DNA Tumor Virus Oncoproteins Research Articles

Paradoxical effects of DNA tumor virus oncogenes on epithelium-derived tumor cell fate during tumor progression and chemotherapy response

Epstein-Barr virus (EBV) and human papillomavirus (HPV) infection is the risk factors for nasopharyngeal carcinoma and cervical carcinoma, respectively. However, clinical analyses demonstrate that EBV or HPV is associated with improved response of patients, although underlying mechanism remains unclear. Here, we reported that the oncoproteins of DNA viruses, such as LMP1 of EBV and E7 of HPV, inhibit PERK activity in cancer cells via the interaction of the viral oncoproteins with PERK through a conserved motif. Inhibition of PERK led to increased level of reactive oxygen species (ROS) that promoted tumor and enhanced the efficacy of chemotherapy in vivo. Consistently, disruption of viral oncoprotein-PERK interactions attenuated tumor growth and chemotherapy in both cancer cells and tumor-bearing mouse models. Our findings uncovered a paradoxical effect of DNA tumor virus oncoproteins on tumors and highlighted that targeting PERK might be an attractive strategy for the treatment of NPC and cervical carcinoma.

Functional and Structural Mimicry of Cellular Protein Kinase A Anchoring Proteins by a Viral Oncoprotein.

The oncoproteins of the small DNA tumor viruses interact with a plethora of cellular regulators to commandeer control of the infected cell. During infection, adenovirus E1A deregulates cAMP signalling and repurposes it for activation of viral gene expression. We show that E1A structurally and functionally mimics a cellular A-kinase anchoring protein (AKAP). E1A interacts with and relocalizes protein kinase A (PKA) to the nucleus, likely to virus replication centres, via an interaction with the regulatory subunits of PKA. Binding to PKA requires the N-terminus of E1A, which bears striking similarity to the amphipathic α-helical domain present in cellular AKAPs. E1A also targets the same docking-dimerization domain of PKA normally bound by cellular AKAPs. In addition, the AKAP like motif within E1A could restore PKA interaction to a cellular AKAP in which its normal interaction motif was deleted. During infection, E1A successfully competes with endogenous cellular AKAPs for PKA interaction. E1A’s role as a viral AKAP contributes to viral transcription, protein expression and progeny production. These data establish HAdV E1A as the first known viral AKAP. This represents a unique example of viral subversion of a crucial cellular regulatory pathway via structural mimicry of the PKA interaction domain of cellular AKAPs.

The retinoblastoma tumor suppressor promotes efficient human cytomegalovirus lytic replication.

The retinoblastoma (Rb) tumor suppressor controls cell cycle, DNA damage, apoptotic, and metabolic pathways. DNA tumor virus oncoproteins reduce Rb function by either inducing Rb degradation or physically disrupting complexes between Rb and its myriad binding proteins. Human cytomegalovirus (HCMV), a betaherpesvirus being investigated for potential roles in human cancers, encodes multiple lytic-phase proteins that inactivate Rb in distinct ways, leading to the hypothesis that reduced Rb levels and/or activity would benefit HCMV lytic infection. Paradoxically, we found that Rb knockdown prior to infection, whether transient or constitutive, impaired HCMV lytic infection at multiple stages, notably viral DNA replication, late protein expression, and infectious virion production. The existence of differentially modified forms of Rb, the temporally and functionally distinct means by which HCMV proteins interact with Rb, and the necessity of Rb for efficient HCMV lytic replication combine to highlight the complex relationship between the virus and this critical tumor suppressor. Initial work examining viral protein modulation of cell cycle progression and oncogenic transformation revealed that these proteins inactivated the function of cellular tumor suppressor proteins. However, subsequent work, including experiments described here using human cytomegalovirus, demonstrate a more nuanced interaction that includes the necessity of cellular tumor suppressors for efficient viral replication. Understanding the positive impacts that cellular tumor suppressors have on viral infections may reveal new activities of these well-studied yet incompletely understood proteins. The basis for oncolytic viral therapy is the selective replication of viruses in transformed cells in which tumor suppressor function may be compromised. Understanding how tumor suppressors support viral infections may allow for the generation of modified oncolytic viruses with greater selective tumor cell replication and killing.

Correlation of Transcription of MALAT-1, a Novel Noncoding RNA, with Deregulated Expression of Tumor Suppressor p53 in Small DNA Tumor Virus Models

Although metastasis-associated lung adenocarcinoma transcript (MALAT)-1 is known to be consistently upregulated in several epithelial malignancies, little is known about its function or regulation. We therefore examined the relationship between MALAT-1 expression and candidate modulators such as DNA tumor virus oncoproteins human papillomavirus (HPV)-16 E6 and E7, BK virus T antigen (BKVTAg), mouse polyoma virus middle T antigen (MPVmTAg) and tumor suppressor genes p53 and pRb. Using suppressive subtractive hybridization (SSH) and real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays, MALAT-1 was shown to be increased in viral oncongene-expressing salivary gland biopsies from humans and mice. The results also indicated that MALAT-1 transcripts and promoter activity were increased in vitro when viral oncongene-expressing plasmids were introduced into different cell types. These same viral oncogenes in addition to increasing MALAT-1 transcription have also been shown to inhibit p53 and/or pRb function. In p53 mutant or inactive cell lines MALAT-1 was also shown to be highly upregulated. We hypothesize that there is a correlation between MALAT-1 over-expression and p53 deregulation. In conclusion, we show that disruption of p53, by both polyoma and papilloma oncoproteins appear to play an important role in the up-regulation of MALAT-1. MALAT-1 might therefore represent a biomarker for p53 deregulation within malignancies.

Interaction of retinoblastoma protein family members with large T-antigen of primate polyomaviruses

The retinoblastoma gene product pRb and other members of the Rb family of pocket proteins have a central role in the regulation of cell cycle progression. Soon after its discovery, pRb was found to interact with the transforming oncoproteins of DNA tumor viruses and this led to rapid advances in our understanding of the mechanisms of viral transformation and cell cycle progression. DNA viruses of the polyomavirus family have small, circular, double-stranded DNA genomes contained within non-enveloped icosahedral capsids and are highly tumorigenic in experimental animals. At least three types of polyomavirus infect humans: JC virus (JCV), BK virus (BKV) and Simian Vacuolating virus-40. The early region of these viruses encodes the transforming proteins large T-antigen and small t-antigen, which are involved in viral replication and also promote transformation of cells in culture and oncogenesis in vivo. Binding of T-antigen to pRb promotes the activation of the E2F family of transcription factors, which induce the expression of cellular genes required for S phase. In the context of lytic infection, this cell cycle progression is necessary for viral replication because polyomaviruses rely on S phase-specific host factors for their DNA synthesis. In the context of cellular transformation and tumorigenesis, T-antigen/pRB interaction is an indispensable event.

Transactivation of the cyclin A promoter by bovine papillomavirus type 4 E5 protein

Bovine papillomavirus type 4 (BPV-4) E5 (formerly E8) is a 42-residue hydrophobic, membrane-localised protein that can transform NIH-3T3 cells by a poorly defined mechanism. In E5-expressing cells, the observed up-regulation of cyclin A is underpinned by transactivation of the cyclin A promoter. Here we show that E5 transactivates the minimal cell cycle-regulated cyclin A promoter in cells both stably and acutely expressing the viral protein. There are no detectable differences between control and E5 cells in protein complexes binding the E2F-like cell cycle-dependent element (CDE)/cell cycle-regulated element (CCRE) of the cyclin A promoter and E5 does not transactivate E2F reporter plasmids in an E2F-dependent manner in vivo. CCAAT box integrity and functional NF-Y complexes are required for E5-mediated transactivation and a M r ∼110 K CCAAT-box binding factor (p110 CBF) associates with NF-YA only in E5 cells. This suggests that E5 sets the extent of cyclin A promoter activation by a mechanism similar to other, structurally unrelated, DNA tumour virus oncoproteins but distinct from the action of serum factors and so is inconsistent with E5 acting through constitutive activation of tyrosine kinase growth factor receptors.

Contribution of p16 INK4a to replicative senescence of human fibroblasts

In standard conditions of tissue culture, human fibroblasts undergo a limited number of population doublings before entering a state of irreversible growth arrest termed replicative senescence or M1. The arrest is triggered by a combination of telomere dysfunction and the stresses inflicted by culture conditions and is implemented, at least in part, by the cyclin-dependent kinase inhibitors p21 CIP1 and p16 INK4a. To investigate the role of p16 INK4a, we have studied fibroblasts from members of melanoma prone kindreds with mutations in one or both copies of the CDKN2A locus. The mutations affect the function of p16 INK4a but not of the alternative product, p14 ARF. The p16 INK4a-defective fibroblasts have an above average life span, compared to the heterozygous and normal age-matched controls, but they arrest with characteristics typical of senescence. Using agents that are known to bypass M1, such as DNA tumor virus oncoproteins or the Bmi1 transcriptional repressor, we provide evidence that p16 INK4a defective cells arrest at a stage that is operationally between M1 and M2 (crisis). As well as indicating that p16 INK4a contributes to but is not essential for replicative senescence of human fibroblasts, our data reveal considerable heterogeneity in the levels and accumulation of p16 INK4a in different strains.

Contribution of p16INK4a to replicative senescence of human fibroblasts

In standard conditions of tissue culture, human fibroblasts undergo a limited number of population doublings before entering a state of irreversible growth arrest termed replicative senescence or M1. The arrest is triggered by a combination of telomere dysfunction and the stresses inflicted by culture conditions and is implemented, at least in part, by the cyclin-dependent kinase inhibitors p21 CIP1 and p16 INK4a . To investigate the role of p16 INK4a , we have studied fibroblasts from members of melanoma prone kindreds with mutations in one or both copies of the CDKN2A locus. The mutations affect the function of p16 INK4a but not of the alternative product, p14 ARF . The p16 INK4a -defective fibroblasts have an above average life span, compared to the heterozygous and normal age-matched controls, but they arrest with characteristics typical of senescence. Using agents that are known to bypass M1, such as DNA tumor virus oncoproteins or the Bmi1 transcriptional repressor, we provide evidence that p16 INK4a defective cells arrest at a stage that is operationally between M1 and M2 (crisis). As well as indicating that p16 INK4a contributes to but is not essential for replicative senescence of human fibroblasts, our data reveal considerable heterogeneity in the levels and accumulation of p16 INK4a in different strains.

Epstein-Barr virus LMP1 blocks p16INK4a-RB pathway by promoting nuclear export of E2F4/5.

The p16INK4a–RB pathway plays a critical role in preventing inappropriate cell proliferation and is often targeted by viral oncoproteins during immortalization. Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is often present in EBV-associated proliferative diseases and is critical for the immortalizing and transforming activity of EBV. Unlike other DNA tumor virus oncoproteins, which possess immortalizing activity, LMP1 does not bind to retinoblastoma tumor suppressor protein, but instead blocks the expression of p16INK4a tumor suppressor gene. However, it has been unclear how LMP1 represses the p16INK4a gene expression. Here, we report that LMP1 promotes the CRM1-dependent nuclear export of Ets2, which is an important transcription factor for p16INK4a gene expression, thereby reducing the level of p16INK4a expression. We further demonstrate that LMP1 also blocks the function of E2F4 and E2F5 (E2F4/5) transcription factors through promoting their nuclear export in a CRM1-dependent manner. As E2F4/5 are essential downstream mediators for a p16INK4a-induced cell cycle arrest, these results indicate that the action of LMP1 on nuclear export has two effects on the p16INK4a–RB pathway: (1) repression of p16INK4a expression and (2) blocking the downstream mediator of the p16INK4a–RB pathway. These results reveal a novel activity of LMP1 and increase an understanding of how viral oncoproteins perturb the p16INK4a–RB pathway.

Human cytomegalovirus pp71 stimulates cell cycle progression by inducing the proteasome-dependent degradation of the retinoblastoma family of tumor suppressors.

The oncoproteins of the DNA tumor viruses, adenovirus E1A, simian virus 40 T antigen, and papillomavirus E7, each interact with the retinoblastoma family of tumor suppressors, leading to cell cycle stimulation, apoptosis induction, and cellular transformation. These proteins utilize a conserved LXCXE motif, which is also found in cellular proteins, to target the retinoblastoma family. Here, we describe a herpesvirus protein that shares a subset of the properties of the DNA tumor virus oncoproteins but maintains important differences as well. The human cytomegalovirus pp71 protein employs an LXCXD motif to attack the retinoblastoma family members and induce DNA synthesis in quiescent cells. pp71 binds to and induces the degradation of the hypophosphorylated forms of the retinoblastoma protein and its family members p107 and p130 in a proteasome-dependent manner. However, pp71 does not induce apoptosis and fails to transform cells. Thus, the similarities and differences in comparison to E1A, T antigen, and E7 make pp71 an interesting new tool with which to further dissect the role of the retinoblastoma/E2F pathway in cellular growth control and carcinogenesis.

Mechanisms by which DNA tumor virus oncoproteins target the Rb family of pocket proteins

Small DNA tumor viruses have evolved different mechanisms to abrogate the function of the retinoblastoma tumor suppressor (pRb). Studies of these viruses have been invaluable in uncovering the central role of the Rb family of pocket proteins in cell cycle control. While the molecular mechanisms by which the viral oncoproteins inactivate the Rb family are still being elucidated, it is clear that targeting of this family is required both for viral replication and for virus-induced transformation of mammalian cells. This review compares and contrasts the approaches DNA tumor viruses have evolved to antagonize Rb family members--ranging from relatively simple equilibrium dissociation of pRb from cellular pRb-binding factors to chaperone-mediated alterations in pocket protein stability and phosphorylation levels. The review will focus on the viral oncoproteins adenovirus E1A, human papillomavirus E7 and the large T antigens of several polyomaviruses. An understanding of these mechanisms may provide further insight into the regulation and functions of Rb family members as well as uncover new targets for the development of novel anti-viral agents, particularly against human papillomavirus, which is a significant cause of human cancer.

Che-1 affects cell growth by interfering with the recruitment of HDAC1 by Rb.

DNA tumor virus oncoproteins bind and inactivate Rb by interfering with the Rb/HDAC1 interaction. Che-1 is a recently identified human Rb binding protein that inhibits the Rb growth suppressing function. Here we show that Che-1 contacts the Rb pocket region and competes with HDAC1 for Rb binding site, removing HDAC1 from the Rb/E2F complex in vitro and from the E2F target promoters in vivo. Che-1 overexpression activates DNA synthesis in quiescent NIH-3T3 cells through HDAC1 displacement. Consistently, Che-1-specific RNA interference affects E2F activity and cell proliferation in human fibroblasts but not in the pocket protein-defective 293 cells. These findings indicate the existence of a pathway of Rb regulation supporting Che-1 as the cellular counterpart of DNA tumor virus oncoproteins.

RB and cyclin dependent kinase pathways: defining a distinction between RB and p16 loss in lung cancer.

The genetic components of the RB:CDK:cyclin:p16 tumor suppressor pathway undergo mutational and epigenetic alterations in a wide range of human cancers and serve as critical targets for inactivation by the transforming oncoproteins of several DNA tumor viruses. Lung cancer has been a useful model system for these studies as it was the first tumor to demonstrate an important role for RB in the genesis of a common adult malignancy and was also the first human cancer to demonstrate genetic evidence for a multi-component RB:p16 tumor suppressor pathway. Lung tumorigenesis, however, is a complex disease process that requires longstanding carcinogen exposure in order to acquire somatic alterations at many distinct genetic loci. Understanding the multifunctional properties of RB to regulate cell proliferation, differentiation, and apoptosis and how they relate to the sequential accumulation of other clonal gene defects will be essential in order to understand the specific patterns of gene inactivation observed in different subtypes of lung cancer and to fulfill the promise of 'molecular target' therapeutics.

Interactions of the PDZ-protein MAGI-1 with adenovirus E4-ORF1 and high-risk papillomavirus E6 oncoproteins.

The oncoproteins of small DNA tumor viruses promote tumorigenesis by complexing with cellular factors intimately involved in the control of cell proliferation. The major oncogenic determinants for human adenovirus type 9 (Ad9) and high-risk human papillomaviruses (HPV) are the E4-ORF1 and E6 proteins, respectively. These seemingly unrelated viral oncoproteins are similar in that their transforming activities in cells depend, in part, on a carboxyl-terminal PDZ domain-binding motif which mediates interactions with the cellular PDZ-protein DLG. Here we demonstrated that both Ad9 E4-ORF1 and high-risk HPV E6 proteins also bind to the DLG-related PDZ-protein MAGI-1. These interactions resulted in MAGI-1 being aberrantly sequestered in the cytoplasm by the Ad9 E4-ORF1 protein or being targeted for degradation by high-risk HPV E6 proteins. Transformation-defective mutant viral proteins, however, were deficient for these activities. Our findings indicate that MAGI-1 is a member of a select group of cellular PDZ proteins targeted by both adenovirus E4-ORF1 and high-risk HPV E6 proteins and, in addition, suggest that the tumorigenic potentials of these viral oncoproteins depend, in part, on an ability to inhibit the function of MAGI-1 in cells.

RRB1 and RRB2 encode maize retinoblastoma-related proteins that interact with a plant D-type cyclin and geminivirus replication protein.

Unlike mammalian and yeast cells, little is known about how plants regulate G1 progression and entry into the S phase of the cell cycle. In mammalian cells, a key regulator of this process is the retinoblastoma tumor suppressor protein (RB). In contrast, G1 control in Saccharomyces cerevisiae does not utilize an RB-like protein. We report here the cloning of cDNAs from two Zea mays genes, RRB1 and RRB2, that encode RB-related proteins. Further, RRB2 transcripts are alternatively spliced to yield two proteins with different C termini. At least one RRB gene is expressed in all the tissues examined, with the highest levels seen in the shoot apex. RRB1 is a 96-kDa nuclear protein that can physically interact with two mammalian DNA tumor virus oncoproteins, simian virus 40 large-T antigen and adenovirus E1A, and with a plant D-type cyclin. These associations are abolished by mutation of a conserved cysteine residue in RRB1 that is also essential for RB function. RRB1 binding potential is also sensitive to deletions in the conserved A and B domains, although differences exist in these effects compared to those of human RB. RRB1 can also bind to the AL1 protein from tomato golden mosaic virus (TGMV), a protein which is essential for TGMV DNA replication. These results suggest that G1 regulation in plant cells is controlled by a mechanism which is much more similar to that found in mammalian cells than that in yeast.

P300 family members associate with the carboxyl terminus of simian virus 40 large tumor antigen.

Several cellular polypeptides critical for growth regulation interact with DNA tumor virus oncoproteins. p400 is a cellular protein which binds to the adenovirus E1A oncoprotein(s). The biological function of p400 is not yet known, but it is structurally and immunologically closely related to p300 and CREB-binding protein, two known E1A-binding transcription adapters. Like p300, p400 is a phosphoprotein that binds to the simian virus 40 large tumor antigen (T). In anti-T coimmunoprecipitation experiments, staggered deletions spanning the amino-terminal 250 amino acids of T did not abrogate T binding to either p400 or p300. A T species composed of residues 251 to 708 bound both p400 and p300, while a T species defective in p53 binding was unable to bind either detectably. Anti-p53 immunoprecipitates prepared from cells containing wild-type T also contained p400 and p300. Hence, both p400 and p300 can bind (directly or indirectly) to a carboxyl-terminal fragment of T which contains its p53 binding domain. Since the p53 binding domain of T contributes to its immortalizing and transforming activities, T-p400 and/or T-p300 interactions may participate in these functions.

Simian virus 40 large T antigen alters the phosphorylation state of the RB-related proteins p130 and p107.

p130 and p107 are nuclear phosphoproteins related to the retinoblastoma gene product (pRb). pRb, p107, and p130 each undergo cell cycle-dependent phosphorylation, form complexes with the E2F family of transcription factors, and associate with oncoproteins of DNA tumor viruses, including simian virus 40 (SV40) large T antigen (TAg) and adenovirus ElA protein. The results of recent studies with mouse embryo fibroblasts (MEFs) lacking the retinoblastoma gene (Rb-1) have suggested that p130 and p107 may be important targets for SV40 large TAg-mediated transformation (J.B. Christensen and M.J. Imperiale, J. Virol. 65:3945-3948, 1995; J. Zalvide and J.A. DeCaprio, Mol. Cell. Biol. 15:5800-5810, 1995). In this report, we demonstrate that the expression of TAg affects the phosphorylation state of p130 and p107. In cells expressing wild-type TAg, only un(der)phosphorylated p130 and p107 were detected. To determine the domains within TAg that contribute to this effect on the phosphorylation of p130, we performed transient expression assays. While transiently expressed p130 was apparently phosphorylated normally, only un(der)phosphorylated p130 was detected when p130 was coexpressed with TAg. Using this assay, we found that the first 147 amino acids of TAg were sufficient to alter the phosphorylation state of p130. Within this region, the LXCXE domain of TAg, required for binding to the retinoblastoma family of proteins, was necessary but not sufficient to affect p130 phosphorylation. Residues within the first 82 amino acids of TAg were also required. TAg with mutations in the N terminus retained the ability to efficiently associate with p130 but did not affect its phosphorylation state. This demonstrates that the effect of SV40 TAg on p130 is not simply the result of binding and suggests that TAg has a novel effect on p130 and p107 that differs from its effect on pRb.

Modulatory effects of human cytomegalovirus infection on malignant properties of cancer cells.

Although there is no definitive evidence of the association of human cytomegalovirus (HCMV) infection with human cancers, the oncogenic potential of HCMV has been well established by in vitro studies demonstrating the ability of UV-irradiated or infectious virus to transform a variety of cells. After prolonged passaging the transformed cell type was maintained while HCMV DNA sequences were no more detectable. Three morphological transforming regions (mtr) of HCMV have been identified. The effects of HCMV on cellular functions which may be associated with the malignant phenotype include the expression of oncogenes and transcriptional activation of growth factors and interleukin synthesis. In infected cells, HCMV induces cytoskeletal alterations and changes in expression of cell surface receptors for extracellular matrix proteins which could result in increased motility and dissemination of cancer cells. Several human neuroblastoma cell lines undergo maturation in different neural crest derived cell types upon treatment with oncogenic potential agents, i.e. retinoic acid. The persistent HCMV infection of neuroblastoma cells (> 1 year) is accompanied by the increased expression of oncoproteins (i.e. N-myc) and decreased expression of tyrosine hydroxylase and dopamine-beta-hydroxylase. The activation of the cellular metabolism is due to HCMV binding to cellular receptors (prior to virus gene expression) and to the activity of HCMV immediate early (IE) gene products. IE proteins act directly as transcriptional activators or their activity is mediated by a variety of cellular transcription factors. HCMV infection may result in activation of promoters of cellular genes coding for cytokines, replication enzymes, proto-oncogenes and viral promoters. Recently it has been demonstrated that HCMV IE proteins block apoptosis probably by suppressing the ability of the antioncogene p53 to activate a reporter gene. The interactions of HCMV with tumor suppressor proteins such as p53 or retinoblastoma (pRb) susceptibility protein are reminiscent of those mediated by the oncoproteins of DNA tumor viruses. The acquisition of a fully malignant phenotype by normal cells is thought to require several mutations in a number of cellular genes. In this connection, HCMV may play the role of a nonobligate either direct or indirect cofactor for tumor genesis, e.g. by blocking apoptosis, which may be an essential requirement for tumor progression. Due to the stimulation of growth factors and/or inhibition of antioncogenes by its gene products, HCMV may modulate the malignant potential for tumor cells.

Temporally distinct patterns of p53-dependent and p53-independent apoptosis during mouse lens development.

Programmed cell death, or apoptosis, is a critical event in the development of multicellular organisms, and its perturbation is implicated in many diseases including cancer. The tumor suppressor protein p53 is known to mediate apoptosis induced by the DNA tumor virus oncoproteins, adenovirus E1A (AdE1A) and SV40 T antigen (SV40 Tag). We have recently demonstrated that the E6 and E7 oncoproteins of human papillomavirus type 16 (HPV-16) modulate apoptosis when expressed in the lens of transgenic mice. In this study we have identified the pathways that mediate E7 induction and E6 inhibition of apoptosis during different stages in the development of the lens. E7 transgenic mice made p53-null were only partially rescued in their apoptotic phenotype, indicating that both p53-dependent and -independent pathways mediate E7-induced apoptosis in the lens. The E6 transgene and p53-null genotype acted additively to reduce levels of apoptosis induced by E7 in neonatal lenses, indicating that E6 modulates apoptosis at least in part through p53-independent mechanisms. The partial reduction in E7-induced apoptosis by the p53-null genotype correlated with an increased incidence of lens tumors in adult E7 transgenic mice. Analyses of embryonic lenses at E13.5, E15.5, and E17.5 revealed a temporally distinct activation of p53-dependent and -independent apoptosis in the E7 lens. During the early stages of lens development, apoptosis was highly p53-dependent, whereas at later stages, apoptosis occurred through both p53-independent and -dependent pathways. This later time correlates temporally with the time of normal fiber cell denucleation, which can be inhibited by E6 through a p53-independent mechanism. These data suggest a similarity between the mechanism regulating E7-induced, p53-independent apoptosis and the apoptotic-like developmental process of fiber cell denucleation, and the mechanisms through which E6 suppresses both processes.

Aberrations of p16Ink4 and retinoblastoma tumour‐suppressor genes occur in distinct sub‐sets of human cancer cell lines

The p16Ink4/MTS1/CDKN2 is a cell-cycle regulatory inhibitor of cyclin-dependent kinase 4 (cdk4), and a candidate tumour suppressor whose gene on chromosome band 9p21 is frequently deleted or mutated in diverse types of cancer. Cdk4 in association with its D-type cyclin partners, together with p16Ink4, and the product of the retinoblastoma tumour-suppressor gene (pRB), appear to constitute a G1-phase-controlling pathway which can become de-regulated through oncogenic aberrations of any of the components. In an attempt to elucidate the underlying molecular mechanisms, we have now surveyed expression of p16Ink4, at the protein and the mRNA levels, in 21 human cell types expressing normal pRB, as compared with another series of 21 cell lines whose pRB is mutant and/or inactivated through sequestration by DNA tumour virus onco-proteins. In contrast to aberrant lack of p16 expression in the majority of RB-positive cell types, expression of apparently normal (as shown by electrophoretic mobility and/or the ability to form protein-protein complexes with cdk4 in vivo) p16 was uniformly preserved in the cancer cell lines whose RB function was compromised. These data indicate that p16 operates upstream of pRB along the same pathway in G1. The results are discussed in view of the nature of a selective growth advantage potentially gained by cells through de-regulation of this key cell-cycle control mechanism.