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Abstract
Tumor suppressor proteins are believed to play a role in regulating cell cycle control during mammalian development. The E6 and E7 oncoproteins from human papillomavirus type 16 are known to affect cell growth control, at least in part, through their inactivation of cellular tumor suppressor gene products, p53 and Rb, respectively. Therefore, these viral proteins can serve as trans-dominant repressors of tumor suppressor gene function. To study the potential role of p53 and Rb in murine lens morphogenesis, we generated transgenic mice in which the expression of E6 or E7 was directed to the developing lens. Transgenic mice expressing E7 exhibited microphthalmia and cataracts, whereas transgenic mice expressing E6 exhibited cataracts without noticeable microphthalmia. Microscopic analysis of the lenses from neonatal and adult E7 transgenic mice revealed inhibition of lens fiber cell differentiation, induction of cell proliferation in spatially inappropriate regions of the lens, and apoptosis. Transgenic mice expressing a mutant E7 that is defective in Rb/p107 binding exhibited normal eyes, suggesting that the activity of Rb and/or Rb-like proteins is required for the perturbation of lens development and induction of apoptosis in E7 mice. Microscopic analysis of lenses from E6 neonatal and adult transgenic mice indicated the presence of nuclei in elongated fiber cells, suggesting that E6 inhibits lens fiber cell denucleation. Furthermore, expression of E6 inhibited the apoptotic-like DNA degradation observed in the lenses of nontransgenic 15.5-day embryos. In lenses from neonatal E6 x E7 double transgenic mice, the level of apoptosis was reduced compared with that seen in lenses from neonatal E7 mice. In adults E6 x E7 double transgenic mice, lens tumors developed, whereas in E6 or E7 only transgenic mice, tumors did not. Taken together, these results point to specific roles in lens morphogenesis for Rb and p53 and to the necessity of these tumor suppressor gene products in regulating exit from the normal cell division cycle in differentiating lens fiber cells.
... These earlier studies were subsequently confirmed from in vivo studies by different groups including our own work [10][11][12][13][14][15]. Moreover, interruption of normal lens physiology by overexpressing exogenous genes or silence of endogenous genes all induces apoptosis followed by lens pathology [16][17][18][19][20][21][22][23][24][25][26][27]. ...
... Failed undergoing apoptosis of the lens stalk leads to cataract and microphthalmia [78]. Disruption of normal lens development through overexpression of exogenous genes or silence of endogenous genes all causes lens pathology [16][17][18][19][20][21][22][23][24][25][26][27]. In the adult lens, we and others have shown that induced apoptosis by environment stresses cause non-congenital cataract in both human and different animals [6][7][8][9][10][11][12][13][14][15]. ...
Deleted in breast cancer 1 (DBC1) was initially identified from a homozygously deleted region in human chromosome 8p21. It has been well established that DBC1 plays a dual role during cancer development. Depending on the physiological context, it can promote or inhibit tumorigenesis. Whether it plays a role in lens pathogenesis remains elusive. In the present study, we demonstrated that DBC1 is highly expressed in lens epithelial cells from different vertebrates and in retina pigment epithelial cells as well. Moreover, DBC1 is SUMOylated through SUMO1 conjugation at K591 residue in human and mouse lens epithelial cells. The SUMOylated DBC1 is localized in the nucleus and plays an essential role in promoting stress-induced apoptosis. Silence of DBC1 attenuates oxidative stress-induced apoptosis. In contrast, overexpression of DBC1 enhances oxidative stress-induced apoptosis, and this process depends on p53. Mechanistically, DBC1 interacts with p53 to regulate its phosphorylation status at multiple sites and the SUMOylation of DBC1 enhances its interaction with p53. Together, our results identify that DBC1 is an important regulator mediating stress-induced apoptosis in lens, and thus participates in control of lens cataractogenesis.
... This model established the feasibility of using transgenic mice to study PV-associated diseases. The high-risk mucosotropic HPV16 E6 and E7 oncogenes were then studied using transgenic mice (73)(74)(75)(76), providing key insights into their contributions to tumorigenesis, including the abilities of E6 to inhibit apoptosis through p53-dependent and -independent means (74,77,78) and E7 to induce hyperplasia through its inactivation of the tumor suppressor pRb and dysregulation of E2F-dependent gene expression (77,79). Epidermis-specific expression of E6 and E7 also induced skin tumors (76). ...
... This model established the feasibility of using transgenic mice to study PV-associated diseases. The high-risk mucosotropic HPV16 E6 and E7 oncogenes were then studied using transgenic mice (73)(74)(75)(76), providing key insights into their contributions to tumorigenesis, including the abilities of E6 to inhibit apoptosis through p53-dependent and -independent means (74,77,78) and E7 to induce hyperplasia through its inactivation of the tumor suppressor pRb and dysregulation of E2F-dependent gene expression (77,79). Epidermis-specific expression of E6 and E7 also induced skin tumors (76). ...
Animal models of viral pathogenesis are essential tools in human disease research. Human papillomaviruses (HPVs) are a significant public health issue due to their widespread sexual transmission and oncogenic potential. Infection-based models of papillomavirus pathogenesis have been complicated by their strict species and tissue specificity. In this GEM review, we discuss the discovery of a murine papillomavirus, MmuPV1, and how its experimental use represents a major advancement in models of papillomavirus-induced pathogenesis/carcinogenesis, and their transmission.
... 19 Recent studies demonstrated that lens differentiation is regulated by the same set of regulators responsible for the control of apoptosis. 19,20 These regulators include the tumor suppressor p53, [21][22][23][24][25][26][27][28] Bcl-2 family members, 25,[29][30][31][32][33] caspase family members, 19, small heat shock proteins 32,[41][42][43][44][45] and tumor necrosis factors. 46 Tumor suppressor p53 has been implicated in regulating lens development. ...
... 24 Loss of p53 activity through expression of viral genes or the endogenous gene knockout induces posterior subcapsular cataracts. 21,47,48 Besides, overexpressing human p53 in mouse lens led to microphthalmia. 22 At the molecular level, p53 has been shown to regulate both major lens transcription factors c-Maf, Prox-1 26 and differentiation-related crystalline genes. ...
Cataract refers to opacities of the lens that impede the passage of light. Mutations in heat shock transcription factor 4 (HSF4) have been associated with cataract; however, the mechanisms regarding how mutations in HSF4 cause cataract are still obscure. In this study, we generated an hsf4 knockout zebrafish model using TALEN technology. The mutant zebrafish developed an early-onset cataract with multiple developmental defects in lens. The epithelial cells of the lens were overproliferated, resulting in the overabundance of lens fiber cells in hsf4null zebrafish lens. Consequently, the arrangement of the lens fiber cells became more disordered and irregular with age. More importantly, the terminal differentiation of the lens fiber cell was interrupted as the organelles cannot be cleaved in due time. In the cultured human lens epithelial cells, HSF4 could stabilize and retain p53 in the nucleus to activate its target genes such as fas cell surface death receptor (Fas) and Bcl-2-associated X apoptosis regulator (Bax). In the hsf4null fish, both p53 and activated-caspase3 were significantly decreased. Combined with the finding that the denucleation defect could be partially rescued through microinjection of p53, fas and bax mRNA into the mutant embryos, we directly proved that HSF4 promotes lens fiber cell differentiation by activating p53 and its downstream regulators. The data we presented suggest that apoptosis-related genes are involved in the lens fiber cell differentiation. Our finding that HSF4 functions in the upstream to activate these genes highlighted the new regulatory modes of HSF4 in the terminal differentiation of lens fiber cell.
... Similarly, transgenic mice expressing human papilloma virus type 16 (HPV-16) E7 in retinal cells exhibit very high rates of retinal cell apoptosis. However, expression of both E7 and E6 transgenes, or the E7 transgene in p53-mutant mice induces retinal tumors with a reduction or absence of associated apoptosis (Howes et al., 1994;Pan and Griep, 1994). HPV-16 E7-protein binds all Rb-family members suggesting that the failure of retinoblastomas to form in targeted Rbmutant mice is a consequence of functional redundancy amongst the Rb-family. ...
The retinoblastoma-related p130 protein is a member of a conserved family, consisting of Rb, p107 and p130, which are believed to play important roles in cell-cycle control and cellular differentiation. We have generated a null mutation in p130 by gene targeting and crossed the null allele into Balb/cJ and C57BL/6J strains of mice. In an enriched Balb/cJ genetic background, p130(−/−) embryos displayed arrested growth and died between embryonic days 11 and 13. Histological analysis revealed varying degrees of disorganization in neural and dermamyotomal structures. Immunohistochemistry with antibody reactive with Islet-1 indicated markedly reduced numbers of neurons in the spinal cord and dorsal root ganglia. Immunohistochemistry with antibody reactive with desmin indicated a similar reduction in the number of differentiated myocytes in the myotome. The myocardium of mutant embryos was abnormally thin and resembled an earlier staged two-chambered heart consisting of the bulbus cordis and the ventricular chamber. TUNEL analysis indicated the presence of extensive apoptosis in various tissues including the neural tube, the brain, the dermomyotome, but not the heart. Immunohistochemistry with antibody reactive with PCNA revealed increased cellular proliferation in the neural tube and the brain, and decreased proliferation in the heart. The placentas of p130(−/−) embryos did not display elevated apoptosis and were indistinguishable from wild type suggesting that the phenotype was not due to placental failure. Following a single cross with the C57BL/6 mice, p130(−/−) animals were derived that were viable and fertile. These results indicate that p130 in a Balb/cJ genetic background plays an essential role that is required for normal development. Moreover, our experiments establish that second-site modifier genes exist that have an epistatic relationship with p130.
... An impressive number of mouse in vivo models exists that probe cell cyclecoupled differentiation of lens fiber cells using loss-of-function [235,[266][267][268][269][270] and gain-of-function [271][272][273][274][275] models. Nevertheless, a number of open questions remain to be addressed that require large-scale protein purifications and analyses of proteinprotein complexes and their post-translational modifications at different stages of the cell cycle, e.g., to probe formation of E2F-pRb [275,276], Pax6-pRb complexes [277,278], and interactions between FGF receptors, other membrane proteins, and their intracellular components [53,235,[279][280][281][282][283][284][285][286][287][288]. ...
In vitro differentiation of human pluripotent stem cells (hPSCs) into specialized tissues and organs represents a powerful approach to gain insight into those cellular and molecular mechanisms regulating human development. Although normal embryonic eye development is a complex process, generation of ocular organoids and specific ocular tissues from pluripotent stem cells has provided invaluable insights into the formation of lineage-committed progenitor cell populations, signal transduction pathways, and self-organization principles. This review provides a comprehensive summary of recent advances in generation of adenohypophyseal, olfactory, and lens placodes, lens progenitor cells and three-dimensional (3D) primitive lenses, “lentoid bodies”, and “micro-lenses”. These cells are produced alone or “community-grown” with other ocular tissues. Lentoid bodies/micro-lenses generated from human patients carrying mutations in crystallin genes demonstrate proof-of-principle that these cells are suitable for mechanistic studies of cataractogenesis. Taken together, current and emerging advanced in vitro differentiation methods pave the road to understand molecular mechanisms of cataract formation caused by the entire spectrum of mutations in DNA-binding regulatory genes, such as PAX6, SOX2, FOXE3, MAF, PITX3, and HSF4, individual crystallins, and other genes such as BFSP1, BFSP2, EPHA2, GJA3, GJA8, LIM2, MIP, and TDRD7 represented in human cataract patients.
... The first transgenic mouse model to direct expression of HPV viral oncoproteins to epithelial cells used the α-crystallin promoter to express HPV16 E6 and E7 in the ocular lens, an epithelial tissue [227]. These mice developed hyperplasia and tumors of the lens, phenotypes that were subsequently found to result from E7-induced inactivation of pRb [228] and E6-dependent inhibition of apoptosis and other p53-dependent and independent activities of E6 [229,230]. Around the same time, HPV16 E7 expression was targeted to retinal cells in the eye to inactivate pRb, but these mice only developed tumors in p53-null mice [231]. The α-crystallin promoter also resulted in ectopic expression of HPV16 E6 and E7 in the murine epidermis and these transgenic mice developed preneoplastic lesions that progressed to carcinomas, thus providing the first in vivo evidence that high-risk HPV oncoproteins have oncogenic potential in an experimental model system [232]. ...
Human tumor viruses cause various human cancers that account for at least 15% of the global cancer burden. Among the currently identified human tumor viruses, two are small DNA tumor viruses: human papillomaviruses (HPVs) and Merkel cell polyomavirus (MCPyV). The study of small DNA tumor viruses (adenoviruses, polyomaviruses, and papillomaviruses) has facilitated several significant biological discoveries and established some of the first animal models of virus-associated cancers. The development and use of preclinical in vivo models to study HPVs and MCPyV and their role in human cancer is the focus of this review. Important considerations in the design of animal models of small DNA tumor virus infection and disease, including host range, cell tropism, choice of virus isolates, and the ability to recapitulate human disease, are presented. The types of infection-based and transgenic model strategies that are used to study HPVs and MCPyV, including their strengths and limitations, are also discussed. An overview of the current models that exist to study HPV and MCPyV infection and neoplastic disease are highlighted. These comparative models provide valuable platforms to study various aspects of virus-associated human disease and will continue to expand knowledge of human tumor viruses and their relationship with their hosts.
... HPV early proteins, E6 and E7, are oncoproteins affecting cell growth control through inactivation of cellular tumor suppressor gene products: p53 and Rb, respectively [14]. E6 and E7 are widely studied in HPV-associated cancer immunotherapy [9][10][11]. ...
Purpose: Nearly half of penile cancers are related to human papillomavirus (HPV) infection. Investigations of tumor- and HPV-specific T cell reactivity in regional lymph nodes (LNs) from patients with penile cancer are warranted.
Materials and Methods: In this study, single-cell suspensions from LNs and peripheral blood from 11 patients with penile cancer were stained with antibodies for lymphocyte markers and analyzed by fluorescence-activated cell sorting (FACS). DNA was extract-ed from the tumor tissue and HPV status was investigated by PCR.
Results: T cell reactivity against autologous tumor extract and against the HPV vaccine Gardasil was tested by flow-cytometric assay of specific cell-mediated immune response in activated whole blood (FASCIA). CD4+/CD8+ ratios were significantly lower in HPV-positive LNs (p<0.05). Immune responses to tumor extract assessed by blast transformation and expansion in vitro, of either CD4+ or CD8+ T cells, were found in 9 of 13 LNs (69%). Thus, 5 of 6 tested patients demonstrated T cell recognition of tumor-asso-ciated antigen(s). In HPV-positive patients, dose-dependent T cell responses against L1 (late) HPV proteins (Gardasil vaccine) were demonstrated.
Conclusions: LN-derived T cells from patients with penile cancer recognize tumor antigen(s) and in HPV-positive cases, there is a response against L1 (late) HPV proteins, being constituents of the Gardasil vaccine
... Mice, wild-type for p53, expressing the HPV-16 E7 gene in photoreceptor cells exhibited retinal degeneration as a result of apoptosis while the same expression in mice heterozygously mutant for p53, initiated retinal tumour formation (Howes et at., 1994). Transgenic mice expressing HPV E7 in the developing ocular lens exhibited apoptosis in lens cells and this was inhibited by co-expression of HPV E6 (Pan & Griep, 1994). Expression of E la in BRK or REF52 cells induced p53dependent apoptosis (Debbas & White, 1993 ;Lowe & Ruley, 1993c) and RBknockout mice died at day 13.5 of development, showing signs of excessive apoptosis in neural tissues (Jacks et a l, 1992 ;Lee et a l, 1992 ;Clarke et a l, 1992). ...
The transcription factor DRTF1/E2F regulates genes required for cell cycle progression and occupies a central role in the control of cellular proliferation by integrating cell cycle machinery with transcription. DRTF1/E2F transcriptional activity is regulated in part by the binding of the tumour suppressor pRb which is mutationally inactivated in a large range of human cancers. Mutations in the p53 tumour suppressor gene are the most frequently observed genetic alterations in human neoplasia. Recent data suggests that the p53 gene product controls a cell cycle checkpoint responsible for maintaining the integrity of the genome, although the exact mechanism by which this occurs is still unclear. The DNA binding activity of DRTF1/E2F is believed to be a heterodimer composed of one of each of the DP- and E2F- polypeptide families. I present evidence that one member, DP-1, can exist in a hypo- or hyper-phosphorylated state in vivo, which in turn correlates with altered DRTF1/E2F affinity for its DNA binding site. Data indicates that the different phosphorylation state may affect DP-I's ability to heterodimerise with partners such as E2F-1 and E2F-5, an event essential for high affinity DNA binding and subsequent transcriptional trans-activation. These results potentially define a new level of control for DRTF1/E2F in which its DNA binding activity is modulated by cell cycle-regulated phosphorylation events on DP-1. Also presented is evidence suggesting that one phosphoform of DP-1 can form an in vivo complex with p53 and that p53 can repress the DNA binding activity of DP- 1/E2F-1 heterodimers. These results contribute to the establishment of DP-1 as a common cellular target in two distinct and independent pathways of growth control mediated through the activities of the pRb and p53 tumour suppressor proteins. The integration of p53 with DP-1 and the consequent regulation of DRTF1/E2F DNA binding activity defines novel potential pathway through which p53 can influence cell cycle progression.
... In fact it seems that all genes whose deregulated expression can drive cells from quiescence through S-phase (E2F and adenovirus ElA, for example) also prom ote apoptosis (Qin et al., 1994;Rao et al., 1992;W u and Levine, 1994). M utations that remove restraints on cell cycle progression, such as loss of Rb activity, have sim ilar effects (Howes et al., 1994;M orgenbesser et al., 1994;Pan and Griep, 1994;Symonds et al., 1994). This observation im m ediately suggests an additional restraint upon tum our progression, and a further layer of complexity to the Vogelstein model: if all m utations conferring a grow th advantage confer a corresponding propensity to undergo cell death, a m utation that confers a survival advantage m ust also be acquired very early in hyperplasia. ...
The nematode worm Caenorhabditis elegans has proved to be an illuminating model for programmed cell death. Three principal genes are involved in nematode PCD, ced-3, ced-4 and ced-9: each has proved to have vertebrate homologues that are critical regulators of vertebrate apoptosis. Until recently, the molecular functions of CED-4 and CED-9 were largely obscure. I have used the yeast Schizosaccharomyces pombe as a naive model system in which to assay the function of the pro-apoptotic protein CED-4. Expression of wild-type ced-4 is toxic to S. pombe, while expression of the point mutant I258N, which gives rise to a ced-4-null phenotype in the worm, has no effect, suggesting that the toxicity is the result of a bona fide activity of CED-4. Mutation of the putative nucleotide-binding P-loop motif of CED-4 also eliminates the lethal phenotype, demonstrating for the first time the importance of this domain for CED-4 function. The anti-apoptotic protein CED-9 is able to rescue S. pombe from CED-4- induced lethality: the most parsimonious explanation for this observation is that CED-9 directly binds and inhibits CED-4. This is confirmed by immunogold labelling of CED-4 visualised by electron microscopy: CED-4 expressed alone is nuclear, but when co-expressed with CED-9 it is found on mitochondrial and ER membranes, the presumptive location of CED-9. The physical interaction between CED-4 and CED-9 is further confirmed by yeast two-hybrid analysis. In addition, the cloning and characterisation of two C. elegans homologues of baculovirus Inhibitor of Apoptosis Proteins (IAPs) is described. Cellular IAP homologues are found in Drosophila and humans and influence apoptosis in both organisms. The C. elegans IAP homologues do not inhibit the activity of CED-3 or CED-4 in S. pombe. Knockout of their activity in vivo by means of RNA-mediated inhibition reveals no obvious cell death-related phenotype.
... Macleod et a l, 1996; Morgenbesser et a l, 1994;Williams et a l, 1994). Similar observations occur in mice transgenic for HPV E7 or a mutant allele of SV40 T-antigen, that selectively targets pRb, with the apoptotic phenotype being rescued by the introduction of HPV E6 or the disruption of either E2F-1 or p53(Fromm et a l, 1994; Howes et a l, 1994;Pan and Griep, 1994;Pan and Griep, 1995;Pan et a l, 1998;Symonds et a l, 1994; Tsai e ta l, 1998). ...
Normal diploid cells have a limited lifespan in culture culminating in a process known as replicative senescence. Since tumour cells have an extended or indefinite lifespan, senescence is viewed as a mechanism for tumour suppression. Senescent cells arrest in the late G1 phase of the cell cycle with the retinoblastoma protein in a predominantly unphosphorylated state. They also accumulate high levels of the cyclin-dependent kinase inhibitors, p16INK4a and p21CIP1 both of which are likely to contribute to the arrest. To dissect the different contributions p16INK4a and p21CIP1 make to replicative senescence cyclin-CDK-CKI complexes have been examined in senescent human diploid fibroblasts and fibroblasts whose lifespan has been extended by DNA virus oncoproteins. The latter proteins, such as SV40 T-antigen or the E6 and E7 proteins of human papilloma virus, interfere with the functions of p53 and pRb. Abrogating pRb renders cells insensitive to p16INK4a whereas abrogating p53 diminishes the expression of p21CIP1. One strain of human diploid fibroblasts, SVts8, have been analysed in detail. These cells express a temperature sensitive form of SV40 T-antigen and are immortal when grown at the permissive temperature but upon inactivation of T-antigen the cells arrest immediately with the characteristics of senescence. Analysis of the cyclin-CDK complexes by gel filtration has shown that while cyclin D1-CDK6 complexes remain intact in senescent cells, the cyclin Dl- CDK6 complexes are completely disrupted, indicating a potential difference between CDK4 and CDK6 in fibroblast senescence. R24P a variant of p16INK4a able to interact with CDK6 but not CDK4 was utilised to dissect the effects of CDK4 and CDK6. The data confirm the critical roles of p16INK4a and p21CIP1 in the implementation of senescence and has provided important insights into different cell cycle regulators in this process.
... The LECs at the lens periphery have been shown to repair DSBs more slowly [247] and along with the nucleated, differentiating LFCs these are the lens cells that are transcriptionally active. Moreover, crystallin gene transcription is dependent upon both p53 [254] and N-myc [255], providing another potential direct link between transcription and DNA repair [256]. Given the long latency between exposure to low dose IR and the appearance of cataract, this is a potential mechanism contributing to the low dose IRinduced phenotype. ...
Ionizing radiation (IR) damages DNA and other macromolecules, including proteins and lipids. Most cell types can repair DNA damage and cycle continuously their macromolecules as a mechanism to remove defective proteins and lipids. In those cells that lack nuclei and other organelles, such as lens fiber cells and mammalian erythrocytes, IR-induced damage to macromolecules is retained because they cannot be easily replenished. Whilst the life span for an erythrocyte is several months, the life span of a human lens is decades. There is very limited turnover in lens macromolecules, therefore the aging process greatly impacts lens structure and function over its lifetime. The lens is a tissue where biomolecular longevity, lifelong retention of its components and continued growth are integral to its homeostasis. These characteristics make the lens an excellent model to study the contribution of retained macromolecular damage over time. Epidemiological data have revealed a significant association between exposure to IR, the loss of lens optical function and the formation of cataracts (cataractogenesis) later in life. Lifestyle, genetic and environmental factors all contribute to cataractogenesis due to their effect on the aging process. Cataract is an iconic age-related disease in humans. IR is a recognised cause of cataract and the occupational lens dose limit is reduced from 150 to 20 mGy / year averaged over 5 years (ICRP Publication 118). Understanding the effects of low dose IR on the lens and its role in cataractogenesis is therefore very important. So we redefine “cataractogenic load” as a term to account for the combined lifestyle, genetic and environmental processes that increase biomolecular damage to lens macromolecules. These processes weaken metabolic defenses, increase post-translational protein modifications, and alter the lipid structure and content of the lens. IR exposure is a significant insult to the lens because of free radical generation and the ensuing oxidative stress. We support the concept that damage caused by IR compounds the aging process by increasing the cataractogenic load, hereby accelerating lens aging and its loss of function.
... Ionizing radiation induces DSBs randomly in the genome, but the genes most likely to retain mutations are those that are transcriptionally active and in the lens such as crystallins (Khan et al. 2015). It is the epithelial cells in the lens periphery that are slower to repair DSBs (Markiewicz et al. 2015) as supported in this study, and both these peripheral epithelial cells and the nucleated, differentiating lens fiber cells are where this transcriptional activity is located and also dependent upon tumor protein p53 (TP53) (Pan and Griep 1994) and N-myc (Cavalheiro et al. 2017). The inter-relationship of DNA repair and transcriptional regulation (Petroni et al. 2016;Lieberman et al. 2017) is, therefore, an important consideration for understanding the results of this study and the wider mechanism(s) of IR-induced cataract. ...
Purpose: Epidemiological evidence regarding the radiosensitivity of the lens of the eye and radiation cataract development has led to changes in the EU Basic Safety Standards for protection of the lens against ionizing radiation. However, mechanistic details of lens radiation response pathways and their significance for cataractogenesis remain unclear. Radiation-induced DNA damage and the potential impairment of repair pathways within the lens epithelium, a cell monolayer that covers the anterior hemisphere of the lens, are likely to be involved.
Materials and Methods: In this work, the lens epithelium has been analyzed for its DNA double-strand break (DSB) repair response to ionizing radiation. The responses of epithelial cells located at the anterior pole (central region) have been compared to at the very periphery of the monolayer (germinative and transitional zones). Described here are the different responses in the two regions and across four strains (C57BL/6, 129S2, BALB/c and CBA/Ca) over a low dose (0–25 mGy) in-vivo whole body X-irradiation range up to 24 hours post exposure.
Results: DNA damage and repair as visualized through 53BP1 staining was present across the lens epithelium, although repair kinetics appeared non-uniform. Epithelial cells in the central region have significantly more 53BP1 foci. The sensitivities of different mouse strains have also been compared.
Conclusions: 129S2 and BALB/c showed higher levels of DNA damage, with BALB/c showing significantly less inter-individual variability and appearing to be a more robust model for future DNA damage and repair studies. As a result of this study, BALB/c was identified as a suitable radiosensitive lens strain to detect and quantify early low dose ionizing radiation DNA damage effects in the mouse eye lens specifically, as an indicator of cataract formation.
... Other cells types, however, undergo rapid apoptotic death following wild-type p53 expression and the regulation of the decision to undergo cell-cycle arrest or programmed cell death is not fully understood (Oren, 1994). Nevertheless, there is substantial evidence that apoptosis is exhibited by cells containing wild-type p53 which suffer additional perturbations to normal cell-cycle control, such as loss of the tumor-suppressor protein pRB function (Debbas and White, 1993;Howes et al., 1994;Pan and Griep, 1994;Haupt et al., 1995a) and deregulated expression of the transcription factor E2F (Qin et al., 1994;Shan and Lee, 1994;Wu and Levine, 1994). Removal of exogenous survival factors has also been associated with p53-directed apoptosis rather than growth arrest (Collins et al., 1992;Gottlieb et al., 1994;Canman et al., 1995). ...
The p53 tumor‐suppressor gene product is frequently inactivated in malignancies by point mutation. Although most tumor‐derived p53 mutants show loss of sequence specific transcriptional activation, some mutants have been identified which retain this activity. One such mutant, p53175P, is defective for the suppression of transformation in rodent cells, despite retaining the ability to suppress the growth of p53‐null human cells. We now demonstrate that p53175P can induce a cell‐cycle arrest in appropriate cell types but shows loss of apoptotic function. Our results therefore support a direct role of p53 transcriptional activation in mediating a cell‐cycle arrest and demonstrate that such activity is not sufficient for the full apoptotic response. These data suggest that either p53 can induce apoptosis through a transcriptionally independent mechanism, a function lost by p53175P, or that this mutant has specifically lost the ability to activate genes which contribute to cell death, despite activation of genes responsible for the G1 arrest. This dissociation of the cell‐cycle arrest and apoptotic activities of p53 indicates that inactivation of p53 apoptotic function without concomitant loss of growth inhibition can suffice to relieve p53‐dependent tumor‐suppression in vivo and thereby contribute to tumor development.
... In conclusion, the data presented here suggest that mdm2 may act as a physiological modulator of p53mediated apoptosis. This capacity of mdm2, along with its ability to relieve p53-mediated growth inhibition (Finlay, 1993;Otto and Deppert, 1993;Chen et al., 1994), may constitute the basis for its oncogenic effects, in particular since the apoptotic activity of p53 has been suggested to mediate its ability to suppress tumorigenesis (Howes et al., 1994;Morgenbesser et al., 1994;Pan and Griep, 1994;Symonds et al., 1994). Inhibition of p53-mediated apoptosis by mdm2 is consistent with the clinical observation that, in many of the tumors containing amplified mdm2, p53 is not mutated (Oliner et al., 1992;Leach et al., 1993;Reifenberger et al., 1993;Cordon-Cardo et al., 1994). ...
The effect of excess mdm2 on p53‐mediated apoptosis was investigated in two human‐derived cell lines, H1299 and HeLa. In H1299 cells, overexpression of mdm2 resulted in effective protection from apoptosis. This protective effect was seen only under conditions allowing the formation of p53‐Mdm2 complexes. In contrast, excess mdm2 failed to abolish p53‐mediated apoptosis in HeLa cells, despite a complete abrogation of p53‐dependent sequence‐specific transcriptional activation (SST). These data strongly support the contention that SST is dispensable for at least some types of p53‐mediated apoptosis. Further, they suggest that one of the roles of mdm2 may be to modulate the apoptotic activity of p53, in a manner which is dictated by the pathway through which p53 induced apoptosis in a given cell type
... Human papillomaviruses (HPV), particularly HPV16, are associated with most cervical cancers [1]. The E6 and E7 proteins, which are usually expressed in all cervical cancers, are able to disrupt cell-cycle control by inactivating the tumor suppressors p53 and pRB via the ubiquitin-dependent proteolytic pathway [2,3]. Hence, HPV16 E6 and E7 are appropriate target antigens for developing vaccines. ...
Aim: The present study aimed to investigate the effect of codon optimization on E6 recombinant protein production in Lactococcus lactis. Method: Here we define the construction of shuttle vector harboring wild-type and codon-optimized HPV16 E6 oncogene, with maximum number of infrequent codons exchanged with codons that are frequently used in Lactococcus lactis subsp. cremoris MG1363. Results: Hence, the codons encoding 159 amino acids were modified, in which a total of 91 codons were changed, resulting in approximately threefold increase in protein expression of recombinant E6 (rE6). Conclusion: Our data revealed that codon usage optimization according to L. lactis desired codon usage can dramatically increase the expression of HPV16 E6, suggesting that this strategy is a valuable approach for immunization through DNA vaccine.
... 1 The known carcinogenic mechanisms of HPV infection in cervical carcinoma include: chronic viral infection, deregulation of cell cycle and chromosomal instability caused by viral oncoprotein E6/E7, and the integration of HPV DNA in human genome. [2][3][4][5] Indeed, our previous study demonstrated that alteration of human genome and functional aberration of integration-targeted genes by HPV integration might play an important role in cervical oncogenesis, which provided additional evidences supporting that integrated viral DNA fragments may affect the expression and function of host genes near integration sites, and thereby resulting in malignant transformation of cervical cells. 6 HeLa cell is one of the most widely used cell model in biomedical research of cervical cancer. ...
Human papillomavirus (HPV) infection is the most important risk factor for cervical cancer development. In HeLa cell line, the HPV viral genome is integrated at 8q24 in one allele of chromosome 8. It has been reported that the HPV fragment integrated in HeLa genome can cis-activate the expression of proto-oncogene MYC, which is located at 500 kb downstream of the integrated site. However, the underlying molecular mechanism of this regulation is unknown. A recent study reported that MYC was highly expressed exclusively from the HPV-integrated haplotype, and a long-range chromatin interaction between the integrated HPV fragment and MYC gene has been hypothesized. In this study, we provided the experimental evidences supporting this long-range chromatin interaction in HeLa cells by using Chromosome Conformation Capture (3C) method. We found that the integrated HPV fragment, MYC and 8q24.22 was close to each other and might form a trimer in spatial location. When knocking out the integrated HPV fragment or 8q24.22 region from chromosome 8 by CRISPR/Cas9 system, the expression of MYC reduced dramatically in HeLa cells. Interestingly, decreased expression was only observed in three from eight cell clones, when only one 8q24.22 allele was knocked out. Functionally, HPV knockout caused senescence-associated acidic β-gal activity in HeLa cells. These data indicate a long-distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region, providing an alternative mechanism relevant to the carcinogenicity of HPV integration. This article is protected by copyright. All rights reserved.
... Our previous study showed that cell cycle genes were a main functional GO category in the G64 module. Consistently, dysregulation of the cell cycle has long been proven to be a critical process causing cancers [19][20][21][22]. However, to our knowledge, it was the first finding ever that G64 genes were co-regulated in various cancers, and patient prognosis can be differentiated by these genes. ...
Studies of cancer heterogeneity have received considerable attention recently, because the presence or absence of resistant sub-clones may determine whether or not certain therapeutic treatments are effective. Previously, we have reported G64, a co-regulated gene module composed of 64 different genes, can differentiate tumor intra- or inter-subpopulations in lung adenocarcinomas (LADCs). Here, we investigated whether the G64 module genes were also expressed distinctively in different subpopulations of other cancers. RNA sequencing-based transcriptome data derived from 22 cancers, except LADC, were downloaded from The Cancer Genome Atlas (TCGA). Interestingly, the 22 cancers also expressed the G64 genes in a correlated manner, as observed previously in an LADC study. Considering that gene expression levels were continuous among different tumor samples, tumor subpopulations were investigated using extreme expressional ranges of G64—i.e., tumor subpopulation with the lowest 15% of G64 expression, tumor subpopulation with the highest 15% of G64 expression, and tumor subpopulation with intermediate expression. In each of the 22 cancers, we examined whether patient survival was different among the three different subgroups and found that G64 could differentiate tumor subpopulations in six other cancers, including sarcoma, kidney, brain, liver, and esophageal cancers.
... This difference in phenotype is likely to be derived from the timing of Prox1 loss, as the Prox1 cKO lenses do not lose all Prox1 protein until after LV closure (Fig. 1K), whereas Prox1 was absent from all tissues in the prior report . The observation that a lack of cell cycle exit is insufficient to explain the Prox1 null lens phenotype is consistent with prior reports of retinoblastoma mutant (Fromm et al., 1994;Pan and Griep, 1994) and p27 Kip1−/− ;p57 Kip2+/− (Zhang et al., 1998) lenses, which do not phenocopy the morphology of lenses lacking Prox1, despite maintaining cell proliferation in the posterior lens. ...
Lens epithelial cells differentiate into lens fibers (LF) in response to a fibroblast growth factor (FGF) gradient. This cell fate decision requires the transcription factor Prox1, which was hypothesized to promote cell cycle exit in differentiating lens fibers. However, conditional deletion of Prox1 from mouse lenses resulted in a failure in lens fiber differentiation despite maintenance of normal cell cycle exit. Instead, RNAseq demonstrated that Prox1 functions as a global regulator of lens fiber cell gene expression. Intriguingly, Prox1 also controlled the expression of fibroblast growth factor receptors (FGFRs) and could bind to their promoters, correlating with decreased downstream signaling through MAPK and AKT in Prox1 mutant lenses. Further, culturing rat lens explants in FGF increased their expression of Prox1, and this was attenuated by the addition of inhibitors of MAPK. Together these results describe a novel feedback loop required for lens differentiation and morphogenesis, where Prox1 and FGFR interact to mediate LF differentiation in response to FGF.
Lens epithelial explants are comprised of lens epithelial cells cultured in vitro on their native basement membrane, the lens capsule. Biologists have used lens epithelial explants to study many different cellular processes including lens fiber cell differentiation. In these studies, fiber differentiation is typically measured by cellular elongation and the expression of a few proteins characteristically expressed by lens fiber cells in situ. Chromatin and RNA was collected from lens epithelial explants cultured in either un-supplemented media or media containing 50% bovine vitreous humor for one or five days. Chromatin for ATAC-sequencing and RNA for RNA-sequencing was prepared from explants to assess regions of accessible chromatin and to quantitatively measure gene expression, respectively. Vitreous humor increased chromatin accessibility in promoter regions of genes associated with fiber differentiation and, surprisingly, an immune response, and this was associated with increased transcript levels for these genes. In contrast, vitreous had little effect on the accessibility of the genes highly expressed in the lens epithelium despite dramatic reductions in their mRNA transcripts. An unbiased analysis of differentially accessible regions revealed an enrichment of cis-regulatory motifs for RUNX, SOX and TEAD transcription factors that may drive differential gene expression in response to vitreous.
Citation: Upreti, A.; Padula, S.L.; Tangeman, J.A.; Wagner, B.D.; O'Connell, M.J.; Jaquish, T.J.; Palko, R.K.; Mantz, C.J.; Anand, D.; Lovicu, F.J.; et al. Lens Epithelial Explants Treated with Vitreous Humor
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.
The crystalline lens is a transparent and refractive biconvex structure formed by lens epithelial cells (LECs) and lens fibers. Lens opacity, also known as cataracts, is the leading cause of blindness in the world.
LECs are the principal cells of lens throughout human life, exhibiting different physiological properties and functions. During the embryonic stage, LECs proliferate and differentiate into lens fibers, which form the crystalline lens. Genetics and environment are vital factors that influence normal lens development. During maturation, LECs help maintain lens homeostasis through material transport, synthesis and metabolism as well as mitosis and proliferation. If disturbed, this will result in loss of lens transparency. After cataract surgery, the repair potential of LECs is activated and the structure and transparency of the regenerative tissue depends on postoperative microenvironment.
This review summarizes recent research advances on the role of LECs in lens development, homeostasis, and regeneration, with a particular focus on the role of cholesterol synthesis (eg., lanosterol synthase) in lens development and homeostasis maintenance, and how the regenerative potential of LECs can be harnessed to develop surgical strategies and improve the outcomes of cataract surgery (Fig. 1). These new insights suggest that LECs are a major determinant of the physiological and pathological state of the lens. Further studies on their molecular biology will offer possibility to explore new approaches for cataract prevention and treatment.
To study the molecular mechanisms of oncogene cooperation, rat Schwann cells were used in which oncogenic Ras or Raf alone cause a cell cycle arrest, whereas cooperating oncogenes lead to transformation (Ridley et al., 1988; Lloyd et al., 1997). This model system was used to search for cooperating oncogenes and to investigate the underlying biochemical mechanisms of oncogene cooperation. Interestingly, mutants of the tumour suppressor p53 efficiently cooperate with oncogenic Ras or Raf to transform Schwann cells. This result prompted me to further investigate the role of p53 in Schwann cells expressing an inducible activated Raf (ARafER). Activation of ARafER led to the induction of the cell cycle inhibitor p21WAF1/Cip1. induction of p21WAF1/Cip1 is p53-dependent, since dominant negative mutants of p53 abolish this induction and subsequently the G1 cell cycle arrest. The pathway leading to the p53-dependent induction of p21WAF1/Cip1 by activated Raf was investigated by addressing the possible role of the MAP-kinase pathway. The contribution of MAP-kinase kinase and MAP-kinase in the signalling pathway triggering the G1 cell cycle arrest were examined using dominant negative and constitutively active mutants as well as a specific chemical inhibitor. My results indicate that MAP-kinase kinase is necessary for p21WAF1/Cip1 induction in Schwann cells. The contribution of the JNK-kinase pathway in the Raf-initiated cell cycle arrest was addressed.
Human papillomavirus (HPV) infection is the most common sexually transmitted viral infection. The majority of genital HPV infections are transient and do not induce any clinical manifestations, because they are rapidly and efficiently cleared by the host immune system. Rarely, a persistent infection is established by high-risk (HR) mucosal HPV genotypes (e.g., HPV16), so that pre-neoplastic and neoplastic lesions may develop. HR-HPVs are involved in the development of about 5% of all human cancers, especially at anogenital sites but also in the head and neck region. Two viral oncoproteins, E6 and E7, are the main drivers of HPV-mediated carcinogenesis, because of their ability to bind to a surprisingly high number of cellular targets, including p53 and pRb tumor suppressors, and to subvert pathways involved in cell cycle regulation, immune response, and genome integrity. In addition, E6 and E7 profoundly alter the host epigenome, further deregulating the expression of cellular tumor suppressors, oncogenes, and immune-related genes. Over time, the interplay between these oncoproteins and the consequent disruption of key cellular pathways lead to the irreversible acquisition of a malignant phenotype. The monoclonal expansion of the HPV-infected cell(s) with the greatest growth advantage will ultimately lead to cancer. The complex and intimately linked series of events of mucosal HPV-induced tumorigenesis, which is mostly clarified for HPV-associated cervical cancer, is still largely unknown for other HPV-driven cancers. Certain cutaneous HPVs, particularly beta HPVs, appear to play a role in the etiology of skin cancer, although major gaps of knowledge remain about their carcinogenic mechanisms.
Genes upregulated by p53 were screened using an erythroleukemic cell line (1-2-3) that expresses only the temperature-sensitive p53 by the mRNA differential display method. One of the upregulated genes was identified as the elongation factor-1α (EF-1α) gene, an essential component of the eukaryotic translation apparatus. Three p53-responsive elements were found in the mouse EF-1α gene and in the corresponding human, rat, and frog genes. These elements conferred the capacity for induction by p53. EF-1α is also a microtubule-severing protein. Upon the temperature-shift, the cells developed the morphology and the localization of α-tubulin similar to those of the cells treated with vincristine, a drug that affects microtubules. The microtubule-severing associated with upregulation of EF-1α by p53 may be a cause of the cell death.
Purpose:
The tumor suppressor p53 is a master regulator of apoptosis and also plays a key role in cell cycle checking. In our previous studies, we demonstrated that p53 directly regulates Bak in mouse JB6 cells and that p53-Bak signaling axis plays an important role in mediating EGCG-induced apoptosis. Furthermore, we have recently demonstrated that the same p53-Bak apoptotic signaling axis executes an essential role in regulating lens cell differentiation. In addition, we have also shown that p53 controls both transcription factors, C-Maf and Prox-1 as well as lens crystallin genes, αA, β- and γ-crystallins. Here, we have examined whether p53 also regulates other known target genes during its modulation of lens differentiation. The human and mouse lens epithelial cells, FHL124 and αTN4-1 were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin.
Methods:
Mice used in this study were handled in compliance with the "Protocol for the Care and Use of Laboratory Animals" (Sun Yat-sen University). Adult mice were used for the collection of lens cells. These samples were used for extraction of total proteins. A total of 32 embryonic mice {8 at 14.5 ED, 8 at 17.5 ED and 8 newborns for wild type} were used for immunohistochemistry, which were used for co-localization study. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J.
Results:
Immunohistochemistry revealed that both the cell cycle checking genes, p21 and Gadd45α and the apoptotic genes, Bcl-2 and PUMA, display developmental changes associated with p53 during mouse lens development. Knockdown of p53 in the mouse lens epithelial cells caused inhibition of lens differentiation. Associated with this inhibition, the cell cycle genes displayed significant downreglation, the apoptotic genes was also attenuated but to a much less degree. In addition, we found that bFGF can induce dose-dependent upregulation of the upstream kinases, CHK1/2 and ERK1/2, both known to phosphorylate p53 and activate the later. Furthermore, We showed that in both developing lens and human lens epithelial cells, p53 can be co-localized with the catalytic subunit of the protein phoshphatase-1 (PP-1), suggesting that PP-1 regulates p53 phosphorylation status both in vivo and in vitro.
Conclusion:
Taken together, our results suggest that during mouse lens development, p53 activity is regulated by ERK and CHK kinases-mediated activation, and by PP-1-mediated inactivation. p53 can regulate multiple groups of genes to mediate lens differentiation.
It has been said that no matter which direction cancer research turns, the p53 tumour suppressor protein comes into view. If there is truth in this statement, then it has much to do with the ability of this protein to induce programmed cell death, or apoptosis. First discovered in 1979 as a protein complexed with the large T antigen of SV40 virus (Lane and Crawford, 1979; Linzer and Levine, 1979), the complete cDNA sequence of p53 was elucidated in 1984 (Matlashewski et al. 1984), and its activity as a tumour suppressor gene was first suspected in 1985 when deletions of the gene were detected in transformed cells infected with Friend leukemia virus (Mowat et al. 1985). Later it was found that expression of p53 could inhibit the neoplastic transformation of primary cells (Finlay et al. 1989), and that this gene was subject to frequent mutations in human tumours (Baker et al. 1989; Hollstein et al. 1991). Germline mutations of the p53 gene were found in individuals suffering from Li-Fraumeni disorder, which is an autosomal dominant disorder predisposing affected individuals to osteosarcomas as well as tumours of the brain, breast and adrenal cortex (Malkin et al. 1990). Tumours from affected individuals were found to invariably lose expression of the wild type p53 allele, thereby identifying p53 as a classical tumour suppressor gene. Analogously, mice genetically engineered to be nullizygous for p53 (p53 “knock-out” mice) died from tumours at 3-6 months of age, although these were chiefly lymphoid tumours (Donehower et al. 1992). Analyses of p53 mutations in human tumour samples to date indicate that this tumour suppressor gene is mutated in over 50% of human tumours, derived from a wide variety of tissues. The p53 gene continues to hold the distinction as the most frequently mutated gene in human cancer.
Cancer is a genetic disease that results from multiple genomic changes. These ultimately lead to the deregulation of the cell cycle machinery and to autonomous cell proliferation. Neoplastic transformation involves four sets of genes: 1) oncogenes, 2) tumor-suppressor genes, 3) mutator genes, and 4) apoptotic genes. In the hematopoietic system, the first step in oncogenesis is the activation of an oncogene that may then be followed by the activation of an additional oncogene and/or the loss of function of a tumor-suppressor gene. The activation of oncogenes may play a predominant role in the formation of sarcomas as well. Tumors of both the hematopoietic system and soft tissues exhibit a karyotype close to normal. On the other hand, carcinomas, the most prevalent forms of cancer, are predominantly due to the loss of function of tumor-suppressor genes with multiple sites of loss of heterozygosity (LOH), and they have dramatic alterations in the karyotype (Rabbitts, 1994).
The laboratory mouse offers tremendous opportunities for studies on the genetic basis of cancer. No other model system is so easily and widely used to test the effect of potentially oncogenic mutations in a living animal. Mouse strains differ extraordinarily in their innate susceptibilty to various forms of cancer (see Jackson Laboratories website: http:// tumor. informatics. jax. org/ straingrid. html). The reasons for these differences are thought to lie in allelic variation in cancer susceptibility genes between strains. Breeding experiments have allowed researchers to localize some of these susceptibility genes regionally (1,2). The molecular identification of these susceptibility genes has proved elusive. However, cancer research has yielded many other types of cancer genes whose function has been tested using the power to manipulate the mouse germline. A large number of oncogenes have been discovered by studying oncogenic retroviruses, from transfection of tumor DNAs into mouse embryo fibroblast cell lines, and by molecular analyses of human tumors. Tumor suppressor genes have been discovered from linkage studies and positional cloning in human families segregating highly penetrant predisposition to specific tumor types (3). Mouse transgenesis has proved to be the most powerful and widely used system for the in vivo analysis of the function of cancer genes discovered by these methods. Several excellent reviews have been written on this subject (4–6). In this chapter, I briefly describe the history of mouse transgenesis in cancer research, some widely utilized models, new technologies, and future directions for mouse genetics in cancer research.
The tumor suppressor p53 is a master regulator of apoptosis and also plays a key role in cell cycle progress and cell differentiation. It mainly acts as a transcription factor. In addition, it can also directly interact with apoptosis regulators in mitochondria to control apoptosis. Recent studies from our laboratory and others have shown that p53 plays an active role in regulating lens differentiation. It does so by modulating developmental apoptosis and also controlling expression of lens differentiation-specific genes. In this chapter, we summary the current progresses in this field.
Genetically engineered mice (GEMs) have provided valuable insights into the carcinogenic properties of various human tumor viruses, which, in aggregate, are etiologically associated with over 15% of all human cancers. This review provides an overview of seminal discoveries made through the use of GEM models for human DNA tumor viruses. Emphasis is placed on the discoveries made in the study of human papillomaviruses, Merkel cell carcinoma–associated polyomavirus, Epstein-Barr virus, and Kaposi's sarcoma–associated herpesvirus, because GEMs have contributed extensively to our understanding of how these DNA tumor viruses directly contribute to human cancers.
Birth and death, the extremes of life, conjure images of antithetic and counteracting events necessary for maintaining a balance among living organisms. Their cellular counterparts, cell division and cell death, work in similar opposition to ensure proper development of an organism and to maintain homeostasis and normal function. A breakdown of either process can be detrimental and, in humans, may lead to birth defects, cancer, autoimmune disease, and neurological disorders. Although researchers have been well aware of the importance of proper cell division for decades, only recently has the significance of cell death attracted widespread appreciation. Consequently, we now have a relatively sophisticated understanding of the machinery that controls cell division; in comparison, we have only a rudimentary knowledge of the mechanisms that underlie cell death. But surprisingly, given our inclination to categorize birth and death as opposing and distinct forces, much of what we currently know about cell death has been fueled by research into cell cycle control. In this chapter, the hypothesis that the control of cell death and the cell cycle overlap will be discussed.
The p53 gene is an important tumor suppressor gene, whose inactivation appears to play a pivotal role in many types of cancer1–3. The p53 protein, encoded by this gene, is a potent sequence-specific transcriptional activator4,5. Binding of the p53 protein to genes which contain consensus p53 binding sites results in a pronounced increase in their transcription rates6–11. Transcriptional activation of relevant target genes, mediated through high affinity sequence-specific DNA binding, is believed to be responsible for many of the biological effects of the wild-type p53 (wt p53) protein. This is supported by the fact that the overwhelming majority of p53 mutations found in human tumors abrogate DNA binding, either by altering direct DNA contact residues or through destabilizing the structure of the DNA binding domain12,13.
Because of the clinical heterogeneity of human inherited retinal degenerations, we have initiated a study to seek common themes in the pathogenesis of cell death of photoreceptors by apoptosis. The interstitial retinol binding protein (IRBP) promoter was used to drive expression of the human papilloma virus 16 (HPV 16) E7 gene in the retina and other ocular tissues in mice. The result is the death of photoreceptors as they undergo terminal differentiation. Lens fiber cells also die after a period of inappropriate proliferation and abnormal differentiation to form cataracts. Cross-breeding these transgenic mice to mice lacking the p53 gene leads to formation of several ocular tumors by one month of age if both copies of the p53 gene are missing. With one copy of the gene, the mice develop retinal tumors after a much longer latency and at a lower incidence; the tumors that do arise have lost their normal copy of the gene. The lack of the p53 gene does not eliminate apoptosis of either the retina or the lens in these transgenic mice although the rate of destruction of photoreceptors is slightly delayed. The retinal tumors apparently arise from precursors that survive amid a dying cell layer.
When functioning normally, oncosuppressor genes, also known as tumour suppressor genes or antioncogenes, prevent the development of one or more types of cancer (reviewed by Knudson, 1993). A powerful way of investigating the mechanism of action of such genes is to examine the consequences of inactivating them in an experimental animal. This can be done via gene targeting in embryonal stem (ES) cells (reviewed by Hooper, 1992). These cells, established in culture from the pluripotent inner cell mass cells of the peri-implantation embryo, retain the capacity to colonise the somatic tissues and germ line of chimaeras produced by injecting them into blastocyst-stage embryos. Tailored mutations can be introduced into chosen genes by introducing into the cultured cells a gene targeting DNA vector and isolating cells in which the vector has undergone homologous recombination with the endogenous gene. These mutations can then be introduced via chimaeras into the mouse germ line. Here I review the study of the Rb-1 and p53 oncosuppressor genes using this approach.
The first evidence for an involvement of the E7 protein in malignant transformation came from studies on HPV-infected cells. In several cervical carcinoma-derived cell lines, such as SiHa, CaSki and HeLa, the HPV DNA is integrated in the cellular genome as single or multiple copies. This viral DNA integration results in the disruption of several viral genes with consistent preservation of only the early E6 and E7 genes (for more detail see chapter 2). The involvement of the E7 protein in immortalization and transformation of the host cell was confirmed by a number of in vitro assays and by transgenic mouse models. Biochemical studies have provided further evidence that the E7 protein is directly involved in the induction of immortalization and malignant transformation of the host cells. E7 proteins from the “high risk” HPV types have the ability to interact with and alter the function of a number of cellular proteins which play a key role in controlling the proliferative program of the cell.
HPV DNA is found integrated into the host genome in a high percentage of cervical carcinomas. While integration appears to be random with respect to the host genome, there appears to be some specificity with respect to the viral genome in that integration frequently occurs within the El or E2 ORFs (see also chapter 2). As a consequence of this integration the only viral genes that are regularly expressed in cervical cancers are E6 and E7. This observation implies that the E6 and the E7 ORF encode for the major viral oncoproteins. This hypothesis has been corroborated by several lines of evidence. Most importantly, both E6 and E7 have oncogenic properties in various in vitro cell culture systems as well as in transgenic animal models, and inhibition of E6/E7 expression results in growth arrest and reversion of the malignant phenotype of cell lines derived form cervical cancers. To understand why expression of these viral proteins can contribute to, or can induce, immortalization/transformation of infected cells, it is important to identify and characterize both the properties of E6 and E7 involved in immortalization/transformation and the properties that are necessary for the viral life cycle. However, due to the lack of an appropriate cell culture system for HPV propagation, studies so far have mostly been limited to the characterization of the oncogenic properties of E6 and E7.
The papillomaviruses (PVs) are a large family of DNA viruses indigenous to many, if not all, vertebrate species. The unique feature of these viruses is that they all induce primarily benign proliferations of epithelial cells in their natural hosts. Individual members of this family show a high degree of both species specificity and tissue specificity. Squamous or mucosal epithelial cells are the targets of infection. The life cycle of the virus is intimately coupled to the differentiation program of infected tissue. Conversely, expression of the early viral genes interferes with the normal pattern of keratin expression, cell cycle regulation, and terminal differentiation, which ultimately gives rise to aberrant cells. With some viral types, however, these benign proliferations progress into aggressive malignancies.
Cancer cells often contain mutations that lead to the loss of retinoblastoma tumor suppressor (Rb) function and the activation of E2F-dependent transcription. As a result, proliferation is deregulated, and sensitivity to apoptotic stimuli is increased. In cell culture studies, the transcription factor E2F1 has been shown to be equally adept at inducing proliferation and apoptosis. Several groups using mouse models have been examining how these E2F1-regulated processes impact the development of cancer. The conclusion from these studies is that E2F1 can function as both oncogene and tumor suppressor gene and that both positive and negative effects on tumorigenesis can be observed whether E2F1 is absent or overexpressed. These findings are discussed in the context of a model in which pathways controlling cell-cycle progression and apoptosis are intimately linked. Mol. Carcinog. 27:151-157, 2000. (C) 2000 Wiley-Liss, Inc.
Purpose: The chicken embryo lens is a classical model system for developmental and cell biology studies. To understand the molecular mechanisms that underlie the morphological changes that occur during lens development, it is important to develop an effective gene transfer method that permits the analysis of gene functions in vivo. In ovo electroporation has been successfully used for introducing DNA into neural and mesenchymal tissues of chicken embryos. In this study, we explored the possibility of using this technique to manipulate gene expression in lens epithelial and fiber cells, as well as in other cells of the chicken eye. Methods: Two DNA constructs were used in this study. pCAX contains a chicken beta-actin promoter fused to the CMV IE enhancer to drive enhanced green fluorescent protein (EGFP) expression. pMES-cNf2 uses the same chimeric promoter to drive the expression of the chicken neurofibromatosis 2 (cNf2) and EGFP proteins in the same cell. Plasmid DNA was injected into the lumen of the lens vesicle in chicken embryos at stage 15. For corneal epithelial and retinal cell electroporation, DNA was placed near the surface ectoderm in the eye region or injected into the vitreous cavity, respectively. Electroporation was performed with one electrode above the eye and the other underneath the head of the embryo. Chicken embryos were harvested at different time points for EGFP expression analysis by immunohistochemistry. 5-bromo-2'-deoxyuridine (BrdU) incorporation assays were used to evaluate the effects of cNf2 on lens epithelial cell proliferation. Results: A strong EGFP signal can be detected in lens cells 4 h after electroporation. The transfected cells maintain high levels of EGFP expression for at least 5 days. Overexpressing cNf2 in lens epithelial cells significantly inhibits cell proliferation. Ectopic expression of EGFP in corneal epithelial and retinal cells was also achieved by in ovo electroporation. Conclusions: We have demonstrated that exogenous DNA can be effectively introduced into lens, corneal and retinal cells in the living embryo by in ovo electroporation. In comparison to viral infection and transgenic mouse approaches, in ovo electroporation offers an easier and quicker way to manipulate gene expression during embryonic development. This technique will be a useful tool for exploring the molecular mechanisms of lens and eye development.
The human papillomavirus type 16 (HPV-16) genome is commonly present in human cervical carcinoma, in which a subset of the viral genes, E6 and E7, are expressed. The HPV-16 E6 and E7 gene products can associate with and inactivate the tumor suppressor proteins p53 and Rb (the retinoblastoma susceptibility gene product), and in tissue culture cells, these viral genes display oncogenic properties. These findings have led to the hypothesis that E6 and E7 contribute to cervical carcinogenesis. This hypothesis has recently been tested by using transgenic mice as an animal model. HPV-16 E6 and E7 together were found to induce cancers in multiple tissues in which they were expressed, including squamous cell carcinoma, the cancer type most commonly associated with HPV-16 in the human cervix, We have extended these studies to investigate the in vivo activities of HPV-16 E7 when expressed in squamous epithelia of transgenic mice. Grossly, E7 transgenic mice had multiple phenotypes, including wrinkled skin that was apparent prior to the appearance of hair on neonates, thickened ears, and loss of hair in adults. In lines of mice expressing higher levels of E7, we observed stunted growth and mortality at an early age, potentially caused by an incapacity to feed. Histological analysis demonstrated that E7 causes epidermal hyperplasia in multiple transgenic lineages with high penetrance. This epithelial hyperplasia was characterized by an expansion of the proliferating compartment and an expansion of the keratin 10-positive layer of cells and was associated with hyperkeratosis. Hyperplasia was found at multiple sites in the animals in addition to the skin, including the mouth palate, esophagus, forestomach, and exocervix, In multiple transgenic lineages, adult animals developed skin tumors late in life with low penetrance. These tumors arose from the squamous epithelia and from sebaceous glands and were characterized histologically to be highly differentiated, locally invasive, and aggressive in their growth properties. On the basis of these phenotypes, we conclude that HPV-16 E7 can alter epithelial cell growth parameters sufficiently to potentiate tumorigenesis in mice.
Various experiments have demonstrated a role for the E2F transcription factor in the regulation of cell growth during the G(0)/G(1)/S phase transition. Indeed, overexpression of the E2F1 product, a component of the cellular E2F activity, induces DNA synthesis in quiescent fibroblasts. To provide an approach to a more detailed biochemical analysis of these events, we have made use of a recombinant adenovirus containing the E2F1 cDNA in order to efficiently express the E2F1 product in an entire population of cells. We demonstrate an induction of DNA synthesis when quiescent cells are infected with the E2F1 recombinant virus. However, we also find that the induction does not lead to a complete replication of the cellular genome, as revealed by flow cytometry. The incomplete nature of cellular DNA replication is due, at least in part, to the fact that E2F1 overexpression leads to massive cell death that is characteristic of apoptosis. This E2F1-mediated induction of apoptosis is largely dependent on endogenous wild-type p53 activity and can be subverted by introducing mutant forms of p53 into these cells or by overexpressing E2F1 in fibroblasts derived from p53-null mouse embryos. We conclude that E2F1 can induce events leading to S phase but that the process is not normal and appears to result from the activation of a cell death pathway.
This chapter describes identification, function, and structure of retinoblastoma gene (RB1). RB1 and RB2 are members of the retinoblastoma gene family. RBI has regions of homology with p107 and the transcription factor TFIIB. pRb functions as a signal transducer, connecting the cell cycle clock with transcriptional control mechanisms mediating progression through the first two-thirds of G1 phase. pRb binds to double-stranded DNA in a nonsequence-specific manner. The broad transcriptional effects of pRb are mediated by its inhibition of factors such as E2F that are required for the expression of genes involved in DNA replication and by repressing transcriptional activation by RNA polymerases I and III. pRb contains at least 12 distinct serine or threonine phosphorylation sites and phosphorylation of pRb appears to be essential for progression through the cell cycle, inhibition of pRb phosphorylation causing cell cycle arrest. It is found that major deletions in the gene occur in 15–40% of retinoblastomas, point mutations being scattered throughout the gene with exons 3, 8, 18, and 19 preferentially altered.
The orderly progression through the cell cycle is mediated by the sequential activation of several cyclin/cyclin-dependent kinase (cdk) complexes. These kinases phosphorylate a number of cellular substrates, among which are the product of the retinoblastoma gene, pRB, and the pRB-related proteins p107 and p130. Phosphorylation of these proteins in late G1 causes their release from the heterodimeric transcription factors E2F/DP. This results in the transcriptional activation of E2F-responsive genes which encode proteins that either directly control cell-cycle progression or function in metabolic processes linked to the cell cycle. Thus E2F/DP complexes are key signal transducers connecting the cell cycle machinery with the transcriptional control of sets of genes that mediate the passage through the G1 and S phases of the cell cycle. This critical regulatory pathway, which gates cell cycle progression, is often disrupted during the pathogenesis of many mammalian tumors. It also plays a pivotal role in animal development and in promoting cellular differentiation. We review the current state of knowledge regarding this pRB/E2F pathway. (Figure 1.1)
Adenoviruses and other small DNA tumor viruses, e.g., SV40 virus and papillomaviruses, have evolved efficient strategies for replicating their genomes on entry into their host cells. Because these viruses have limited coding capacities, they cannot produce all of the protein products required to synthesize their new double-stranded DNA genomes and, therefore, must utilize many components of the host-cell machinery. In naturally occurring infections, adenovirus frequently enters noncycling, differentiated epithelial cells. This presents adenovirus with the problem of how to stimulate these quiescent cells to an active growth state in order to create a cellular environment in which viral propagation can take place. The small DNA viruses have devised strategies in which certain of their early viral proteins, i.e., adenovirus E1A and E1B, SV40 T antigen, and papilloma E6 and E7, physically target specific cellular proteins whose function it is to regulate cellular growth.
One of the major challenges facing teratologists is the elucidation of mechanisms by which birth defects occur. This requires an understanding of the primary targets of physiological and environmental agents that affect the health and well-being of the conceptus, the cellular events of pathogenesis, and the genes which place the embryo at risk. Cell death is gene-directed event in the developing embryo. Its’ broad phylogenetic representation is consistent with a selective advantage in development for the involution of vestigial structures which have importance in phylogenetic but not ontogenetic development, reduction of superfluous cells, disposal of cells that have already completed their functions, elimination of cells which differentiate inappropriately, and suppression of potentially harmful or abnormal cells (Saunders 1966; Ellis et al 1991).
The high risk genital human papillomaviruses are the clearest examples of human tumour viruses, having a role in the development of virtually all cervical cancers. These viruses encode several proteins with oncogenic activity, of which E6 and E7 most clearly participate in the appearance of premalignant lesions. Both E6 and E7 function by perturbing the normal activity of cell proteins essential for regulated cell growth, and best understood are the interactions of E6 with the TP53 protein and E7 with the RB protein family. TP53 and RB are important tumour suppressor proteins, and loss of their function is a common theme in most human cancers, regardless of origin. The ability of E6 and E7 to prevent normal TP53 and RB function is therefore likely to be central in the oncogenic activity of the HPVs. However, both E6 and E7 appear to have additional functions that could contribute to malignant development. Identification of virally encoded proteins that appear to have a role in both the development and the maintenance of the transformed phenotype presents some exciting possibilities for the treatment of HPV associated cancers.
Background: Oxidative damage is a major cause of age-related cataracts, and the ubiquitin-proteasome system is involved in lens differentiation and development. Ubiquitin carboxy-terminal hydrolase L1 (UCHL1), one of key enzymes of ubiquitin-proteasome system, was discovered to participate in the age related diseases and oxidative stress damage. Objective: This study was to investigate the effects of UCHL1 on the formation and development of age-related cataract. Methods: Lens capsule were collected from 24 patients with age-related cataract (including 12 cases of cortical cataract and 12 cases of nuclear cataract) during the surgery. Five normal lens capsule membranes were obtained from eye bank of Tongji University. Human lens epithelial cells (LECs) line (SRA01/04) was also collected in this study. Expression of UCHL1 in the lens epithelial layer of different samples was assayed using immunofluorescence technology. UCHL1 eukaryotic expressing vector was constructed and transfected into cultured SRA01/04 by liposome, and green fluorescent protein (GFP) eukaryotic expressing vector was transfected at the same method as the control group. UCHL1 over-expressing cells were then exposed to different concentrations (0.2, 0.3, 0.4 and 0.5 mol/L) of tert-butyl hydroperoxide (TBHP) for 24 hours and subsequently monitored for cell viability evaluation by MTT assay. Results: Immunofluorescence showed that UCHL1 was expressed in human lens epithelial layer, but significantly different expressing levels were seen among normal lens capsular membrane, cortical cataract and nuclear cataract (F=13.411, P=0.000), and UCHL1 expressing levels were lower in cortical cataract and nuclear cataract than the normal lens (P=0.000, P=0.000). No significant difference was found in UCHL1 expressing level between cortical cataract and nuclear cataract (P=0.164). Western blot analysis verified that UCHL1 exhibited a stranger expression in the UCHL1 transfected group compared with the GFP transfected group, illuminating a successful transfection of UCHL1 in SRA01/04 cells. MMT assay revealed that the A 570/630 value in UCHL1 transfected cells was significantly elevated in comparison with GFP transfected cells following the treatment of 0.3 mol/L TBHP. Conclusions: UCHL1 has an antioxidative ability, and it might plays an important role in the progress of age-related cataract.
During the terminal differentiation of lens fiber cells, nuclear DNA is known to accumulate free 3'-OH ends and is progressively lost from the nucleus. Toward the end of this process, nuclei undergo pycnosis and disappear. The size of the DNA in the epithelia and in early and late stages of fiber cell development was examined by electrophoresis on nondenaturing agarose/polyacrylamide gels. Low molecular weight DNA of discrete sizes appears only at the final stages of nuclear degeneration in central fiber cells and persists after the disappearance of the nuclei. These low molecular weight DNA fragments appear as multiples of a monomeric unit and are similar to the fragments produced by the digestion of epithelial cell nuclei by micrococcal nuclease. The data indicate that in lens fiber nuclei the double-strand breaks in vivo affect the chromatin during nuclear degeneration, and the data suggest that the DNA of these cells is organized into chromatin composed of discrete subunits.
Binding of the retinoblastoma gene product (pRB) by viral oncoproteins, including the E7 of human papillomavirus type 16 (HPV 16), is thought to be important in transformation of cells. One of the steps in transformation is the immortalization process. Here we show that mutations in E7 within the full-length genome which inhibit binding of pRB do not abrogate the ability of the HPV 16 DNA to immortalize primary human epithelial (keratinocyte) cells. A mutation in one of the cysteines of a Cys-X-X-Cys motif which is contained in the carboxy half of the E7 and is part of a zinc finger arrangement completely eliminates the ability of HPV 16 DNA to immortalize cells. The results indicate the importance of E7 in the immortalization of primary keratinocytes but suggest that the binding of pRB is not essential.
The E7 gene of human papillomavirus type 16 encodes a multifunctional nuclear phosphoprotein that is functionally and structurally similar to the adenovirus (Ad) E1A proteins and the T antigens of other papovaviruses. E7 can cooperate with an activated ras oncogene to transform primary rodent cells, trans activate the Ad E2 promoter, and abrogate transforming growth factor beta-mediated repression of c-myc. Recent studies suggest that these functions may in part be a consequence of the ability of E7 to associate with the product of the retinoblastoma tumor suppressor gene (pRB). In this study, a series of site-specific mutations of the human papillomavirus type 16 E7 gene product were constructed and assessed for their effects on intracellular protein stability, ras cooperativity, transcriptional trans activation, pRB association, and phosphorylation. The results of these studies indicate that the transforming and trans-activating domains extensively overlap within a region of the protein analogous to conserved region 2 of Ad E1A, suggesting that pRB binding is necessary for both activities. Deletion of sequences in conserved region 1 abrogates cellular transformation but has only a marginal effect on trans activation. These data suggest that E7 trans activation and cellular transformation are interrelated but separable functions.
The adenovirus E1A gene product, the simian virus 40 large tumor antigen, and the human papillomavirus E7 protein share a short amino acid sequence that constitutes a domain required for the transforming activity of these proteins. These sequences are also required for these proteins to bind to the retinoblastoma gene product (pRb). Recent experiments have shown that E1A can dissociate complexes containing the transcription factor E2F bound to pRb, dependent on this conserved sequence element. We now show that the E7 protein and the simian virus 40 large tumor antigen can dissociate the E2F-pRb complex, dependent on this conserved sequence element. We also find that the E2F-pRb complex is absent in various human cervical carcinoma cell lines that either express the E7 protein or harbor an RB1 mutation, suggesting that the loss of the E2F-pRb interaction may be an important aspect in human cervical carcinogenesis. We suggest that the ability of E1A, the simian virus 40 large tumor antigen, and E7 to dissociate the E2F-pRb complex may be a common activity of these viral proteins that has evolved to stimulate quiescent cells into a proliferating state so that viral replication can proceed efficiently. In circumstances in which a lytic infection does not proceed, the consequence of this action may be to initiate the oncogenic process in a manner analogous to the mutation of the RB1 gene.
Studies of adenovirus E1A oncoprotein mutants suggest that the association of E1A with the retinoblastoma protein (pRB) is necessary for E1A-mediated transformation. Mutational analysis of E1A indicates that two regions of pRB are required for E1A to form stable complexes with the retinoblastoma protein. In addition to pRB binding, these regions are necessary for E1A association with several other cellular proteins, including p130, p107, cyclin A, and p33cdk2. Here we show that short synthetic peptides containing the pRB-binding sequences of E1A are sufficient for interaction with p107, cyclin A, and p130. The E7 protein of human papillomavirus type 16 contains an element that binds to pRB and appears to be functionally homologous to the E1A sequences. Peptides containing this region of the E7 protein were able to interact with p107, cyclin A, and p130 in addition to pRB. These findings suggest that the common mechanism of transformation used by these viral oncogenes involves their association with a set of polypeptides.
Human retinoblastomas can occur both as hereditary and as sporadic cases. Knudson's proposal that they result from two mutational events, of which one is present in the germ line in hereditary cases, has been confirmed by more recent molecular analysis, which has shown both events to involve loss or mutational inactivation of the same gene, RB-1 (ref. 2). RB-1 heterozygosity also predisposes to osteosarcoma, and RB-1 allele losses are seen in sporadic lung, breast, prostate and bladder carcinomas. RB-1 is expressed in most, if not all, tissues and codes for a nuclear phosphoprotein which becomes hypophosphorylated in the G0 growth arrest state and in the G1 phase of the cell cycle. To gain a further insight into the role of RB-1 we and other groups have generated mice carrying an inactivated allele of the homologous gene, Rb-1 (ref. 10), by gene targeting. We report here that young heterozygous mice do not appear abnormal and do not develop retinoblastoma at a detectable frequency. However, homozygous mutant embryos fail to reach term and show a number of abnormalities in neural and haematopoietic development. Broadly similar results are reported by the other groups.
The observed interaction between p53 and the oncoproteins encoded by several DNA tumor viruses suggests that these viruses mediate their transforming activities at least in part by altering the normal growth regulatory function of p53. In this study we examined the effect of viral oncoprotein expression on the transcriptional transactivation function of wild-type p53 in human cells. Plasmids expressing human p53 were cotransfected with either SV40 large T-antigen or human papillomavirus (HPV) type 16 E6 expression plasmids and assayed for transactivation function using a reporter gene driven by a p53-responsive promoter containing multiple copies of the consensus p53 DNA binding motif, TGCCT. Both large T-antigen and E6 were able to inhibit transactivation by wild-type p53. Furthermore, SV40 T-antigen mutants that are defective for p53 binding were not able to inhibit transactivation and HPV E6 proteins that were either mutant or derived from non-oncogenic HPV types and unable to bind p53, had no effect on p53 transactivation. These results demonstrate the physiological relevance of the interaction of SV40 T-antigen and HPV E6 oncoproteins with p53 in vivo and suggest that the transforming functions of these viral oncoproteins may be linked to their ability to inhibit p53-mediated transcriptional activation.
Mutations in the p53 tumour-suppressor gene are the most frequently observed genetic lesions in human cancers. To investigate the role of the p53 gene in mammalian development and tumorigenesis, a null mutation was introduced into the gene by homologous recombination in murine embryonic stem cells. Mice homozygous for the null allele appear normal but are prone to the spontaneous development of a variety of neoplasms by 6 months of age. These observations indicate that a normal p53 gene is dispensable for embryonic development, that its absence predisposes the animal to neoplastic disease, and that an oncogenic mutant form of p53 is not obligatory for the genesis of many types of tumours.
The wild-type E6 and E7 genes of human papillomavirus type 16 (HPV16) can cooperate to immortalize normal human keratinocytes in culture. The E6 open reading frame of HPV16 and other HPV types highly associated with cervical cancer has the potential of encoding both full-length E6 and two truncated E6* proteins, the latter being generated via splicing within the E6 open reading frame portion of the E6-E7 polycistronic transcript. Those types, such as HPV6, that are infrequently associated with cervical carcinoma lack the splice site and encode only a full-length E6. We have now found that, in addition to cooperating with E7 to immortalize keratinocytes, HPV16 E6 can induce anchorage-independent growth in NIH 3T3 cells and trans-activate the adenovirus E2 promoter. HPV6 E6 was also able to trans-activate the adenovirus E2 promoter, although it was inactive in both cell transformation assays. An HPV16 splice site mutant which expressed only the full-length HPV16 E6 was active in all three assays, indicating that the E6* proteins are not required for these activities. The plasmid which encodes the E6* proteins was inactive and did not potentiate the activity of the HPV16 splice site mutant. The mutation that prevented splicing in E6-E7 mRNA severely reduced the level of E7 protein and increased E6 protein. Taken together, the results suggest that the primary function of the splice within E6 is to facilitate the translation of E7 and reduce translation of full-length E6, rather than to generate biologically active E6* proteins.
The RB gene product is a nuclear phosphoprotein that undergoes cell cycle-dependent changes in its phosphorylation status. To test whether RB regulates cell cycle progression, purified RB proteins, either full-length or a truncated form containing the T antigen-binding region, were injected into cells. Injection of either protein early in G1 inhibits progression into S phase. Co-injection of anti-RB antibodies antagonizes this effect. Injection of RB into cells arrested at G1/S or late in G1 has no effect on BrdU incorporation, suggesting that RB does not inhibit DNA synthesis in S phase. These results indicate that RB regulates cell proliferation by restricting cell cycle progression at a specific point in G1 and establish a biological assay for RB activity. Neither co-injection of RB with a T antigen peptide nor injection into cells expressing T antigen prevents cells from progressing into S phase, which supports the hypothesis that T antigen binding has functional consequences for RB.
The p53 gene is a frequent target of mutation in a wide variety of human cancers. Previously, it was reported that conditional expression of wild-type p53 protein in a cell line (GM47.23) derived from a human glioblastoma multiform tumor had a negative effect on cell proliferation. We have now investigated the effect that induction of wild-type p53 protein in this cell line has on the expression of the proliferating-cell nuclear antigen gene. The proliferating-cell nuclear antigen gene encodes a nuclear protein that is an auxiliary factor of DNA polymerase delta and part of the DNA replication machinery of the cell. We show that inhibition of cell cycle progression into S-phase after induction of wild-type p53 protein is accompanied by selective down-regulation of proliferating-cell nuclear antigen mRNA and protein expression.
By in situ hybridisation we have examined the expression of p53 during mouse embryogenesis from day 8.5 to day 18.5 post coitum (p.c.). High levels of p53 mRNA were detected in all cells of the day 8.5 p.c. and 10.5 p.c. mouse embryo. However, at later stages of development, expression became more pronounced during differentiation of specific tissues e.g. of the brain, liver, lung, thymus, intestine, salivary gland and kidney. In cells undergoing terminal differentiation, the level of p53 mRNA declined strongly. In the brain, hybridisation signals were also observed in postmitotic but not yet terminally differentiated cells. Therefore, gene expression of p53 does not appear to be linked with cellular proliferation in this organ. A proposed role for p53 in cellular differentiation is discussed.
Although it is generally believed that the product of the retinoblastoma susceptibility gene (RB1) is an important regulator of cell proliferation, the biochemical mechanism for its action is unclear. We now show that the RB protein is found in a complex with the E2F transcription factor and that only the under phosphorylated form of RB is in the E2F complex. Moreover, the adenovirus E1A protein can dissociate the E2F-RB complex, dependent on E1A sequence also critical for E1A to bind to RB. These sequences are also critical for E1A to immortalize primary cell cultures and to transform in conjunction with other oncogenes. Taken together, these results suggest that the interaction of RB with E2F is an important event in the control of cellular proliferation and that the dissociation of the complex is part of the mechanism by which E1A inactivates RB function.
The E6-E7 region of human papillomavirus types 16 and 18 is selectively retained and expressed in cervical carcinoma cells. In cultured human keratinocytes, expression of the E6 and E7 open reading frames of human papillomavirus type 18, under the control of its homologous promoter, resulted in high-frequency immortalization. Furthermore, by using a system that allows for stratification of keratinocytes in vitro (raft system), we observed that the morphological differentiation of these E6-E7 immortalized cells was altered such that parabasal cells extended throughout most of the epithelium, with abnormal nuclei present in the upper regions. Examination of E6-E7-expressing cell lines in the raft system at a later passage revealed that complete loss of morphological differentiation had occurred. E7 alone was a much less effective immortalizing agent than E6 and E7 together and acted only minimally to alter morphological differentiation in vitro. No such activities were found for E6 alone. High-frequency transformation of human epithelial cells thus appears to require expression of both E6 and E7 gene products.
Stable protein complexes between the large T antigens of mouse, monkey, baboon, or human polyomaviruses and the retinoblastoma protein were detected by an in vitro coimmunoprecipitation assay. All of the large T antigens tested were able to bind to both human and mouse retinoblastoma polypeptides, showing that these interactions have been conserved during evolution.
The association of certain human papillomavirus (HPV) types with the majority of human cervical carcinomas suggests a role for the virus in the development of this type of cancer. In this paper, we have examined the transforming properties of several HPV types where the early region genes of the virus are under the control of a strong heterologous promoter and show that major differences exist between the HPV types in their ability to transform primary rat kidney epithelial cells in conjunction with an activated ras oncogene. Those HPV types most commonly found in carcinomas--types 16, 18, 31 and 33--are capable of co-operating with ras to transform primary cells, but those types most commonly found in benign lesions--types 6 and 11--are not. We further demonstrate that the E7 gene of HPV16 by itself is sufficient to co-operate with activated ras to produce transformed cells which are tumorigenic in immunocompetent animals.
The early human papillomavirus type 16 genes that directly participate in the in vitro transformation of primary human keratinocytes have been defined. In the context of the full viral genome, mutations in either the E6 or E7 open reading frame completely abrogated transformation of these cells. Mutations in the E1, E2, and E2-E4 open reading frames, on the other hand, had no effect. Thus, both the full-length E6 and E7 genes were required for the induction of keratinocyte immortalization and resistance to terminal differentiation. The E6 and E7 genes expressed together from the human beta-actin promoter were sufficient for this transformation; mutation of either gene in the context of this recombinant plasmid eliminated the ability to induce stable differentiation-resistant transformants.
The proliferating cell nuclear antigen (PCNA), also known as cyclin and DNA polymerase delta auxiliary factor, is present in reduced amounts in nongrowing cells and is synthesized at a greater rate in the S phase of growing cells. The recently discovered involvement of PCNA in DNA replication suggested that this pattern of expression functions to regulate DNA synthesis. We have investigated this possibility further by examining the synthesis, stability, and accumulation of PCNA in HeLa cells fractionated by centrifugal elutriation into nearly synchronous populations of cells at various positions in the cell cycle. In these fractionated cells we found that there is an increase in the rate of PCNA synthesis with a peak in early S phase of the cell cycle, but the magnitude of the increase is only 2-3-fold. This change reflects similar changes in the amount of PCNA mRNA. The fluctuating synthesis of PCNA maintains this protein at a roughly constant proportion of the total cell protein, although the amount doubles/cell in the cell cycle. Consistent with this observation, the stability of PCNA does not differ significantly from that of total cellular protein in synchronized HeLa cells. We also observed that a maximum of one-third of the total PCNA is tightly associated with the nucleus, presumably in replication complexes, at the peak of S phase. We conclude that the cyclic synthesis of PCNA in cycling HeLa cells maintains PCNA in excess of the amount involved directly in DNA replication and the amount of the protein neither fluctuates significantly with the cell cycle nor is limiting for DNA synthesis.
We have isolated a cDNA clone of the murine homologue of the human retinoblastoma (Rb) susceptibility gene. DNA sequence analysis reveals a high degree of conservation with the human Rb sequence, both in the coding and in the noncoding regions. The predicted amino acid sequence of the mouse Rb protein shows 91% identity to that of the human protein. Both proteins were found to contain a peptide sequence reminiscent of a leucine-repeat motif ("leucine-zipper") that is also found in the myc, fos, and jun oncogenes. Synthetic peptide antiserum directed against a portion of the mouse Rb protein detects three proteins of 104-110 kDa in cells that were transiently transfected with a mouse Rb gene expression construct. In the mouse embryo the expression of Rb mRNA was ubiquitous, with maximal expression being observed around 13 days of gestation. In the embryo, the highest level of expression was observed in liver and brain. In contrast, the Rb gene was found to be expressed at a very low level in adult mouse liver with high levels being found in lung, thymus, and spleen. A shorter Rb transcript was detected in mouse testes.
The close association of human papillomavirus type 16 DNA with a majority of cervical carcinomas implies some role for the virus in this type of cancer. To define the transforming properties of HPV‐16 DNA in vitro we have now performed transfection experiments on baby rat kidney cells using HPV‐16 DNA in conjunction with an activated ras gene. We have demonstrated that a 6.6‐kb DNA fragment, containing the early genes of HPV‐16 under the control of Moloney murine leukaemia virus long terminal repeats (MoMuLV‐LTRs), cooperates with EJ‐ras in transforming these cells. Both DNAs are required and neither alone is effective. The cooperating activity appears to reside in a protein or proteins derived from the E6/E7 region of the HPV‐16 genome.
The E7 proteins encoded by the human papillomaviruses (HPVs) associated with anogenital lesions share significant amino acid sequence homology. The E7 proteins of these different HPVs were assessed for their ability to form complexes with the retinoblastoma tumor suppressor gene product (p105-RB). Similar to the E7 protein of HPV-16, the E7 proteins of HPV-18, HBV-6b and HPV-11 were found to associate with p105-RB in vitro. The E7 proteins of HPV types associated with a high risk of malignant progression (HPV-16 and HPV-18) formed complexes with p105-RB with equal affinities. The E7 proteins encoded by HPV types 6b and 11, which are associated with clinical lesions with a lower risk for progression, bound to p105-RB with lower affinities. The E7 protein of the bovine papillomavirus type 1 (BPV-1), which does not share structural similarity in the amino terminal region with the HPV E7 proteins, was unable to form a detectable complex with p105-RB. The amino acid sequences of the HPV-16 E7 protein involved in complex formation with p105-RB in vitro have been mapped. Only a portion of the sequences that are conserved between the HPV E7 proteins and AdE1A were necessary for association with p105-RB. Furthermore, the HPV-16 E7-p105-RB complex was detected in an HPV-16-transformed human keratinocyte cell line.
In near-physiological concentrations, glucocorticoid hormones cause the death of several types of normal and neoplastic lymphoid cell, but the mechanisms involved are unknown. One of the earliest structural changes in the dying cell is widespread chromatin condensation, of the type characteristic of apoptosis, the mode of death frequently observed where cell deletion seems to be 'programmed'. It is shown here that this morphological change is closely associated with excision of nucleosome chains from nuclear chromatin, apparently through activation of an intracellular, but non-lysosomal, endonuclease.
The E7 gene of human papillomavirus type 16 encodes a multifunctional nuclear phosphoprotein that is functionally and structurally similar to the adenovirus (Ad) E1A proteins and the T antigens of other papovaviruses. E7 can cooperate with an activated ras oncogene to transform primary rodent cells, trans activate the Ad E2 promoter, and abrogate transforming growth factor beta-mediated repression of c-myc. Recent studies suggest that these functions may in part be a consequence of the ability of E7 to associate with the product of the retinoblastoma tumor suppressor gene (pRB). In this study, a series of site-specific mutations of the human papillomavirus type 16 E7 gene product were constructed and assessed for their effects on intracellular protein stability, ras cooperativity, transcriptional trans activation, pRB association, and phosphorylation. The results of these studies indicate that the transforming and trans-activating domains extensively overlap within a region of the protein analogous to conserved region 2 of Ad E1A, suggesting that pRB binding is necessary for both activities. Deletion of sequences in conserved region 1 abrogates cellular transformation but has only a marginal effect on trans activation. These data suggest that E7 trans activation and cellular transformation are interrelated but separable functions.
A preparative method for isolating high-molecular-weight DNA from animal cells is described. This method is based on the use of proteinase K, a powerful proteolytic enzyme with a broad action spectrum, which is very active in the presence of sodium dodecylsulfate and ethylene-diamine tetraacetate. The DNA preparation is free of RNA, protein and degrading enzymes. The number-average molecular weight of the native DNA is 190 × 106, whereas it is 90 × 106 for single-stranded DNA, indicating that the DNA molecules do not contain single-stranded nicks. The native DNA molecules range in molecular weight from 40 × 106 to more than 500 × 106.
THE early region of the small DNA tumour virus, simian virus 40 (SV40),
is known to code for at least two polypeptides, the t and T antigens
(`small t' and `large T'). Both these polypeptides are expressed in
cells transformed by the virus1-4, and the T antigen has been
shown to be essential for both the initiation and maintenance of the
transformed state5-9. We therefore need to know how this T
protein interacts with components of the host cell in order to
understand the mechanism of SV40-induced transformation. We report here
that the T antigen in a line of SV40-transformed mouse cells forms an
oligomeric complex with a specific cell coded protein.
SV40 infection or transformation of murine cells stimulated the production of a 54K dalton protein that was specifically immunoprecipitated, along with SV40 large T and small t antigens, with sera from mice or hamsters bearing SV40-induced tumors. The same SV40 anti-T sera immunoprecipitated a 54K dalton protein from two different, uninfected murine embryonal carcinoma cell lines. These 54K proteins from SV40-transformed mouse cells and the uninfected embryonal carcinomas cells had identical partial peptide maps which were completely different from the partial peptide map of SV40 large T antigen. An Ad2+ND4-transformed hamster cell line also expressed a 54K protein that was specifically immunoprecipitated by SV40 T sera. The partial peptide maps of the mouse and hamster 54K protein were different, showing the host cell species specificity of these proteins. The 54K hamster protein was also unrelated to the Ad2+ND4 SV40 T antigen. Analogous proteins immunoprecipitated by SV40 T sera, ranging in molecular weight from 44K to 60K, were detected in human and monkey SV40-infected or -transformed cells. A wide variety of sera from hamsters and mice bearing SV40-induced tumors immunoprecipitated the 54K protein of SV40-transformed cells and murine embryonal carcinoma cells. Antibody produced by somatic cell hybrids between a B cell and a myeloma cell (hybridoma) against SV40 large T antigen also immunoprecipitated the 54K protein in virus-infected and -transformed cells, but did not do so in the embryonal carcinoma cell lines. We conclude that SV40 infection or transformation of mouse cells stimulates the synthesis or enhances the stability of a 54K protein. This protein appears to be associated with SV40 T antigen in SV40-infected and -transformed cells, and is co-immunoprecipitated by hybridomas sera to SV40 large T antigen. The 54K protein either shares antigenic determinants with SV40 T antigen or is itself immunogenic when in association with SV40 large T antigen. The protein varies with host cell species, and analogous proteins were observed in hamster, monkey and human cells. The role of this protein in transformation is unclear at present.
A quantitative analysis of cell division and cell elongation was carried out during lens morphogenesis in the rat. At 13 days of development elongating cells in the posterior part of the lens vesicle (presumptive fibre cells) have a lower mitotic activity than cells in the anterior vesicle. By 14 days these elongating cells do not divide. Thus at 14 days of development the lens can be separated into two compartments; a proliferation compartment in the anterior lens and an elongation compartment in the posterior lens.
The three main groups of lens-specific proteins, α-,β- and γ-crystallins, were localized by immunofiuorescence. Alpha-crystallin is the first crystallin to be detected and is localized in some lens pit cells at 12 days of development. By 14 days all lens cells contain α-crystallin. Beta- and β-crystallins are detected later at 12½ days and are localized in some cells situated primarily in the posterior part of the lens vesicle. At later stages of development these crystallins are restricted to cells of the elongation compartment, i.e. presumptive fibre and fibre cells. Possible mechanisms that govern the temporal and spatial distribution of crystallins are discussed.
Human papillomavirus type 16 (HPV16) E6 and E7 are selectively retained and expressed in HPV16-associated human genital tumors. E6 is active in several cell culture assays, including transformation of NIH 3T3 cells, trans activation of the adenovirus E2 promoter, and cooperation with E7 to immortalize normal human keratinocytes. Biochemically, the HPV16 E6 protein has been shown to bind to tumor suppressor protein p53 in vitro and induce its degradation in a rabbit reticulocyte lysate. To examine the relationship between the various biological activities of E6 and inactivation of p53, we tested the abilities of dominant negative mutants of p53 to substitute functionally for E6 in the three cell culture assays. While wild-type p53 inhibited keratinocyte proliferation, both mouse and human mutant p53s, in conjunction with E7, increased proliferation of the keratinocytes, resulting in generation of immortalized lines. However, in contrast to E6, mutant p53 was unable to induce transformation or trans activate the adenovirus E2 promoter in NIH 3T3 cells. These results suggest that inactivation of wild-type p53 is necessary for HPV-induced immortalization of human keratinocytes and that different or additional activities are required for E6-dependent transformation and trans activation of NIH 3T3 cells.
Wild-type p53 has recently been shown to repress transcription from several cellular and viral promoters. Since p53 mutations are the most frequently reported genetic defects in human cancers, it becomes important to study the effects of mutations of p53 on promoter functions. We, therefore, have studied the effects of wild-type and mutant human p53 on the human proliferating-cell nuclear antigen (PCNA) promoter and on several viral promoters, including the herpes simplex virus type 1 UL9 promoter, the human cytomegalovirus major immediate-early promoter-enhancer, and the long terminal repeat promoters of Rous sarcoma virus and human T-cell lymphotropic virus type I. HeLa cells were cotransfected with a wild-type or mutant p53 expression vector and a plasmid containing a chloramphenicol acetyltransferase reporter gene under viral (or cellular) promoter control. As expected, expression of the wild-type p53 inhibited promoter function. Expression of a p53 with a mutation at any one of the four amino acid positions 175, 248, 273, or 281, however, correlated with a significant increase of the PCNA promoter activity (2- to 11-fold). The viral promoters were also activated, although to a somewhat lesser extent. We also showed that activation by a mutant p53 requires a minimal promoter containing a lone TATA box. A more significant increase (25-fold) in activation occurs when the promoter contains a binding site for the activating transcription factor or cyclic AMP response element-binding protein. Using Saos-2 cells that do not express p53, we showed that activation by a mutant p53 was a direct enhancement. The mutant forms of p53 used in this study are found in various cancer cells. The activation of PCNA by mutant p53s may indicate a way to increase cell proliferation by the mutant p53s. Thus, our data indicate a possible functional role for the mutants of p53 found in cancer cells in activating several important loci, including PCNA.
The retinoblastoma gene is mutated in several types of human cancer and is the best characterized of the tumour-suppressor genes. A mouse strain has been constructed in which one allele of Rb is disrupted. These heterozygous animals are not predisposed to retinoblastoma, but some display pituitary tumours arising from cells in which the wild-type Rb allele is absent. Embryos homozygous for the mutation die between days 14 and 15 of gestation, exhibiting neuronal cell death and defective erythropoiesis.
The retinoblastoma gene, a prototypic tumour-suppressor gene, encodes a nuclear phosphoprotein (Rb). To understand better the role of Rb in development and in tumorigenesis, mice with an insertional mutation in exon 20 of the Rb-1 locus were generated. Homozygous mutants die before the 16th embryonic day with multiple defects. The haematopoietic system is abnormal; there is a significant increase in the number of immature nucleated erythrocytes. In the nervous system, ectopic mitoses and massive cell death are found, particularly in the hindbrain. All spinal ganglion cells die, but the neural retina is unaffected. Transfer of the human retinoblastoma (RB) mini-transgene into the mutant mice corrects the developmental defects. Thus, Rb is essential for normal mouse development.
The cellular transcription factor E2F, previously identified as a component of early adenovirus transcription, has now been
shown to be important in cell proliferation control. E2F appears to be a functional target for the action of the tumor suppressor
protein Rb that is encoded by the retinoblastoma susceptibility gene. The disruption of this E2F-Rb interaction, as well as
a complex involving E2F in association with the cell cycle-regulated cyclin A-cdk2 kinase complex, may be a common mechanism
of action for the oncoproteins encoded by the DNA tumor viruses.
Cooperation between the adenovirus E1A and E1B oncogenes is required for transformation of primary quiescent rodent cells. Although expression of E1A alone will stimulate cell proliferation sufficient to initiate transformed focus formation, proliferation fails to be sustained and foci degenerate. Coexpression of either the 19-kDa or 55-kDa E1B oncoproteins with E1A permits high-frequency transformation by overcoming this cytotoxic response. Without E1B 19-kDa protein expression, however, transformants remain susceptible to induction of cell death. Rapid loss of viability is coincident with nucleolytic cleavage of DNA in intranucleosomal regions and chromatin condensation, hallmarks of programmed cell death (apoptosis). Furthermore, overexpression of a known suppressor of apoptosis, the Bcl-2 protooncogene, can rescue E1A-induced focus degeneration. Thus E1A-dependent stimulation of cell proliferation is accompanied by apoptosis and thereby insufficient to singly induce transformation. High-frequency transformation requires a second function encoded by the E1B 19-kDa protein to block apoptosis.
The cellular phosphoprotein p53 inhibits progression through the mammalian cell cycle. Both p53 alleles are frequently mutated in human tumours, indicating that p53 is a tumour suppressor. Recent studies have suggested that p53 functions as a transcriptional activator, but the significance of this activity in cell-cycle control has not been established. The adenovirus 2 (Ad2) early 1B (E1B) 55K protein binds to p53 in transformed cells and contributes to oncogenic transformation by Ad2 (refs 10-12). Here we report that mutants of E1B 55K and wild-type Ad12 E1B 54K proteins show a strong correlation between their ability to inhibit p53-mediated transcriptional activation and their ability to cooperate with adenovirus E1A protein in the transformation of primary cells. These results indicate that p53 probably inhibits cell cycling by functioning as a transcription factor.
Although Rat-1 fibroblasts expressing c-myc constitutively are unable to arrest growth in low serum, their numbers do not increase in culture because of substantial cell death. We show this cell death to be dependent upon expression of c-myc protein and to occur by apoptosis. Regions of the c-myc protein required for induction of apoptosis overlap with regions necessary for cotransformation, autoregulation, and inhibition of differentiation, suggesting that the apoptotic function of c-myc protein is related to its other functions. Moreover, cells with higher levels of c-myc protein are more prone to cell death upon serum deprivation. Finally, we demonstrate that deregulated c-myc expression induces apoptosis in cells growth arrested by a variety of means and at various points in the cell cycle.
Human papillomavirus (HPV) DNAs are detected in approximately 90% of anogenital carcinomas. To assess directly the effect of HPV on squamous differentiation, normal human cervical and foreskin epithelial cells and cells immortalized by recombinant HPV DNAs were transplanted beneath a skin-muscle flap in athymic mice. Xenografts containing normal cells formed well-differentiated stratified squamous epithelia 2 to 3 weeks after transplantation, but cell lines immortalized by four HPV types (HPV16, HPV18, HPV31, and HPV33) detected in anogenital cancer exhibited dysplastic morphology and molecular alterations in gene expression characteristic of intraepithelial neoplasia. Morphological alterations were accompanied by delayed commitment to terminal differentiation, alterations in the pattern of involucrin expression, and reductions in levels of involucrin and keratin 1 RNAs. HPV18-immortalized cells developed dysplastic changes more rapidly than cells immortalized by HPV16 DNA. These results show that human genital epithelial cells immortalized by HPV DNAs detected in genital cancers undergo dysplastic differentiation in vivo.
A DNA-binding site selection and enrichment procedure revealed a sequence-specific DNA-binding activity selectively associated with glutathione S-transferase-retinoblastoma protein chimeras (GST-RB) that had been incubated with a human cell extract. Appropriate mutant forms of GST-RB, incubated in equivalent extracts, did not associate with this specific DNA-binding activity, and a peptide replica of the HPV E7 RB-binding segment selectively inhibited the association of GST-RB with the sequence-specific DNA-binding protein(s). Sequence analysis of oligonucleotides with high affinity for GST-RB complexes, as well as the results of competition binding studies, strongly suggest that RB can associate specifically with the transcription factor E2F or with a protein having closely related DNA-binding properties.
Human papillomavirus type 16 (HPV16) is frequently found in carcinomas and precancerous lesions of the uterine cervix and is thought to be closely associated with carcinogenesis in these regions. However, the transforming activity of the E6 and E7 genes in vivo has not been characterized. To investigate this function, we produced transgenic mice carrying HPV16 E6 and E7 open reading frames. We obtained five transgenic founders and established three transgenic lineages. We observed testicular tumors of germ cell origin in mice of all three lineages. Morphological studies showed that these tumors were a type of seminoma. Both testes of all tumor-bearing mice were affected with this type of tumor. Strikingly, in one lineage, all of the male mice developed this tumor. On Northern (RNA) analysis, a high level of expression of HPV mRNA was detected in these tumors. These results suggest that transforming genes of HPV16 have transforming activity in vivo and preferential effects on germ cells in the testis.
Human papillomavirus type 16 (HPV-16) is a DNA tumor virus that is associated with human anogenital cancers and encodes two
transforming proteins, E6 and E7. The E7 protein has been shown to bind to the retinoblastoma tumor suppressor gene product,
pRB. This study shows that the E6 protein of HPV-16 is capable of binding to the cellular p53 protein. The ability of the
E6 proteins from different human papillomaviruses to form complexes with p53 was assayed and found to correlate with the in
vivo clinical behavior and the in vitro transforming activity of these different papillomaviruses. The wild-type p53 protein
has tumor suppressor properties and has also been found in association with large T antigen and the E1B 55-kilodalton protein
in cells transformed by SV40 and by adenovirus type 5, respectively, providing further evidence that the human papillomaviruses,
the adenoviruses, and SV40 may effect similar cellular pathways in transformation.
The E6 protein encoded by the oncogenic human papillomavirus types 16 and 18 is one of two viral products expressed in HPV-associated cancers. E6 is an oncoprotein which cooperates with E7 to immortalize primary human keratinocytes. Insight into the mechanism by which E6 functions in oncogenesis is provided by the observation that the E6 protein encoded by HPV-16 and HPV-18 can complex the wild-type p53 protein in vitro. Wild-type p53 gene has tumor suppressor properties, and is a target for several of the oncoproteins encoded by DNA tumor viruses. In this study we demonstrate that the E6 proteins of the oncogenic HPVs that bind p53 stimulate the degradation of p53. The E6-promoted degradation of p53 is ATP dependent and involves the ubiquitin-dependent protease system. Selective degradation of cellular proteins such as p53 with negative regulatory functions provides a novel mechanism of action for dominant-acting oncoproteins.
Deletions or mutations of the retinoblastoma gene, RB1, are common features of many tumors and tumor cell lines. Recently, the RB1 gene product, p105-RB, has been shown to form stable protein/protein complexes with the oncoproteins of two DNA tumor viruses, the adenovirus E1A proteins and the simian virus 40 (SV40) large T antigen. Neither of these viruses is thought to be associated with human cancer, but they can cause tumors in rodents. Binding between the RB anti-oncoprotein and the adenovirus or SV40 oncoprotein can be recapitulated in vitro with coimmunoprecipitation mixing assays. These assays have been used to demonstrate that the E7 oncoprotein of the human papilloma virus type-16 can form similar complexes with p105-RB. Human papilloma virus-16 is found associated with approximately 50 percent of cervical carcinomas. These results suggest that these three DNA viruses may utilize similar mechanisms in transformation and implicate RB binding as a possible step in human papilloma virus-associated carcinogenesis.
To study how the oncogenic process may involve effects on differentiation, we overexpressed an immortalizing oncogene in a developing tissue in transgenic mice. By use of a gene fusion of the alpha A-crystallin promoter to the viral immortalizing oncogene, polyoma large T antigen (PyLT), we created transgenic mice that express PyLT specifically in ocular lens. Expression of large T antigen during embryonic development led to a perturbation in lens development, specifically, an interference with the normal program of fiber cell differentiation. This resulted in microphthalmia, which persisted throughout the life of the animal. Histological analysis revealed impairment of cell elongation, denucleation, and mitotic senescence in both primary and secondary fiber cell differentiation. Strikingly, there was no evidence for hyperplasia or for tumor development in vivo, unlike the consequences of many immortalizing oncogenes on tissues in other transgenic mice. In vitro, however, the developmentally perturbed cells derived from the transgenic lens showed high proliferative capacity. Our results suggest that a primary effect of aberrant expression of an immortalizing gene is an interference with normal tissue development; however, this interference may not necessarily induce proliferation or lead to tumor formation.
The human papillomavirus types (HPVs) most often associated with cancer of the cervix, such as HPV16, have been reported previously to immortalize normal human foreskin keratinocytes in vitro, while the types that are primarily associated with benign cervical lesions failed to do so. In this study we have determined the HPV16 genes that are responsible for the immortalizing activity of the viral genome. Transfection with a plasmid in which E6 and E7 were the only intact open reading frames (ORFs) induced an indefinite life-span in the keratinocytes with an efficiency similar to that of the entire early region of the viral DNA. Mutants in the E6E7 clone with inactivating lesions in E6 or E7 failed to induce immortalization. When transfected alone, E7 could induce hyperproliferation, but these cells eventually senesced. By itself, E6 exhibited no activity, Co-transfection of a plasmid with an intact E6 ORF and a second plasmid with an intact E7 ORF generated keratinocyte lines with indefinite growth potential. The E6 and E7 proteins were detected in the lines induced by the E6E7 DNA and by co-transfection of the E6 and E7 plasmids. Therefore, we conclude that HPV16 E6 and E7 cooperative to immortalize human keratinocytes in vitro. Changes in cellular gene expression are probably also required for immortalization since all of the keratinocyte lines examined were aneuploid. Serum and calcium resistant sublines were isolated from the E6E7 induced lines, indicating that other HPV genes do not play an obligatory role in the generation of resistance to differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)
Introduction of an exogenous retinoblastoma (RB) gene in RB-deficient retinoblastoma or osteosarcoma cells has been shown to suppress their neoplastic phenotype. In experiments designed to explore the potential mechanism of RB tumor suppression, we report here that the phosphorylation state of RB protein is modulated during normal cellular events. In resting cells, RB protein is present in its least phosphorylated form; in rapidly proliferating cells, RB protein is highly phosphorylated. Maximal phosphorylation is associated with S phase of the cell cycle. Induction of differentiation in several human leukemia cell lines by treatment with phorbol ester or retinoic acid leads to dephosphorylation of RB. Time course studies indicate that RB dephosphorylation precedes the total arrest of cell growth during differentiation. These observations strongly suggest that the function of RB protein is modulated by a phosphorylation/dephosphorylation mechanism during cell proliferation and differentiation.
p105-RB is the product of the retinoblastoma tumor suppressor gene. It is a nuclear phosphoprotein hypothesized to act as an inhibitor of cellular proliferation, yet surprisingly it is present in actively dividing cells. To look for changes in p105-RB that may regulate its activity during the cell cycle, we generated synchronized cell populations and followed their progression through the cell cycle. p105-RB is synthesized throughout the cycle, but is phosphorylated in a phase-specific manner. In the G0 and G1 phases of the cell cycle, an unphosphorylated species of the protein is the only detectable form, whereas in the S and G2/M phases, multiple phosphorylated species of p105-RB are detected.
The retinoblastoma susceptibility gene product, Rb, is suspected to suppress cell growth. Rb is a 110-114 kd nuclear phosphoprotein. We have previously demonstrated that SV40 T antigen binds only to unphosphorylated Rb, and not pp112-114Rb, the family of phosphorylated Rb. Here we demonstrate the cell cycle-dependent phosphorylation of Rb. In G0/G1 cells, virtually all the Rb is unphosphorylated. In contrast, during S and G2, it is largely, if not exclusively, phosphorylated. Rb phosphorylation occurs at the G1/S boundary in several cell types tested. A 14 residue peptide, corresponding to the SV40 T domain required for transformation, is able to compete effectively with SV40 T for binding to p110Rb. We propose a model to explain how Rb may suppress cell growth by acting as a cell cycle regulatory element.
Clinical and epidemiological data have implicated the human papillomaviruses (HPVs) as having an etiologic role in some anogenital malignancies, with HPV-16 being most frequently (greater than 60%) detected in cervical carcinoma. HPV-16 is actively transcribed in the cancers; the most abundant transcripts map to the E6 and E7 early open reading frames. Evidence is presented that the HPV-16 E7 open reading frame encodes transcriptional transactivation and cellular transformation functions analogous to those of adenovirus E1A proteins. Specifically, the HPV-16 E7 gene product could transactivate the adenovirus E2 promoter and cooperate with an activated ras oncogene to transform primary baby rat kidney cells. The E7 transforming function differed somewhat from that of adenovirus E1A in that E7 was also able to transform established mouse cells. Examination of the amino acid sequence of HPV-16 E7 revealed striking similarities with conserved domains 1 and 2 of adenovirus E1A proteins.
Monkey cells synthesizing SV40 large T antigen were lysed and the extracts immunoprecipitated with either monoclonal anti-T antibody or monoclonal antibody to p110-114, the product of the retinoblastoma susceptibility gene (Rb). T and p110-114 coprecipitated in each case, implying that the proteins are complexed with each other. Substitution and internal deletion mutants of T that contain structural alterations in a ten residue, transformation-controlling domain failed to complex with p110-114. In contrast, T mutants bearing structural changes outside of this domain bound to p110-114. These results are consistent with a model for transformation by SV40 which, at least in part, involves T/p110-114 complex formation and the perturbation of Rb protein and/or T function.
One of the cellular targets implicated in the process of transformation by the adenovirus E1A proteins is a 105K cellular protein. Previously, this protein had been shown to form stable protein/protein complexes with the E1A polypeptides but its identity was unknown. Here, we demonstrate that it is the product of the retinoblastoma gene. The interaction between E1A and the retinoblastoma gene product is the first demonstration of a physical link between an oncogene and an anti-oncogene.