoint, which can be activated by multiple cellular stresses [80]. ...
In this review, we emphasized important biomarkers, pathogenesis, and newly developed therapeutic approaches in the treatment of colorectal cancer (CRC). This includes a complete description of small-molecule inhibitors, phytopharmaceuticals with antiproliferative potential, monoclonal antibodies for targeted therapy, vaccinations as immunotherapeutic agents, and many innovative strategies to intervene in the interaction of oncogenic proteins. Many factors combine to determine the clinical behavior of colorectal cancer and it is still difficult to comprehend the molecular causes of a person's vulnerability to CRC. It is also challenging to identify the causes of the tumor's onset, progression, and responsiveness or resistance to antitumor treatment. Current recommendations for targeted medications are being updated by guidelines throughout the world in light of the growing number of high-quality clinical studies. So, being concerned about the aforementioned aspects, we have tried to present a summarized pathogenic view, including a brief description of biomarkers and an update of compounds with their underlying mechanisms that are currently under various stages of clinical testing. This will help to identify gaps or shortfalls that can be addressed in upcoming colorectal cancer research.
... N-terminally and C-terminally Flag/HA tagged MmuPV1 E7 and HPV16 E7 were cloned into pCMV Bam/neo vectors [82]. The MmuPV1 E7 K81S mutant was PCR amplified with primers containing the K81S mutation and Q5 High Fidelity Polymerase (NEB). ...
Human papillomaviruses (HPVs) contribute to approximately 5% of all human cancers. Species-specific barriers limit the ability to study HPV pathogenesis in animal models. Murine papillomavirus (MmuPV1) provides a powerful tool to study the roles of papillomavirus genes in pathogenesis arising from a natural infection. We previously identified Protein Tyrosine Phosphatase Non-Receptor Type 14 (PTPN14), a tumor suppressor targeted by HPV E7 proteins, as a putative cellular target of MmuPV1 E7. Here, we confirmed the MmuPV1 E7-PTPN14 interaction. Based on the published structure of the HPV18 E7/PTPN14 complex, we generated a MmuPV1 E7 mutant, E7K81S, that was defective for binding PTPN14. Wild-type (WT) and E7K81S mutant viral genomes replicated as extrachromosomal circular DNAs to comparable levels in mouse keratinocytes. E7K81S mutant virus (E7K81S MmuPV1) was generated and used to infect FoxN/Nude mice. E7K81S MmuPV1 caused neoplastic lesions at a frequency similar to that of WT MmuPV1, but the lesions arose later and were smaller than WT-induced lesions. The E7K81S MmuPV1-induced lesions also had a trend towards a less severe grade of neoplastic disease. In the lesions, E7K81S MmuPV1 supported the late (productive) stage of the viral life cycle and promoted E2F activity and cellular DNA synthesis in suprabasal epithelial cells to similar degrees as WT MmuPV1. There was a similar frequency of lateral spread of infections among mice infected with E7K81S or WT MmuPV1. Compared to WT MmuPV1-induced lesions, E7K81S MmuPV1-induced lesions had a significant expansion of cells expressing differentiation markers, Keratin 10 and Involucrin. We conclude that an intact PTPN14 binding site is necessary for MmuPV1 E7's ability to contribute to papillomavirus-induced pathogenesis and this correlates with MmuPV1 E7 causing a delay in epithelial differentiation, which is a hallmark of papillomavirus-induced neoplasia.
... To explore the role of TRRAP in regulating endogenous NANOG levels, TRRAP expression was silenced using TRRAP shRNA in HCT-15 spheroid cells. TRRAP knockdown reduced the protein levels of NANOG, CD44, and cyclin D1, in contrast to the increase in the protein level of P53, a well-known tumor suppressor gene in colorectal cancer [22], in response to TRRAP silencing ( Figure 4B). It has been reported that NANOG [23] or CD44 [24] play key roles in developing resistance to cisplatin. ...
NANOG, a stemness-associated transcription factor, is highly expressed in many cancers and plays a critical role in regulating tumorigenicity. Transformation/transcription domain-associated protein (TRRAP) has been reported to stimulate the tumorigenic potential of cancer cells and induce the gene transcription of NANOG. This study aimed to investigate the role of the TRRAP-NANOG signaling pathway in the tumorigenicity of cancer stem cells. We found that TRRAP overexpression specifically increases NANOG protein stability by interfering with NANOG ubiquitination mediated by FBXW8, an E3 ubiquitin ligase. Mapping of NANOG-binding sites using deletion mutants of TRRAP revealed that a domain of TRRAP (amino acids 1898–2400) is responsible for binding to NANOG and that the overexpression of this TRRAP domain abrogated the FBXW8-mediated ubiquitination of NANOG. TRRAP knockdown decreased the expression of CD44, a cancer stem cell marker, and increased the expression of P53, a tumor suppressor gene, in HCT-15 colon cancer cells. TRRAP depletion attenuated spheroid-forming ability and cisplatin resistance in HCT-15 cells, which could be rescued by NANOG overexpression. Furthermore, TRRAP knockdown significantly reduced tumor growth in a murine xenograft transplantation model, which could be reversed by NANOG overexpression. Together, these results suggest that TRRAP plays a pivotal role in the regulation of the tumorigenic potential of colon cancer cells by modulating NANOG protein stability.
... Ectopic p53 expression and functional analyses LN18 cells (2.5x10 5 cells/well ) were co-transfected with the plasmid carrying the gene encoding a wildtype p53 under the CMV promoter (pC53-SN3, 0.1 µg) (25) or empty vector (pcDNA3.1, 0.3 µg) and the plasmid encoding EGFP protein (pEGFP-N1, 0.3 µg) using Lipofectamine 2000 (Invitrogen, USA). ...
Most of anti-tumour therapies eliminate neoplastic cells by introducing DNA damage which ultimately triggers cell death. These effects are counteracted by activated DNA repair pathways and RECQL family helicases, including BLM, participate in replication stress, DNA damage and repair. Glioblastoma (GBM) is a common, malignant brain tumour that inevitably recurs despite surgical resection, radiotherapy, and chemotherapy with temozolomide (TMZ). Expression and functions of the BLM helicase in therapy resistant GBMs have not been elucidated. We analysed expression and localisation of BLM in human gliomas and several glioma cell lines using TCGA datasets, immunostaining and Western blotting. BLM depleted human glioma cells were generated with CRISPR/Cas9. Effects of chemotherapeutics on cell proliferation, DNA damage and apoptosis were determined with flow cytometry, immunofluorescence Western blotting and RNA sequencing. We found upregulated BLM mRNA levels in malignant gliomas, increased cytosolic localisation and poor survival of BLM highly expressing GBM patients. BLM deficiency in LN18 and LN229 glioma cells resulted in profound transcriptomic alterations, reduced cell proliferation, and altered cell responses to chemotherapeutics. BLM-deficient glioma cells were resistant to the TMZ and PARP inhibitor treatment and underwent polyploidy or senescence depending on the TP53 activity. These dependencies were not detected in RECQL4-deficient cells. Our findings of high BLM expression in GBMs and its roles in responses to chemotherapeutics provide a rationale for targeting BLM helicase in those tumours. BLM deficiency affects responses of glioma cells to chemotherapeutics targeting PARP-1 dependent pathways, while these effects are not detected in RECQL4 deficient cells implying distinct functions of these helicases.
... Since its critical role as a tumor suppressor was uncovered [75][76][77][78][79], there has been a significant research effort being directed to understanding the complexities of p53 signaling in cancer and in normal cell biology [33]. Most of this research has been focused on understanding p53 signaling at the protein level and not on the transcriptional regulation of p53 itself [34]. ...
While chemotherapy is a key treatment strategy for many solid tumors, it is rarely curative, and most tumor cells eventually become resistant. Because of this, there is an unmet need to develop systemic treatments that capitalize on the unique mutational landscape of each patient’s tumor. The most frequently mutated protein in cancer, p53, has a role in nearly all cancer subtypes and tumorigenesis stages and therefore is one of the most promising molecular targets for cancer treatment. Unfortunately, drugs targeting p53 have seen little clinical success despite promising preclinical data. Most of these drug compounds target specific aspects of p53 inactivation, such as through inhibiting negative regulation by the mouse double minute (MDM) family of proteins. These treatment strategies fail to address cancer cells’ adaptation mechanisms and ignore the impact that p53 loss has on the entire p53 network. However, recent gene therapy successes show that targeting the p53 network and cellular dysfunction caused by p53 inactivation is now possible and may soon translate into successful clinical responses. In this review, we discuss p53 signaling complexities in cancer that have hindered the development and use of p53-targeted drugs. We also describe several current therapeutics reporting promising preclinical and clinical results.
... net/addgene:16440, RRID:Addgene_16436, and Addgene plasmid # 16437, http://n2t.net/addgene:16437, RRID:Addgene_16437) were gifts from Dr Bert Vogelstein (33). ...
Recent studies suggested that aggregates of mutant p53 proteins may propagate and impair normal p53 functioning in recipient cells. Our previous study showed that cancer cell-derived p53 aggregates that cells internalized interfered with p53-dependent apoptosis in recipient cells. However, involvement of p53 aggregate propagation in cancer pathology has not been fully elucidated. Here, we screened patients with high-grade serous ovarian carcinoma, which is characterized by an extremely high frequency of TP53 gene mutations, to show that patients with cytoplasmic p53 deposits have a poor prognosis compared with patients with complete p53 absence or strong nuclear p53 positivity. Cytoplasmic p53 in the patients with poor prognosis consisted of protein aggregates, which suggests that p53 aggregates are oncogenic drivers. Indeed, an inhibitor of p53 aggregation restored cellular apoptosis, a proper p53 function, in p53 aggregate-bearing patient-derived tumor organoids. In cell-based assays, endogenous and exogenous mutant p53 aggregates hindered chemotherapeutic activity of cisplatin, which depends on normal p53 functions. This inhibition was reduced by blocking p53 aggregation or internalization of p53 aggregates. Our study thus indicates the involvement of p53 aggregate transmission in poor prognosis and in chemotherapy resistance in cancers.
... Previous studies have found that a variety of molecules promote or inhibit neutrophil apoptosis 35,36 . P53 is a tetrameric transcription factor that is heavily regulated by post-transcriptional modifications and was initially identified as a tumor suppressor gene 68 . However, increasing evidence shows that it halts cell proliferation and induces apoptosis 69,70 . ...
Neutrophils are the earliest master inflammatory regulator cells recruited to target tissues after direct infection or injury. Although inflammatory factors are present in muscle that has been indirectly disturbed by peripheral nerve injury, whether neutrophils are present and play a role in the associated inflammatory process remains unclear. Here, intravital imaging analysis using spinning-disk confocal intravital microscopy was employed to dynamically identify neutrophils in denervated muscle. Slice digital scanning and 3D-view reconstruction analyses demonstrated that neutrophils escape from vessels and migrate into denervated muscle tissue. Analyses using reactive oxygen species (ROS) inhibitors and flow cytometry demonstrated that enhanced ROS activate neutrophils after denervation. Transcriptome analysis revealed that the vast majority of neutrophils in denervated muscle were of the CXCR2 subtype and were recruited by CXCL1. Most of these cells gradually disappeared within 1 week via P53-mediated apoptosis. Experiments using specific blockers confirmed that neutrophils slow the process of denervated muscle atrophy. Collectively, these results indicate that activated neutrophils are recruited via chemotaxis to muscle tissue that has been indirectly damaged by denervation, where they function in delaying atrophy.
... BamBl and the fragment cloned into the EcoRl and BamBl sites of pGex-2TK. The sequence of the cloned product was confirmed using pGex sequencing primers ( Friedlander et al. 1996 The p53 binding site of the Hu mdm-2 promoter cloned into pGL3-luc cmvbamneo SN-3 Dr X Lu Baker et al. 1990 Hup53 expression plasmid CMV-P-gal Invitrogen -P-galactosidase reporter plasmid ...
p> BAG-1 is a multifunctional protein that binds a wide range of cellular targets including heat shock proteins and some nuclear hormone receptors. BAG-1 exists as three isoforms, BAG-1L, BAG-1M and BAG-IS. BAG-1L contains a nuclear localisation signal, which is not present in the other isoforms and is predominantly localised in the cell nucleus.
To determine the significance of BAG-1 expression in breast cancer, tumours from 138 patients with breast cancer treated with hormonal therapy were analysed by immunohistochemistry. Nuclear BAG-1 immunostaining was associated with expression of oestrogen receptor alpha and progesterone receptor and with improved survival. Reporter gene assays were used to determine the effects of BAG-1 isoforms on oestrogen dependent transcription, and coimmunoprecipitation assays to analyse the interaction of BAG-1 with oestrogen receptors. The nuclear BAG-1 isoform, BAG-1L, interacted with oestrogen receptor alpha and beta and increased oestrogen dependent transcription in breast cancer cells. BAG-IS is also highly expressed in some breast cancers, and to investigate its role in protecting breast cancer cells from apoptosis reporter assays and microarray analysis were used. BAG-IS overexpression reduced p53 dependent transcription and candidate BAG-1 target genes that may be involved in protecting breast cancer cells from apoptosis were identified.
BAG-1 protects breast cancer cells from apoptosis and interferes with p53 function. Importantly, since high levels of BAG-1 L can increase responsiveness to oestrogens in breast cancer cells, BAG-1 may be a marker of responsiveness to hormonal therapy, via direct effects on receptor function. These findings support the hypothesis that BAG-1 is an important molecule in breast cancer and suggest that BAG-1 may prove to be a novel target for cancer therapy. </p
... Mutations in the tumor suppressor gene, p53, account for ~ 50% of human cancers (Doniger et al. 2008;Finlay et al. 1989;Baker et al. 1990;Hamzehloie et al. 2012;Zhang et al. 2020). p53 is a critical regulator of tissue homeostasis (Baugh et al. 2018;Diller et al. 1990;Chng et al. 2007), which further binds to stabilize DNA as a tetramer, leading to the regulation of genes. ...
Background
A variety of accessible data, including those of single-nucleotide polymorphisms (SNPs) on the human p53 gene, are made widely available on a global scale. Owing to this, our investigation aimed to deal with the detrimental SNPs in the p53 gene by executing various valid computational tools, including—Filter, SIFT, PredictSNP, Fathmm, UTRScan, ConSurf, SWISS-MODEL, Amber 16 package, Tm-Adjust, I-Mutant, Task Seek, GEPIA2 after practical and basic appraisal, dissolvable openness, atomic progression, analyzing the energy minimization and assessing the gene expression pattern.
Results
Out of the total 581 p53 SNPs, 420 SNPs were found to be missense or non-synonymous, 435 SNPs were in the three prime UTR, and 112 SNPs were in the five prime UTR from which 16 non-synonymous SNPs (nsSNPs) were predicted to be non-tolerable while PredictSNP package predicted 14. Concentrating on six bioinformatics tools of various dimensions, a combined output was generated, where 14 nsSNPs could exert a deleterious effect. We found 5 missense SNPs in the DNA binding domain's three crucial amino acid positions, using diverse SNP analyzing tools. The underlying discoveries were fortified by microsecond molecular dynamics (MD) simulations, TM-align, I-Mutant, and Project HOPE. The ExPASy-PROSITE tools characterized whether the mutations were located in the functional part of the protein or not. This study provides a decisive outcome, concluding the accessible SNPs' information by recognizing the five unfavorable nsSNPs—rs28934573 (S241F), rs11540652 (R248Q), rs121913342 (R248W), rs121913343 (R273C), and rs28934576 (R273H). By utilizing Heatmapper and GEPIA2, several visualization plots, including heat maps, box plots, and survival plots, were produced.
Conclusions
These plots disclosed differential expression patterns of the p53 gene in humans. The investigation focused on recognizing the detrimental nsSNPs, which augmented the danger posed by various oncogenesis in patients of different populations, including within the genome-wide studies (GWS).
... However, whereas fasting-selective transcription factors have been well-described in the liver (Goldstein and Hager, 2015), the transcriptional regulation of the thermogenic program upon fasting in adipocytes has received little attention. The transcription factor p53 was originally described as main player in cancer development, as evidenced by the high prevalence of TP53 mutations in several cancer types in humans (Baker et al., 1990a;Baker et al., 1990b;Olivier et al., 2002). However, besides its vital function as tumor suppressor, more recent studies have established p53 as an important regulator of metabolism and tissue homeostasis in non-cancer contexts (Lacroix et al., 2020). ...
Active thermogenic adipocytes avidly consume energy substrates like fatty acids and glucose to maintain body temperature upon cold exposure. Despite strong evidence for the involvement of brown adipose tissue (BAT) in controlling systemic energy homeostasis upon nutrient excess, it is unclear how the activity of brown adipocytes is regulated in times of nutrient scarcity. Therefore, this study aimed to scrutinize factors that modulate BAT activity to balance thermogenic and energetic needs upon simultaneous fasting and cold stress. For an unbiased view, we performed transcriptomic and miRNA sequencing analyses of BAT from acutely fasted (24 h) mice under mild cold exposure. Combining these data with in-depth bioinformatic analyses and in vitro gain-of-function experiments, we define a previously undescribed axis of p53 inducing miR-92a-1-5p transcription that is highly upregulated by fasting in thermogenic adipocytes. p53, a fasting-responsive transcription factor, was previously shown to control genes involved in the thermogenic program and miR-92a-1-5p was found to negatively correlate with human BAT activity. Here, we identify fructose transporter Slc2a5 as one direct downstream target of this axis and show that fructose can be taken up by and metabolized in brown adipocytes. In sum, this study delineates a fasting-induced pathway involving p53 that transactivates miR-92a-1-5p, which in turn decreases Slc2a5 expression, and suggests fructose as an energy substrate in thermogenic adipocytes.
... When mutated, they unleash their oncogenic potential mainly by remaining in a permanently activated state (Furney et al., 2006;Pon and Marra, 2015), as for example TAL1 or c-Myc (Sanda et al., 2012;Littlewood et al., 2012;Nie et al., 2012;Lin et al., 2012). Loss-of-function mutations in TFs can act as tumor suppressors and are often the drivers of or are associated with various cancers (Sherr, 2004), with p53 being the most prominent (Olivier et al., 2002;Baker et al., 1990;Malkin et al., 1990). ...
Transcription factors play key roles in orchestrating a plethora of cellular mechanisms and controlling cellular homeostasis. Transcription factors share distinct DNA binding domains, which allows to group them into protein families. Among them, the Forkhead box O (FOXO) family contains transcription factors crucial for cellular homeostasis, longevity and response to stress. The dysregulation of FOXO signaling is linked to drug resistance in cancer therapy or cellular senescence, however, selective drugs targeting FOXOs are limited, thus knowledge about structure and dynamics of FOXO proteins is essential. Here, we provide an extensive study of structure and dynamics of all FOXO family members. We identify residues accounting for different dynamic and structural features. Furthermore, we show that the auto-inhibition of FOXO proteins by their C-terminal trans-activation domain is conserved throughout the family and that these interactions are not only possible intra-, but also inter-molecularly. This indicates a model in which FOXO transcription factors would co-regulate their activities.
... 37 At cell level, the p53 protein was observed to be equipped for hindering cell development. 38 Later, the TP53 gene that encodes the p53 protein was set up as a growth silencer suppressor and was observed to be the most mutated gene in diseases, with roughly 50% penetrance. 39,40 The cancer suppressor p53 is a phosphoprotein scarcely perceivable inside the core of typical cells. ...
Screening tests for panels of biomarkers such as the panel comprising p53, CEA, and CA19-9 facilitated early diagnosis of cancers and improvement of the quality of life. In this review, diverse electrochemical sensors used for the assay of p53, CEA, and CA 19-9 in biological samples are shown. Different methods of analysis such as differential pulse voltammetry, electrochemical impendance spectroscopy, chronoamperometry, and stochastic method were used for the assay of one biomarker (differential pulse voltammetry, chronoamperometry) or for the assay of a panel of biomarkers comprising p53, CA19-9, and CEA using stochastic method. The lowest limits of determination were obtained using the stochastic method; they were of fg mL-1/fU mL-1 magnitude order.
... A truncated protein is formed as a result of APC mutations in a region of open reading frame called MCR (mutation cluster region).The changes in question at APC have been characterized by the inactivation of Axin binding sites (SAMP), C-terminal basic region, nuclear localization signals, and multiple β-catenin binding regions (20R)(Parker & Neufeld, 2020). The lack of β-catenin reaching the nucleus as a result of continuous elimination leads to suppression of Wnt target genes by DNA-bound TCF (T cell factor) /LEF (lymphoid enhancer factor) family proteins(MacDonald et al., 2009).The tumor suppressor p53 protein known to be mutated abundantly in CRC(Baker et al., 1990), is a TF that triggers apoptosis and affects the normal developmental process of the cell by stopping the cell cycle under cellular stress or by inducing aging(Li et al., 2015). There are several clinical trials and laboratory evidence supporting that restoration or reactivation of p53 by various small molecule inhibitors or medicines can induce apoptotic pathways such as death-receptorinduced or mitochondrial(Tan et al., 2005; Zhou et al., 2008). ...
Integrated and comprehensive systems biology approaches are essential in terms of being approaches that can reveal the possible causes and mechanisms of cancer as well as treatment candidates. Making sense of transcriptome-based big data with biostatistical calculations contributes to revealing the mechanisms underlying the disease pathogenesis, revealing the clinically actionable genes, and identifying new actions that can be taken for the treatment of the disease.
Since the design of a drug for disease requires long processes and heavy costs, the importance of drug repurposing, which can create treatment options for diseases, is well understood, especially during the COVID-19 pandemic. Considering these important benefits of drug repositioning, we proposed drug candidates that could be a treatment option for CRC, which still suffers from people all over the world, with an integrated approach that brings together many disciplines.
... Further, c-MET promoter (1850 bp) analysis reveals the binding sites for transcription factors known to interact or regulate through hTERT like p53 and c-Myc. p53 is a tumor suppressor protein, targets the genes associated with growth, survivability, angiogenesis, and metastasis [48][49][50][51]. Activation of p53 occurs in response to DNA damage, which further stimulates the transcription of cyclin-dependent kinase (Cdk) inhibitory protein p21 and cell cycle arrest [52]. ...
Human telomerase reverse transcriptase (hTERT), the essential catalytic subunit of telomerase, is associated with telomere homeostasis to prevent replicative senescence and cellular aging. However, hTERT reactivation also has been linked to the acquisition of several hallmarks of cancer, although the underlying mechanism beyond telomere extension remains elusive. This study demonstrated that hTERT overexpression promotes, whereas its inhibition by shRNA suppresses, epithelial-mesenchymal transition (EMT) in lung cancer cells (A549 and H1299). We found that hTERT modulates the expression of EMT markers E-cadherin, vimentin, and cytokeratin-18a through upregulation of the c-MET. Ectopic expression of hTERT induces expression of c-MET, while hTERT-shRNA treatment significantly decreases the c-MET level in A549 and H1299 through differential expression of p53 and c-Myc. Reporter assay suggests the regulation of c-MET expression by hTERT to be at the promoter level. An increase in c-MET level significantly promotes the expression of mesenchymal markers, including vimentin and N-cadherin, while a notable increase in epithelial markers E-cadherin and cytokeratin-18a is observed after the c-MET knockdown in A549.
... The estimated incidence rate of colorectal carcinoma in Bangladesh is 2.3%. 6 Males are affected slightly more than females. 7 Cancer occurring in the young (<40 years of age) are usually located on the distal colon and rectum which tend to show features associated with aggressive behavior. ...
Background: This cross- sectional observational study was carried out with an aim to look for microsatellite instability (MSI) status in colorectal carcinoma and their association with different histomorphological patterns and biological behavior of colorectal carcinoma.Methods: This cross-sectional observational study was done in the Department of Pathology, Bangabandhu Sheikh Mujib Medical University Hospital (BSMMU), Dhaka, Bangladesh during September 2014 to October 2015. A total of 39 surgically resected sample of colorectal carcinoma were included. Consent from each patient was taken. The samples were histopathologically evaluated according to the standard protocol. The statistical analyses were done using Statistical packages for social sciences (SPSS 15) for Windows.Results: A total of 39 cases of colorectal carcinoma were included in this study. Majority of the patients (55.5%) was in 6th decade in MSI and 29.1% were MSI absent group. The mean age was found 47.67±10.97 years in present group and 47.84±14.26 years in absent group. The difference was not statistically significant (p>0.05). TNM stage with MSI was observed. The mean CEA level was 100.74±103.66 and 60.43±91.72. The mean Hb was 9.72±1.99 % and 9.92±2.17, the range was 7.2-12.2 and 4.6-13.4 among the groups. The mean difference was not statistically significant (p>0.05). Ulcerated was 3 (33.3%) and 19 (64.5%). Stage 3 tumor was 4 (44.4%) and 16 (51.6%). Grade 2 tumor was 5 (55.6%) and 17 (58.0%).Conclusions: For the first time in Bangladesh, this study was undertaken to evaluate the microsatellite instability (MSI) status in colorectal cancer tissue and their association with different histomorphological patterns of colorectal carcinoma.
... The estimated incidence rate of colorectal carcinoma in Bangladesh is 2.3%. 6 Males are affected slightly more than females. 7 Cancer occurring in the young (<40 years of age) are usually located on the distal colon and rectum which tend to show features associated with aggressive behavior. ...
INTRODUCTION The incidence of colorectal carcinoma varies considerably throughout the world, being one of the leading cancer sites in the developed countries. 1 Approximately 6% of the population will develop colorectal cancer during their course of life. 2 In 2013 it was estimated that there will be 142,820 new cases of colon and rectum cancer and an estimated 50,830 will be die of this diseases. 3 With respect ABSTRACT Background: This cross-sectional observational study was carried out with an aim to look for microsatellite instability (MSI) status in colorectal carcinoma and their association with different histomorphological patterns and biological behavior of colorectal carcinoma. Methods: This cross-sectional observational study was done in the Department of Pathology, Bangabandhu Sheikh Mujib Medical University Hospital (BSMMU), Dhaka, Bangladesh during September 2014 to October 2015. A total of 39 surgically resected sample of colorectal carcinoma were included. Consent from each patient was taken. The samples were histopathologically evaluated according to the standard protocol. The statistical analyses were done using Statistical packages for social sciences (SPSS 15) for Windows. Results: A total of 39 cases of colorectal carcinoma were included in this study. Majority of the patients (55.5%) was in 6th decade in MSI and 29.1% were MSI absent group. The mean age was found 47.67±10.97 years in present group and 47.84±14.26 years in absent group. The difference was not statistically significant (p>0.05). TNM stage with MSI was observed. The mean CEA level was 100.74±103.66 and 60.43±91.72. The mean Hb was 9.72±1.99 % and 9.92±2.17, the range was 7.2-12.2 and 4.6-13.4 among the groups. The mean difference was not statistically significant (p>0.05). Ulcerated was 3 (33.3%) and 19 (64.5%). Stage 3 tumor was 4 (44.4%) and 16 (51.6%). Grade 2 tumor was 5 (55.6%) and 17 (58.0%). Conclusions: For the first time in Bangladesh, this study was undertaken to evaluate the microsatellite instability (MSI) status in colorectal cancer tissue and their association with different histomorphological patterns of colorectal carcinoma.
... The luciferase reporter assay was performed as described recently [4]. In short, U-2 OS cells were co-transfected using FuGENE 6 (Promega) with a combination of reporter vector, encoding firefly luciferase under the control of BLNK promoter (wild type or mutant), and expression vector pC53-SN3, encoding wild-type p53 or pC53-SCX3 encoding Val143Ala p53 mutant (a gift from Dr. Bert Vogelstein and Dr. Kenneth W. Kinzler from Johns Hopkins University, Baltimore, MD, USA) [39]. As a negative control, the p53 plasmid was replaced by empty vector. ...
Co-treatment with actinomycin D and nutlin-3a (A + N) strongly activates p53. Previously we reported that CHIR-98014 (GSK-3 kinase inhibitor), acting in cells exposed to A + N, prevents activation of TREM2-an innate immunity and p53-regulated gene associated with Alzheimer’s disease. In order to find novel candidate p53-target genes and genes regulated by CHIR-98014, we performed RNA-Seq of control A549 cells and the cells exposed to A + N, A + N with CHIR-98014 or to CHIR-98014. We validated the data for selected genes using RT-PCR and/or Western blotting. Using CRISPR/Cas9 technology we generated p53-deficient cells. These tools enabled us to identify dozens of candidate p53-regulated genes. We confirmed that p53 participates in upregulation of BLNK, APOE and IRF1. BLNK assists in activation of immune cells, APOE codes for apolipoprotein associated with Alzheimer’s disease and IRF1 is activated by interferon gamma and regulates expression of antiviral genes. CHIR-98014 prevented or inhibited the upregulation of a fraction of genes stimulated by A + N. Downregulation of GSK-3 did not mimic the activity of CHIR-98014. Our data generate the hypothesis, that an unidentified kinase inhibited by CHIR-98014, participates in modification of p53 and enables it to activate a subset of its target genes, e.g., the ones associated with innate immunity.
... pGL13, a firefly luciferase reporter plasmid containing 13 p53 binding elements, pCMV-Neo-Bam, and vectors expressing wild-type and dominant negative mutant p53 pCMV-Neo-Bam-p53WT and pCMV-Neo-Bam-p53R273H (pC53-4.2N3), respectively, were described elsewhere (50,51). pGL3 control vector expressing firefly luciferase under SV40 promoter was purchased from Promega. ...
Kaposi Sarcoma-associated herpesvirus (KSHV) is a carcinogenic double-stranded DNA virus and the etiological agent of Kaposi’s Sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s Disease (MCD). To prevent premature apoptosis and support its replication cycle, KSHV expresses a series of open reading frames (ORFs) that regulate signaling by the p53 tumor suppressor protein. Here we describe a novel viral inhibitor of p53 encoded by KSHV ORF45 and identify its mechanism of action. ORF45 binds to p53 and prevents its interactions with USP7, a p53 deubiquitinase. This results in decreased accumulation, localization of p53 to the cytoplasm, and diminished transcriptional activity.
IMPORTANCE
Unlike in other cancers, the tumor suppressor protein p53 is rarely mutated in Kaposi Sarcoma (KS). Rather, Kaposi Sarcoma-associated herpesvirus (KSHV) inactivates p53 through multiple viral proteins. One possible therapeutic approach to KS is the activation of p53, which would result in apoptosis and tumor regression. In this regard, it is important to understand all the mechanisms used by KSHV to modulate p53 signaling. This work describes a novel inhibitor of p53 signaling and a potential drug target, ORF45, and identifies the mechanisms of its action.
Overview
Cancer is a genetic disease. Mutations and other alterations in growth promoting genes (oncogenes) and tumor suppressor genes can accumulate during the lifetime of a normal cell resulting in cancer. Unlike oncogenes, tumor suppressor genes generally require biallelic inactivation in order to demonstrate a cancerous phenotype. Importantly, the discovery of heritable tumor suppressor gene mutations that lead to familial forms of cancer has revealed great insight into tumor suppressor function. This has clinical screening implications for individuals with a family history of cancer and has led to newer therapies to target cancer cells with loss of specific tumor suppressors.
The success of precision oncology—which aims to match the right therapies to the right patients based on molecular status—is predicated on a robust pipeline of molecular targets against which therapies can be developed. Recent advances in genomics and functional genetics have enabled the unbiased discovery of novel molecular targets at scale. We summarize the promise and challenges in integrating genomic and functional genetic landscapes of cancer to establish the next generation of cancer targets.
Acute ultraviolet (UV)-B radiation is the major external factor causing photodamage. In this study, we aimed to determine the effects of Dendrobium nobile Lindl. polysaccharides (DNPs) on photodamage in HaCaT keratinocytes after UVB irradiation and the underlying mechanisms. We found that DNPs significantly attenuated the decline in the viability and proliferation of HaCaT cells after UVB irradiation. Moreover, DNPs scavenged reactive oxygen species (ROS), improved the activities of endogenous antioxidant enzymes, including superoxide dismutase, catalase, and glutathione peroxidase, and reduced the levels of malondialdehyde, while partially attenuating cell cycle arrest, suggesting their antioxidant and anti-apoptotic properties. The mitogen-activated protein kinase (MAPK) pathway was found to be important for the attenuation of UVB-induced photodamage in the HaCaT cells. Furthermore, DNPs exerted cytoprotective effects by downregulating UVB-induced ROS-mediated phosphorylation of MAPKs, including p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase, and by inhibiting p53 expression as well as the apoptotic cascade response. Therefore, DNPs ameliorated UVB-induced oxidative damage and apoptosis in HaCaT cells via the regulation of MAPKs. Our findings thus highlight the Dendrobium nobile Lindl polysaccharides as promising therapeutic candidates for UVB-induced photodamage.
Somatic cell reprogramming using the microRNAs miR-200c, miR-302s, and miR-369s leads to increased expression of cyclin-dependent kinase inhibitors in human colorectal cancer (CRC) cells and suppressed tumor growth. Here, we investigated whether these microRNAs inhibit colorectal tumorigenesis in CPC;Apc mice, which are prone to colon and rectal polyps. Repeated administration of microRNAs inhibited polyp formation. Microarray analysis indicated that c-MAF, which reportedly shows oncogene-like behavior in multiple myeloma and T cell lymphoma, decreased in tumor samples but increased in microRNA-treated normal mucosa. Immunohistochemistry identified downregulation of c-MAF as an early tumorigenesis event in CRC, with low c-MAF expression associated with poor prognosis. Of note, c-MAF expression and p53 protein levels were inversely correlated in CRC samples. c-MAF knockout led to enhanced tumor formation in azoxymethane/dextran sodium sulfate–treated mice, with activation of cancer-promoting genes. c-MAF may play a tumor-suppressive role in CRC development.
Most of anti-tumour therapies eliminate neoplastic cells by introducing DNA damage which ultimately triggers cell death. These effects are counteracted by activated DNA repair pathways to sustain tumour proliferation capacity. RECQL helicases family, including BLM, participate in DNA damage and repair, and prevent the replication stress. Glioblastoma (GBM) is a common, malignant brain tumour that inevitably recurs despite surgical resection, radiotherapy, and chemotherapy with temozolomide (TMZ). Expression and functions of the BLM helicase in GBM therapy resistance have not been elucidated. We analysed expression and localisation of BLM in human gliomas and several glioma cell lines using TCGA datasets, immunostaining and Western blotting. BLM depleted human glioma cells were generated with CRISPR/Cas9. Effects of chemotherapeutics on cell proliferation, DNA damage and apoptosis were determined with flow cytometry, immunofluorescence, Western blotting and RNA sequencing. We found upregulated BLM mRNA levels in malignant gliomas, increased cytosolic localisation and poor survival of BLM highly expressing GBM patients. BLM deficiency in LN18 and LN229 glioma cells resulted in profound transcriptomic alterations, reduced cell proliferation, and altered cell responses to chemotherapeutics. BLM-deficient glioma cells were resistant to the TMZ and PARP inhibitor treatment and underwent polyploidy or senescence depending on the TP53 activity. Our findings of high BLM expression in GBMs and its roles in responses to chemotherapeutics provide a rationale for targeting BLM helicase in those tumours. BLM deficiency affects responses of glioma cells to chemotherapeutics targeting PARP1 dependent pathways.
With over 200 types of cancer diagnosed to date, researchers the world over have been forced to rapidly update their understanding of the biology of cancer. In fact, only the study of the basic cellular processes, and how these are altered in cancer cells, can ultimately provide a background for rational therapies. Bringing together the state-of-the-art contributions of international experts, Systems Biology of Cancer proposes an ultimate research goal for the whole scientific community: exploiting systems biology to generate in-depth knowledge based on blueprints that are unique to each type of cancer. Readers are provided with a realistic view of what is known and what is yet to be uncovered on the aberrations in the fundamental biological processes, deregulation of major signaling networks, alterations in major cancers and the strategies for using the scientific knowledge for effective diagnosis, prognosis and drug discovery to improve public health.
With over 200 types of cancer diagnosed to date, researchers the world over have been forced to rapidly update their understanding of the biology of cancer. In fact, only the study of the basic cellular processes, and how these are altered in cancer cells, can ultimately provide a background for rational therapies. Bringing together the state-of-the-art contributions of international experts, Systems Biology of Cancer proposes an ultimate research goal for the whole scientific community: exploiting systems biology to generate in-depth knowledge based on blueprints that are unique to each type of cancer. Readers are provided with a realistic view of what is known and what is yet to be uncovered on the aberrations in the fundamental biological processes, deregulation of major signaling networks, alterations in major cancers and the strategies for using the scientific knowledge for effective diagnosis, prognosis and drug discovery to improve public health.
With over 200 types of cancer diagnosed to date, researchers the world over have been forced to rapidly update their understanding of the biology of cancer. In fact, only the study of the basic cellular processes, and how these are altered in cancer cells, can ultimately provide a background for rational therapies. Bringing together the state-of-the-art contributions of international experts, Systems Biology of Cancer proposes an ultimate research goal for the whole scientific community: exploiting systems biology to generate in-depth knowledge based on blueprints that are unique to each type of cancer. Readers are provided with a realistic view of what is known and what is yet to be uncovered on the aberrations in the fundamental biological processes, deregulation of major signaling networks, alterations in major cancers and the strategies for using the scientific knowledge for effective diagnosis, prognosis and drug discovery to improve public health.
With over 200 types of cancer diagnosed to date, researchers the world over have been forced to rapidly update their understanding of the biology of cancer. In fact, only the study of the basic cellular processes, and how these are altered in cancer cells, can ultimately provide a background for rational therapies. Bringing together the state-of-the-art contributions of international experts, Systems Biology of Cancer proposes an ultimate research goal for the whole scientific community: exploiting systems biology to generate in-depth knowledge based on blueprints that are unique to each type of cancer. Readers are provided with a realistic view of what is known and what is yet to be uncovered on the aberrations in the fundamental biological processes, deregulation of major signaling networks, alterations in major cancers and the strategies for using the scientific knowledge for effective diagnosis, prognosis and drug discovery to improve public health.
With over 200 types of cancer diagnosed to date, researchers the world over have been forced to rapidly update their understanding of the biology of cancer. In fact, only the study of the basic cellular processes, and how these are altered in cancer cells, can ultimately provide a background for rational therapies. Bringing together the state-of-the-art contributions of international experts, Systems Biology of Cancer proposes an ultimate research goal for the whole scientific community: exploiting systems biology to generate in-depth knowledge based on blueprints that are unique to each type of cancer. Readers are provided with a realistic view of what is known and what is yet to be uncovered on the aberrations in the fundamental biological processes, deregulation of major signaling networks, alterations in major cancers and the strategies for using the scientific knowledge for effective diagnosis, prognosis and drug discovery to improve public health.
With over 200 types of cancer diagnosed to date, researchers the world over have been forced to rapidly update their understanding of the biology of cancer. In fact, only the study of the basic cellular processes, and how these are altered in cancer cells, can ultimately provide a background for rational therapies. Bringing together the state-of-the-art contributions of international experts, Systems Biology of Cancer proposes an ultimate research goal for the whole scientific community: exploiting systems biology to generate in-depth knowledge based on blueprints that are unique to each type of cancer. Readers are provided with a realistic view of what is known and what is yet to be uncovered on the aberrations in the fundamental biological processes, deregulation of major signaling networks, alterations in major cancers and the strategies for using the scientific knowledge for effective diagnosis, prognosis and drug discovery to improve public health.
With over 200 types of cancer diagnosed to date, researchers the world over have been forced to rapidly update their understanding of the biology of cancer. In fact, only the study of the basic cellular processes, and how these are altered in cancer cells, can ultimately provide a background for rational therapies. Bringing together the state-of-the-art contributions of international experts, Systems Biology of Cancer proposes an ultimate research goal for the whole scientific community: exploiting systems biology to generate in-depth knowledge based on blueprints that are unique to each type of cancer. Readers are provided with a realistic view of what is known and what is yet to be uncovered on the aberrations in the fundamental biological processes, deregulation of major signaling networks, alterations in major cancers and the strategies for using the scientific knowledge for effective diagnosis, prognosis and drug discovery to improve public health.
With over 200 types of cancer diagnosed to date, researchers the world over have been forced to rapidly update their understanding of the biology of cancer. In fact, only the study of the basic cellular processes, and how these are altered in cancer cells, can ultimately provide a background for rational therapies. Bringing together the state-of-the-art contributions of international experts, Systems Biology of Cancer proposes an ultimate research goal for the whole scientific community: exploiting systems biology to generate in-depth knowledge based on blueprints that are unique to each type of cancer. Readers are provided with a realistic view of what is known and what is yet to be uncovered on the aberrations in the fundamental biological processes, deregulation of major signaling networks, alterations in major cancers and the strategies for using the scientific knowledge for effective diagnosis, prognosis and drug discovery to improve public health.
With over 200 types of cancer diagnosed to date, researchers the world over have been forced to rapidly update their understanding of the biology of cancer. In fact, only the study of the basic cellular processes, and how these are altered in cancer cells, can ultimately provide a background for rational therapies. Bringing together the state-of-the-art contributions of international experts, Systems Biology of Cancer proposes an ultimate research goal for the whole scientific community: exploiting systems biology to generate in-depth knowledge based on blueprints that are unique to each type of cancer. Readers are provided with a realistic view of what is known and what is yet to be uncovered on the aberrations in the fundamental biological processes, deregulation of major signaling networks, alterations in major cancers and the strategies for using the scientific knowledge for effective diagnosis, prognosis and drug discovery to improve public health.
With over 200 types of cancer diagnosed to date, researchers the world over have been forced to rapidly update their understanding of the biology of cancer. In fact, only the study of the basic cellular processes, and how these are altered in cancer cells, can ultimately provide a background for rational therapies. Bringing together the state-of-the-art contributions of international experts, Systems Biology of Cancer proposes an ultimate research goal for the whole scientific community: exploiting systems biology to generate in-depth knowledge based on blueprints that are unique to each type of cancer. Readers are provided with a realistic view of what is known and what is yet to be uncovered on the aberrations in the fundamental biological processes, deregulation of major signaling networks, alterations in major cancers and the strategies for using the scientific knowledge for effective diagnosis, prognosis and drug discovery to improve public health.
With over 200 types of cancer diagnosed to date, researchers the world over have been forced to rapidly update their understanding of the biology of cancer. In fact, only the study of the basic cellular processes, and how these are altered in cancer cells, can ultimately provide a background for rational therapies. Bringing together the state-of-the-art contributions of international experts, Systems Biology of Cancer proposes an ultimate research goal for the whole scientific community: exploiting systems biology to generate in-depth knowledge based on blueprints that are unique to each type of cancer. Readers are provided with a realistic view of what is known and what is yet to be uncovered on the aberrations in the fundamental biological processes, deregulation of major signaling networks, alterations in major cancers and the strategies for using the scientific knowledge for effective diagnosis, prognosis and drug discovery to improve public health.
The protein p53 has been extensively investigated since it was found 43 years ago and has become a “guardian of the genome” that regulates the division of cells by preventing the growth of cells and dividing them, that is, inhibits the development of tumors. Initial proof of protein existence by researchers in the mid‐1970s was found by altering and regulating the SV40 big T antigen termed the A protein. Researchers demonstrated how viruses play a role in cancer by employing viruses' ability to create T‐antigens complex with viral tumors, which was discovered in 1979 following a viral analysis and cancer analog research. Researchers later in the year 1989 explained that in Murine Friend, a virus‐caused erythroleukemia, commonly found that p53 was inactivated to suggest that p53 could be a “tumor suppressor gene.” The TP53 gene, encoding p53, is one of human cancer's most frequently altered genes. The protein‐regulated biological functions of all p53s include cell cycles, apoptosis, senescence, metabolism of the DNA, angiogenesis, cell differentiation, and immunological response. We tried to unfold the history of the p53 protein, which was discovered long back in 1979, that is, 43 years of research on p53, and how p53's function has been developed through time in this article.
p>The p53 tumor suppressor protein has a critical role in the cellular response to stress, and when activated, functions to transactivate genes which induce apoptosis or growth arrest. The hdm2 oncoprotein is the most significant regulator of p53 activity, and it achieves this partly through inhibition of p53 transactivating ability. Hdm2 also interacts with other non-p53 proteins and through its many protein-protein interactions, influences diverse cellular pathways. Work by our group previously identified a putative protein-protein interaction between hdm2 and the redox-sensitive transcriptional co-repressor CtBP2. Based on this observation, the objectives of the current study were: (i) To verify that hdm2 and CtBP2 interact, (ii) To characterise the physical nature of the interaction, (iii) to determine the functional consequences of the interaction and how it may be regulated.
The interaction between hdm2 and CtBP2 was verified using in vitro and in vivo protein binding assays. Deletion mutants were used to identify the interacting domains in the proteins, and the effect of the interaction on p53 activity assessed using reporter gene assays.
Hdm2 participates in a novel protein-protein interaction with CtBP2 both in vitro and in vivo . The acidic domain of hdm2 and the N-terminus of CtBP2 are necessary and sufficient for this interaction. CtBP proteins undergo an NADH-induced conformational change, which we show results in a loss of its hdm2 binding ability. This negative regulation is dependent on the conserved NADH-binding GXGXXG motif in CtBP2.
The recruitment of CtBP2 by hdm2 results in a promoter selective repression of p53-depdnent transcription. Furthermore, hypoxia-mimicking conditions which increase intracellular NADH levels abolish this repression, thus enhancing p53 activity.</p
The oxygenation of polyunsaturated fatty acids such as arachidonic and linoleic acid through enzymes like lipoxygenases (LOXs) are common and often leads to the production of various bioactive lipids that are important both in acute inflammation and its resolution and thus in disease progression. Amongst the several isoforms of LOX that are expressed in mammals, 15-lipoxygenase (15-LOX) has shown to be crucial in the context of inflammation. Moreover, being expressed in cells of the immune system, as well as in epithelial cells; the enzyme has been shown to crosstalk with a number of important signalling pathways. Mounting evidences from recent reports suggest that 15-LOX has anti-cancer activities which are dependent or independent of its metabolites, and is executed through several downstream pathways like cGMP, PPAR, p53, p21 and NAG-1. However, it is still unclear whether the up-regulation of 15-LOX is associated with cancer cell apoptosis. Monoamine oxidase A (MAO-A), on the other hand, is a mitochondrial flavoenzyme which is believed to be involved in the pathogenesis of atherosclerosis and inflammation and in many other neurological disorders. MAO-A has also been reported as a potential therapeutic target in different types of cancers like prostate cancer, lung cancer etc.
In this review, we discussed about the role of fatty acids and their lipid mediators in cancer cell apoptosis. Here we particularly focused on the contribution of oxidative enzymes like 15-LOX and MAO-A in mediating apoptosis in lung cancer cell after fatty acid induction.
Mutant forms of the gene encoding the tumor suppressor p53 are found in numerous human malignancies, but the physiologic function of p53 and the effects of mutations on this function are unknown. The p53 protein binds DNA in a sequence-specific manner and thus may regulate gene transcription. Cotransfection experiments showed that wild-type p53 activated the expression of genes adjacent to a p53 DNA binding site. The level of activation correlated with DNA binding in vitro. Oncogenic forms of p53 lost this activity. Moreover, all mutants inhibited the activity of coexpressed wild-type p53, providing a basis for the selection of such mutants during tumorigenesis.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
A significant percentage of human tumors contain activated ras oncogenes that have acquired oncogenic potential as a result of somatic point mutations at codon 12 or 61 of the encoded ras gene product. We report here a method to detect and characterize mutations in ras genes that is based on the ability of pancreatic ribonuclease (RNase A; EC 3.1.27.5) to cleave RNA heteroduplexes containing single-base mismatches. Using this method, we show that certain human tumor cells contain mutant c-Ki-ras genes, and we define the nature and position of these mutations. At the same time, we describe the presence and estimate the expression of both normal and mutant c-Ki-ras alleles in the same tumor cells. This method should be useful for the diagnostic detection and characterization of single point mutations in expressed genes.
Mutant forms of the p53 cellular tumor antigen elicit neoplastic transformation in vitro. Recent evidence indicated that loss of normal p53 expression is a frequent event in certain types of tumors, raising the possibility that such loss provides transformed cells with a selective growth advantage. Thus, it was conceivable that the mutants might contribute to transformation by abrogating normal p53 function. We therefore studied the effect of plasmids encoding wild-type (wt) p53 on the ability of primary rat embryo fibroblasts to be transformed by a combination of mutant p53 and ras. It was found that wt p53 plasmids indeed caused a marked reduction in the number of transformed foci. Furthermore, wt p53 plasmids also suppressed the induction of transformed foci by combinations of bona fide oncogenes, such as myc plus ras or adenovirus E1A plus ras. On the other hand, plasmids carrying mutations in the p53 coding region totally failed to inhibit oncogene-mediated focus induction and often even slightly stimulated it. Hence, such mutations completely abolished the activity of wt p53 that is responsible for the "suppressor" effect. The latter fact is of special interest, since similar mutations in p53 are often observed in human and rodent tumors. The inhibitory effect of p53 was most pronounced when early-passage cells were used as targets, whereas established cell lines were less sensitive. These data support the notions that wt p53 expression may be restrictive to neoplastic progression and that p53 inactivation may play a crucial role in tumorigenesis.
The p53 gene has been a constant source of fascination since its discovery nearly a decade ago. Originally considered to be an oncogene, several convergent lines of research have indicated that the wild-type gene product actually functions as a tumour suppressor gene. For example, expression of the neoplastic phenotype is inhibited, rather than promoted, when rat cells are transfected with the murine wild-type p53 gene together with mutant p53 genes and/or other oncogenes. Moreover, in human tumours, the short arm of chromosome 17 is often deleted. In colorectal cancers, the smallest common region of deletion is centred at 17p13.1; this region harbours the p53 gene, and in two tumours examined in detail, the remaining (non-deleted) p53 alleles were found to contain mutations. This result was provocative because allelic deletion coupled with mutation of the remaining allele is a theoretical hallmark of tumour-suppressor genes. In the present report, we have attempted to determine the generality of this observation; that is, whether tumours with allelic deletions of chromosome 17p contain mutant p53 genes in the allele that is retained. Our results suggest that (1) most tumours with such allelic deletions contain p53 point mutations resulting in amino-acid substitutions, (2) such mutations are not confined to tumours with allelic deletion, but also occur in at least some tumours that have retained both parental 17p alleles, and (3) p53 gene mutations are clustered in four 'hot-spots' which exactly coincide with the four most highly conserved regions of the gene. These results suggest that p53 mutations play a role in the development of many common human malignancies.
Previous studies have demonstrated that allelic deletions of the short arm of chromosome 17 occur in over 75% of colorectal carcinomas. Twenty chromosome 17p markers were used to localize the common region of deletion in these tumors to a region contained within bands 17p12 to 17p13.3. This region contains the gene for the transformation-associated protein p53. Southern and Northern blot hybridization experiments provided no evidence for gross alterations of the p53 gene or surrounding sequences. As a more rigorous test of the possibility that p53 was a target of the deletions, the p53 coding regions from two tumors were analyzed; these two tumors, like most colorectal carcinomas, had allelic deletions of chromosome 17p and expressed considerable amounts of p53 messenger RNA from the remaining allele. The remaining p53 allele was mutated in both tumors, with an alanine substituted for valine at codon 143 of one tumor and a histidine substituted for arginine at codon 175 of the second tumor. Both mutations occurred in a highly conserved region of the p53 gene that was previously found to be mutated in murine p53 oncogenes. The data suggest that p53 gene mutations may be involved in colorectal neoplasia, perhaps through inactivation of a tumor suppressor function of the wild-type p53 gene.
A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique.
The 11-4 p53 cDNA clone failed to transform primary rat fibroblasts when cotransfected with the ras oncogene. Two linker insertion mutations at amino acid 158 or 215 (of 390 amino acids) activated this p53 cDNA for transformation with ras. These mutant cDNAs produced a p53 protein that lacked an epitope, recognized by monoclonal antibody PAb246 (localized at amino acids 88 to 110 in the protein) and preferentially bound to a heat shock protein, hsc70. In rat cells transformed by a genomic p53 clone plus ras, two populations of p53 proteins were detected, PAb246+ and PAb246-, which did or did not bind to this monoclonal antibody, respectively. The PAb246- p53 preferentially associated with hsc70, and this protein had a half-life 4- to 20-fold longer than free p53 (PAb246+). These data suggest a possible functional role for hsc70 in the transformation process. cDNAs for p53 derived from methylcholanthrene-transformed cells transform rat cells in cooperation with the ras oncogene and produce a protein that bound with the heat shock proteins. Recombinant clones produced between a Meth A cDNA and 11-4 were tested for the ability to transform rat cells. A single amino acid substitution at residue 132 was sufficient to activate the 11-4 p53 cDNA for transformation. These studies have identified a region between amino acids 132 and 215 in the p53 protein which, when mutated, can activate the p53 cDNA. These results also call into question what the correct p53 wild-type sequence is and whether a wild-type p53 gene can transform cells in culture.
Two monoclonal antibodies against the p53 protein, PAb 122 and 200-47, were microinjected into mammalian cells as a probe
to determine the role of the p53 protein in cell proliferation. PAb 122 recognizes the p53 proteins of mouse and human cells
but not of hamster cells, whereas 200-47 recognizes the p53 proteins of mouse and hamster cells but not of human cells. The
ability of these antibodies to inhibit serum-stimulated DNA synthesis of cells in culture correlates with their ability to
recognize the species-specific antigenic determinants. More important, however, is the observation that microinjected PAb
122 inhibits the transition of Swiss 3T3 cells from G0 to S phase, but has no effect on the progression of these cells from
mitosis to the S phase.
We introduced a mouse IL-2 cDNA expression vector into an IL-2-dependent mouse helper T cell line HT-2. Transfected cells secreted substantial amounts of IL-2, to which they themselves responded by proliferating without further requirement for exogenous IL-2. The proliferation was a direct function of the cell density and was inhibitable by antibodies against IL-2 or IL-2-R, indicating the autocrine nature of the proliferation. Those producing higher amounts of IL-2 were found to be tumorigenic when inoculated into nude mice. The latency period of tumor development correlated inversely with the level of IL-2 secreted. Tumor cells proliferated in vitro in an IL-2 autocrine fashion indistinguishable from that of the inoculated cells. We thus provide evidence that the aberrant activation of the IL-2 autocrine circuit can lead T cells to malignant transformation.
We studied the cotransfer and cointegration of several genes transfected into four cell lines of primate origin. Mouse thymidine-kinase-negative LM cells, which had been extensively studied previously, were used as a reference. We found that in monkey kidney Vero cells, on average between 3.5 and 6.0 kb of plasmid sequences was integrated per clone, while in the murine LM cell Une, 9–186 kb of exogenous DNA was integrated per clone. Transformed Vero clones which had integrated more than 6 kb of DNA did not integrate larger DNA fragments in a second transformation assay than had the parental Vero cells. We found that the efficiency of gene cointegration is similar in Vero, HeLa and GM4312A cells, the latter being deficient in the repair of UV-induced damage. The human hepatocarcinoma Hep G2 cells integrated on the average 2 kb more exogenous DNA than the three other primate cell Unes, which resulted in a 4–5 times higher efficiency of gene cointegration. Plasmid penetration and persistence in a free state between 24 h and two weeks after transfection was similar in Vero and LM cells. No major post-integration DNA rearrangement could be demonstrated after the isolation of Vero clones. These observations correlate the low efficiency of gene cointegration in some primate cell lines with a genomic recombination step or with rearrangements taking place during early cell divisions following integration
We tested a population of over 60 patients with chronic myelogenous leukemia (CML) for changes in the structure and expression of the p53 gene, which is located on chromosome 17. Six of 27 (22%) blast crisis samples and 3 of 5 (60%) accelerated phase samples had rearrangements of chromosome 17, whereas only 3 of 42 (7%) chronic phase patients had cytogenetic changes in chromosome 17. There was no loss of heterozygosity during the transition to blastic crisis among seven individuals who were informative for polymorphic probes for regions in or around the p53 gene on 17p. One patient in the chronic phase and one patient in the blastic phase of the 61 CML patients studied exhibited rearrangements of the p53 gene that were detectable by Southern analysis. One p53 allele was rearranged in the chronic phase patient and both p53 alleles were rearranged in the blastic phase patient. The p53 messenger RNA (mRNA) was of normal size (2.8 kb) in chronic phase and blast crisis, and the expression of the p53 gene was at least as high or higher in blast crisis as in the chronic phase of CML. The high incidence of abnormalities of chromosome 17 in blast-crisis CML found in our studies and the discovery of rearrangements of the p53 gene in two CML patients studied suggest that further study with probes for the p53 gene and anonymous polymorphic sites in chromosome 17 should be conducted in CML.
Primary lung cancer samples of the major histological types were examined for expression of the tumor suppressor gene p53 by immunohistochemistry. Abnormalities in p53 expression were found in 28 of 40 carcinomas, 14 of 17 squamous tumours showing abnormal p53 expression, whereas no expression of p53 was detectable in 7 carcinoid tumours or in 10 normal lung samples. Direct evidence for homozygous expression of mutant p53 mRNA in representative carcinomas was obtained by means of an asymmetric polymerase chain reaction mRNA sequencing strategy, which allowed sequencing without any cloning step. All the mutations were G to T transversions resulting in mis-sense mutations in aminoacids highly conserved in evolution. Mutation of the p53 gene is the most frequently identified genetic change in human lung cancer; these findings suggest that simple immunohistological methods can provide strong evidence of such mutation.
The human anti-oncoprotein p53 is shown to be a substrate of cdc2. The primary site of phosphorylation is serine-315. Serine-315 is phosphorylated by both p60-cdc2 and cyclin B-cdc2 enzymes. The phosphorylation of p53 is cell cycle-dependent. The abundance of p53 also oscillates during the cell cycle. The protein is largely absent from cells that have just completed division but accumulates in cells during G1 phase. Phosphorylation by cdc2 might regulate the antiproliferative activity of p53.
The isolation and construction of a complete human p53 cDNA and subsequent expression in monkey cells is described. A set of new anti-(human p53) monoclonal antibodies has also been obtained and used to show the expression of the human p53 cDNA in cos-l cells. These antibodies enable the specific detection of human p53, which is synthesised in the presence of p53 from other species. Fusion proteins of p53 with beta-galactosidase were used firstly as antigen and secondly, in conjunction with competition assays, to localise the determinants recognized by the antibodies. At least two previously unrecognized epitopes are involved and two of the antibodies are human-p53-specific. The epitopes are denaturation-resistant and the antibodies are, therefore, valuable for immunoblotting as well as immunoprecipitation and enzyme-linked immunoassay. Transfection of plasmids containing complete human p53 cDNA into monkey (cos-l) cells cause expression of human p53 recognized by the monoclonal antibodies. Control plasmids did not induce immunoreactive protein.
DNA clones of the wild-type p53 proto-oncogene inhibit the ability of E1A plus ras or mutant p53 plus ras-activated oncogenes to transform primary rat embryo fibroblasts. The rare clones of transformed foci that result from E1A plus ras plus wild-type p53 triple transfections all contain the p53 DNA in their genome, but the great majority fail to express the p53 protein. The three cell lines derived from such foci that express p53 all produce mutant p53 proteins with properties similar or identical to transformation-activated p53 proteins. The p53 mutants selected in this fashion (transformation in vitro) resemble the p53 mutants selected in tumors (in vivo). These results suggest that the p53 proto-oncogene can act negatively to block transformation.
The ability of SV40-transformed human (ataxia-telangiectasia) fibroblasts to maintain Epstein-Barr virus (EBV)-based plasmids and cosmids extrachromosomally has been investigated. Transfection of a culture of cells with two different plasmids gave rise to cell clones which were able to maintain both plasmids extrachromosomally. When an EBV-based cosmid library was transfected into the cells and an individual cell clone was isolated, the extrachromosomal DNA derived from the cosmid contained numerous deletions and rearrangements. When individual cosmids were transfected into the culture, and several cell clones were isolated, the intracellular cosmid-derived DNA again showed the presence of multiple deletions and rearrangements. We conclude that although SV40-transformed cells are able to maintain more than one different EBV-based plasmid extrachromosomally, large EBV-derived molecules are extensively rearranged. SV40-transformed human fibroblasts cannot therefore be usefully used in attempting to clone genes from EBV-based cosmid libraries.
Allele loss is a hallmark of chromosome regions harboring recessive oncogenes. Lung cancer frequently demonstrates loss of heterozygosity on 17p. Recent evidence suggests that the p53 gene located on 17p13 has many features of such an antioncogene. The p53 gene was frequently mutated or inactivated in all types of human lung cancer. The genetic abnormalities of p53 include gross changes such as homozygous deletions and abnormally sized messenger RNAs along with a variety of point or small mutations, which map to the p53 open reading frame and change amino acid sequence in a region highly conserved between mouse and man. In addition, very low or absent expression of p53 messenger RNA in lung cancer cell lines compared to normal lung was seen. These findings, coupled with the previous demonstration of 17p allele loss in lung cancer, strongly implicate p53 as an anti-oncogene whose disruption is involved in the pathogenesis of human lung cancer.
To examine the extent and variation of allelic loss in a common adult tumor, polymorphic DNA markers were studied from every
nonacrocentric autosomal arm in 56 paired colorectal carcinoma and adjacent normal colonic mucosa specimens. This analysis
was termed an allelotype, in analogy with a karyotype. Three major conclusions were drawn from this analysis: (i) Allelic
deletions were remarkably common; one of the alleles of each polymorphic marker tested was lost in at least some tumors, and
some tumors lost more than half of their parental alleles. (ii) In addition to allelic deletions, new DNA fragments not present
in normal tissue were identified in five carcinomas; these new fragments contained repeated sequences of the variable number
of tandem repeat type. (iii) Patients with more than the median percentage of allelic deletions had a considerably worse prognosis
than did the other patients, although the size and stage of the primary tumors were very similar in the two groups. In addition
to its implications concerning the genetic events underlying tumorigenesis, tumor allelotype may provide a molecular tool
for improved estimation of prognosis in patients with colorectal cancer.
Three chromosome regions, i.e., 11p15, 13q, and 17p, were previously reported by three independent groups to be specifically reduced to hemizygosity in human primary breast cancer. We examined the DNA of 64 mammary tumors for loss of heterozygosity (LOH) with 28 polymorphic DNA markers dispersed on 10 arms of 8 different chromosomes. Complete or near-complete absence of LOH was observed on 5 arms (5 chromosomes). LOH at all three previously invoked regions was confirmed, and the highest frequency was found on 17p (67% of heterozygous patients). Allele loss of a marker from chromosome 3 (region p14-p21) was found in 7 of 15 informative cases. Concurrent LOH at 2 to 4 loci was noted in 20 of the 43 tumors showing LOH. Allele losses did not correlate with any of the six clinico-histopathological variables investigated, but in a group of patients in which we were unable to demonstrate LOH, the absence of distant metastases was statistically significant (P less than 0.05). These results suggest that some of the observed allele losses reflect random events, possibly as a result of genetic instability, but are not without biological significance for the progression of particular subclasses of breast tumors.
p53 is a 53-kDa nuclear protein that is associated with malignant transformation in several tumor model systems. In a survey of 134 human carcinomas, sarcomas, leukemias, and lymphomas obtained at surgery or from peripheral blood, we found rearrangements of the p53 gene only in osteogenic sarcomas (3 of 6 osteogenic sarcomas examined). Normal tissue from one of these patients had an unrearranged gene, indicating that the genetic abnormality in the tumor was acquired. Two of the sarcomas with rearranged genes expressed levels of p53 protein that were elevated relative to other tumors. Rearranged p53 genes were also found in human osteogenic sarcoma cell lines.
We observed six major tryptic phosphopeptides in p53 from simian virus 40-transformed and normal NIH 3T3 cells. Analyses of
the phosphopeptides indicated that serines 37, 310 and/or 312, 389 and one or more of serines 7, 9, 12, 18, and 23 were phosphorylated.
Phosphorylation of serines 310 and/or 312 was twofold higher in the simian virus 40-transformed cells as compared with that
in normal NIH 3T3 cells.
Because most colorectal carcinomas appear to arise from adenomas, studies of different stages of colorectal neoplasia may shed light on the genetic alterations involved in tumor progression. We looked for four genetic alterations (ras-gene mutations and allelic deletions of chromosomes 5, 17, and 18) in 172 colorectal-tumor specimens representing various stages of neoplastic development. The specimens consisted of 40 predominantly early-stage adenomas from 7 patients with familial adenomatous polyposis, 40 adenomas (19 without associated foci of carcinoma and 21 with such foci) from 33 patients without familial polyposis, and 92 carcinomas resected from 89 patients. We found that ras-gene mutations occurred in 58 percent of adenomas larger than 1 cm and in 47 percent of carcinomas. However, ras mutations were found in only 9 percent of adenomas under 1 cm in size. Sequences on chromosome 5 that are linked to the gene for familial adenomatous polyposis were not lost in adenomas from the patients with polyposis but were lost in 29 to 35 percent of adenomas and carcinomas, respectively, from other patients. A specific region of chromosome 18 was deleted frequently in carcinomas (73 percent) and in advanced adenomas (47 percent) but only occasionally in earlier-stage adenomas (11 to 13 percent). Chromosome 17p sequences were usually lost only in carcinomas (75 percent). The four molecular alterations accumulated in a fashion that paralleled the clinical progression of tumors. These results are consistent with a model of colorectal tumorigenesis in which the steps required for the development of cancer often involve the mutational activation of an oncogene coupled with the loss of several genes that normally suppress tumorigenesis.
The ability of simian virus 40-transformed human fibroblasts to integrate and maintain transfected genomic DNA has been investigated in two normal and six DNA-repair-deficient human cell lines. These cell lines were transfected with DNA containing two selective markers (G418 and hygromycin (Hyg) resistance) separated by random pieces of human DNA of 0-40 kb in length. The transfection frequency for the selected (G418R) marker was between 2 x 10(-4) and 2 x 10(-3) for all cell lines, comparable to many other mammalian systems. About 50% of the G418R colonies were also initially resistant to Hyg. Analysis of the DNA from individual clones expanded for a further month revealed, however, that about one to three copies of the selected marker but only about 0.1 copy per cell of the unselected marker were maintained. Our results were broadly similar for all eight cell lines. Thus the amount of integrated DNA that is stably maintained in these cells is in general very small (less than 50 kb). This may provide an explanation for the difficulties encountered in many laboratories in attempts to correct the defect in DNA-repair-deficient human cells by transfection with genomic DNA. Our results also show that none of several defects in DNA repair has any obvious effect on either the transfection frequency or the amount of stably integrated foreign DNA.
The Friend virus-transformed erythroleukemic cell line DP16-9B4 has undergone a complex rearrangement of the p53 oncogene and lacks any detectable expression of the p53 protein. We report here characterization of both p53 alleles in this cell line and identify independent integrations of Friend murine leukemia virus sequences into the coding region of both alleles.
The tumor antigen p53 is overproduced in transformed cells of various species, including man. HL-60 is an exceptional human tumor cell line that does not express this protein. Hybridization of polyadenylylated mRNA of these cells with a human p53 cDNA probe (p53-H14), which we cloned, had indicated a total absence of the mature-size (3.0 kilobases) or any aberrant p53 mRNA species. Analysis of the genomic HL-60 DNA indicated that the p53 gene in these cells was significantly altered. Most of the gene was deleted, and the residual p53 sequences of these cells, which show weak homology, mapped to the corresponding 5' region of the p53 gene. In agreement with previously documented results, we found that HL-60 cells have an amplified c-myc gene. We suggest that the deficiency of the p53 protein in HL-60 cells could have been overcome by using an alternative metabolic pathway. The c-myc product is a candidate for such an alternative protein.
The clonal composition of human colorectal tumors was studied by means of restriction fragment length polymorphisms (RFLPs).
First, X-linked RFLPs were used to examine the pattern of X chromosome inactivation in colorectal tumors of females. All 50
tumors examined showed monoclonal patterns of X chromosome inactivation; these tumors included 20 carcinomas as well as 30
adenomas of either familial or spontaneous type. Second, RFLPs of autosomes were used as clonal markers to detect the somatic
loss or gain of specific chromosomal sequences in colorectal tumors. Among other changes, it was found that somatic loss of
chromosome 17p sequences occurred in over 75 percent of the carcinomas examined, but such loss was rare in adenomas. These
data support a monoclonal origin for colorectal neoplasms, and suggest that a gene on the short arm of chromosome 17 may be
associated with progression from the benign to the malignant state.
By a molecular genetic approach using polymorphic DNA markers that detect allelic deletion of specific chromosomal regions, we analyzed for possible loss of chromosomal heterozygosity in five different histological types of lung cancers obtained from 47 patients. In small-cell carcinomas, the incidence of allelic deletions at three different chromosomal loci was extremely high; loss of heterozygosity was detected on chromosomes 3p in 7 of 7 patients (100%), 13q in 10 of 11 patients (91%), and 17p in 5 of 5 patients (100%). The deletions at these loci in small-cell carcinomas were observed even in the tumors without any clinical evidence of metastasis. Furthermore, loss of heterozygosity on chromosomes 3p and 13q occurred prior to NMYC amplification and chromosome 11p deletion. Loss of heterozygosity on chromosome 3p was also detected with high frequency in adenocarcinomas [5 of 6 patients (83%)]. Heterozygosity of chromosomes 13q and 17p was lost in 10 of 31 patients (32%) and in 3 of 12 patients (25%), respectively, of lung cancers other than small-cell carcinomas. These results indicate that recessive genetic changes involving sequences on chromosomes 3p, 13q, and 17p may play important roles in the genesis of small-cell carcinoma, and those on chromosome 3p may play an important role in the genesis of adenocarcinoma.
The oncogene product p53, isolated from SV3T3 cells where it forms a complex with simian virus 40 large tumor antigen (T antigen) in the nucleus, has been found to be phosphorylated at at least four distinct sites on the 390 amino acid protein. Separation of tryptic phosphopeptides has permitted identification of two sites as Ser-312 and Ser-389, and permitted analysis of the types of phosphate bonds. The peptide containing Ser-312 separates electrophoretically into three charged forms; two are resistant to dephosphorylation by both alkaline phosphatase and alkaline hydrolysis, suggesting a phosphodiester. The carboxyl-terminal phosphopeptide containing Ser-389 was alkaline phosphatase-resistant and liberated four ribonucleoside monophosphates upon base or RNase hydrolysis, suggesting that Ser-389 may be covalently linked to RNA. Phosphorylation of Ser-389 decreased markedly at the nonpermissive temperature in simian virus 40 tsA58-transformed cells, indicating a dependence on native T antigen function and a possible role in transformation by T antigen. Two additional phosphorylation sites, one involving serine and one involving threonine, probably reside in the amino-terminal segment of p53 and appear to be peptide-phosphate monoesters.
The suitability of Chinese hamster and human cell lines for DNA-mediated gene transformation was investigated with respect to two parameters: the average quantity of and the integrity of integrated exogenous DNA fragments. No large differences were observed between most cell lines concerning the extent of fragmentation of the transferred DNA molecules. By contrast, the average number of sequences stably incorporated by the human cells (four lines tested) was 20- to 100-fold lower than the average amount inserted in the five Chinese hamster lines investigated. The very low uptake exhibited by the human cells, ranging from less than 100 up to 500 kb, renders these cells less suitable for transfection with genomic DNA to isolate specific genes.
Single base substitutions can be detected and localized by a simple and rapid method that involves ribonuclease cleavage of
single base mismatches in RNA:DNA heteroduplexes. A 32P-labeled RNA probe complementary to wild-type DNA is synthesized in
vitro and annealed to a test DNA containing a single base substitution. The resulting single base mismatch is cleaved by ribonuclease
A, and the location of the mismatch is then determined by analyzing the sizes of the cleavage products by gel electrophoresis.
Analysis of every type of mismatch in many different sequence contexts indicates that more than 50 percent of all single base
substitutions can be detected. The feasibility of this method for localizing base substitutions directly in genomic DNA samples
is demonstrated by the detection of single base mutations in DNA obtained from individuals with beta-thalassemia, a genetic
disorder in beta-globin gene expression.
We have developed a general method for introducing cloned genes into mammalian cells that affords substantial benefits over current technology. It is simple, rapid, and applicable to many (perhaps all) cell types, including those that are refractory to traditional transfection procedures. The method involves exposure of a suspension of cells and cloned DNA to a high-voltage electric discharge. In a model application of this transfection procedure, we have studied the expression of cloned human and mouse Ig kappa genes stably introduced into mouse pre-B cells and fibroblasts. We find that there is a B-cell-specific enhancer-activator region in the J-C intron of the human kappa gene that is necessary for efficient transcription of the cloned gene in mouse pre-B lymphocytes. This suggests that both the DNA element and the proteins required for its regulatory activity have been highly conserved in evolution and that these elements operate at the pre-B-cell stage of immunocyte development, a stage that precedes productive kappa gene rearrangement.
ALLELIC LOSSES OF CHROMOSOME-9
- Y C Tsai