Human papillomavirus type 8 oncoproteins E6 and E7 cooperate in downregulation of the cellular checkpoint kinase-1.

Human papillomavirus 8 (HPV8) is associated with the development of squamous cell carcinoma (SCC) of the skin. HPV-infected keratinocytes are able to override normal checkpoint control mechanisms and sustain cell cycle activity, allowing for synthesis of cellular proteins necessary for viral genome amplification. To study how HPV8 may disrupt cell cycle control, we analyzed the impact of HPV8 early gene expression on one of the key regulators of cell cycle and DNA damage response, checkpoint kinase-1 (CHK1). We found that expression of E1, E1̂E4, E2, E6 or E7 individually did not affect CHK1; however, keratinocytes expressing the complete early genome region (CER) of HPV8 showed a profound loss of CHK1 protein levels, that proved to be mediated by E6E7 co-expression. Neither CHK1 promoter regulation nor the ubiquitin-proteasome pathway are involved in HPV8-mediated CHK1 repression. However, CHK1 protein repression in organotypic skin cultures was paralleled by downregulation of the autophagy marker LC3B. Treatment of HPV8-CER expressing cells with the autophagy inhibitor Bafilomycin A1 rescued CHK1 expression and led to LC3B accumulation. Taken together, our data implicate that CHK1 autophagic degradation is enhanced by HPV8, which may contribute to the oncogenic potential of the virus.

Human papillomavirus mediated inhibition of DNA damage sensing and repair drives skin carcinogenesis.

BACKGROUND: The failure to mount an effective DNA damage response to repair UV induced cyclobutane pyrimidine dimers (CPDs) results in an increased propensity to develop cutaneous squamous cell carcinoma (cSCC). High-risk patient groups, such as organ transplant recipients (OTRs) frequently exhibit field cancerization at UV exposed body sites from which multiple human papillomavirus (HPV)-associated cSCCs develop rapidly, leading to profound morbidity and increased mortality. In vitro molecular evidence indicates that HPV of genus beta-papillomavirus (ß-PV) play an important role in accelerating the early stages of skin tumorigenesis. METHODS: We investigated the effects of UV induced DNA damage in murine models of ß-PV E6 oncoprotein driven skin tumorigenesis by crossing K14-HPV8-E6wt mice (developing skin tumors after UV treatment) with K14-CPD-photolyase animals and by generating the K14-HPV8-E6-K136N mutant mouse strain. Thymine dimers (marker for CPDs) and γH2AX (a marker for DNA double strand breaks) levels were determined in the murine skin and organotypic skin cultures of E6 expressing primary human keratinocytes after UV-irradiation by immunohistochemistry and in cell lines by In Cell Western blotting. Phosphorylation of ATR/Chk1 and ATM were assessed in cell lines and organotypic skin cultures by Western blots and immunohistochemistry. RESULTS: Skin tumor development after UV-irradiation in K14-HPV8-E6wt mice could completely be blocked through expression of CPD-photolyase. Through quantification of thymine dimers after UV irradiation in cells expressing E6 proteins with point mutations at conserved residues we identified a critical lysine in the C-terminal part of the protein for prevention of DNA damage repair and p300 binding. Whereas all K14-HPV8-E6wt animals develop skin tumors after UV expression of the HPV8-E6-K136N mutant significantly blocked skin tumor development after UV treatment. The persistence of CPDs in hyperproliferative epidermis K14-HPV8-E6wt skin resulted in the accumulation of γH2AX foci. DNA damage sensing was impaired in E6 positive cells grown as monolayer culture and in organotypic cultures, due to lack of phosphorylation of ATM, ATR and Chk1. CONCLUSION: We showed that cells expressing E6 fail to sense and mount an effective response to repair UV-induced DNA lesions and demonstrated a physiological relevance of E6-mediated inhibition of DNA damage repair for tumor initiation. These are the first mechanistical in vivo data on the tumorigenicity of HPV8 and demonstrate that the impairment of DNA damage repair pathways by the viral E6 protein is a critical factor in HPV-driven skin carcinogenesis.

Resistance to UV-induced apoptosis by ß-HPV5 E6 involves targeting of activated BAK for proteolysis by recruitment of the HERC1 ubiquitin ligase.

UV exposure is the main etiological agent in the development of non-melanoma skin cancer (NMSC), but mounting evidence suggests a co-factorial role for ß-genus HPV types early in tumor initiation or progression. UV damage initiates an apoptotic response, driven at the mitochondrial level by BCL-2 family proteins, that eliminates damaged cells that may accumulate deleterious mutations and acquire tumorigenic properties. BAK is a pro-apoptotic BCL-2 protein that functions ultimately to form pores that permeabilize the mitochondrial outer membrane, thereby committing a cell to death, a process involving changes in BAK phosphorylation and conformation. The E6 protein of ß-type HPV5 signals BAK for proteasomal degradation, a function that confers protection from UV-induced apoptosis. We find that HPV5 E6 does not constitutively target BAK for proteolysis, but targets the latter stages of BAK activation, following changes in phosphorylation and conformation. A mutational analysis identified the lysine residue on BAK required for proteolysis, and a functional siRNA screen identified the HECT domain E3 ubiquitin ligase HERC1 as being required for E6-mediated BAK degradation. We show that HERC1 interacts with BAK in E6-expressing cells that have been damaged by UV, and provide evidence that the interaction of HERC1 with BAK requires access to a hydrophobic surface on BAK that binds BH3 domains of BCL-2 proteins. We also show that HERC1 contains a putative BH3 domain that can bind to BAK. These findings reveal a specific and unique mechanism used by the HPV5 E6 protein to target BAK.

Tyrosine dephosphorylation is required for Bak activation in apoptosis.

Activation of the cell-death mediator Bak commits a cell to mitochondrial apoptosis. The initial steps that govern Bak activation are poorly understood. To further clarify these pivotal events, we have investigated whether post-translational modifications of Bak impinge on its activation potential. In this study, we report that on apoptotic stimulation Bak undergoes dephosphorylation at tyrosine residue 108 (Y108), a critical event that is necessary but not sufficient for Bak activation, but is required both for early exposure of the occluded N-terminal domain and multimerisation. RNA interference (RNAi) screening identified non-receptor tyrosine phosphatases (PTPNs) required for Bak dephosphorylation and apoptotic induction through chemotherapeutic agents. Specifically, modulation of PTPN5 protein expression by siRNA and overexpression directly affected both Bak-Y108 phosphorylation and the initiation of Bak activation. We further show that MEK/ERK signalling directly affects Bak phosphorylation through inhibition of PTPN5 to promote cell survival. We propose a model of Bak activation in which the regulation of Bak dephosphorylation constitutes the initial step in the activation process, which reveals a previously unsuspected mechanism controlling the initiation of mitochondrial apoptosis.

A conserved C-terminal sequence of high-risk cutaneous beta-human papillomavirus E6 proteins alters localization and signalling of ß1-integrin to promote cell migration.

Beta-human papillomaviruses (ß-HPV) infect cutaneous epithelia, and accumulating evidence suggests that the virus may act as a co-factor with UV-induced DNA damage in the development and progression of non-melanoma skin cancer, although the molecular mechanisms involved are poorly understood. The E6 protein of cutaneous ß-HPV types encodes functions consistent with a role in tumorigenesis, and E6 expression can result in papilloma formation in transgenic animals. The E6 proteins of high-risk α-HPV types, which are associated with the development of anogenital cancers, have a conserved 4 aa motif at their extreme C terminus that binds to specific PDZ domain-containing proteins to promote cell invasion. Likewise, the high-risk ß-HPVs HPV5 and HPV8 E6 proteins also share a conserved C-terminal motif, but this is markedly different from that of α-HPV types, implying functional differences. Using binding and functional studies, we have shown that ß-HPV E6 proteins target ß1-integrin using this C-terminal motif. E6 expression reduced membrane localization of ß1-integrin, but increased overall levels of ß1-integrin protein and its downstream effector focal adhesion kinase in human keratinocytes. Altered ß1-integrin localization due to E6 expression was associated with actin cytoskeleton rearrangement and increased cell migration that was abolished by point mutations in the C-terminal motif of E6. We concluded that modulation of ß1-integrin signalling by E6 proteins may contribute towards the pathogenicity of these ß-HPV types.

Expression of betapapillomavirus oncogenes increases the number of keratinocytes with stem cell-like properties.

Human papillomaviruses (HPV) of genus Betapapillomavirus (betaPV) are associated with nonmelanoma skin cancer development in epidermodysplasia verruciformis (EV) and immunosuppressed patients. Epidemiological and molecular studies suggest a carcinogenic activity of betaPV during early stages of cancer development. Since viral oncoproteins delay and perturb keratinocyte differentiation, they may have the capacity to either retain or confer a "stem cell-like" state on oncogene-expressing cells. The aim of this study was to determine (i) whether betaPV alters the expression of cell surface markers, such as CD44 and epithelial cell adhesion molecule (EpCAM), that have been associated with epithelial stemness, and (ii) whether this confers functional stem cell-like properties to human cutaneous keratinocytes. Fluorescence-activated cell sorter (FACS) analysis revealed an increase in the number of cells with high CD44 and EpCAM expression in keratinocyte cultures expressing HPV type 8 (HPV8) oncogenes E2, E6, and E7. Particularly through E7 expression, a distinct increase in clonogenicity and in the formation and size of tumor spheres was observed, accompanied by reduction of the epithelial differentiation marker Calgranulin B. These stem cell-like properties could be attributed to the pool of CD44(high) EpCAM(high) cells, which was increased within the E7 cultures of HPV5, -8, and -20. Enhanced EpCAM levels were present in organotypic skin cultures of primary keratinocytes expressing E7 of the oncogenic HPV types HPV5, -8, and -16 and in clinical samples from EV patients. In conclusion, our data show that betaPV may increase the number of stem cell-like cells present during early carcinogenesis and thus enable the persistence and accumulation of DNA damage necessary to generate malignant stem cells.

Chk1 activity is required for BAK multimerization in association with PUMA during mitochondrial apoptosis.

BACKGROUND: The Bcl-2 protein BAK is a key player in mitochondrial apoptosis and responds to a myriad of different death signals. Activation of BAK is a multistep process that involves a number of conformational changes mediated by BH3-only proteins or p53 which leads to BAK multimerization and pore formation in the mitochondrial outer membrane. We previously reported that BAK activation is dependent upon dephosphorylation of both tyrosine and serine residues. Further, recent reports demonstrated that PP2A activity is required for BAK multimerization. Since Chk1, a checkpoint kinase involved in the activation of G2 checkpoint, is regulated by PP2A, we therefore hypothesized that Chk1 is involved in BAK multimerization during cell cycle arrest upon severe DNA damage. FINDINGS: We now show that treatment of HCT116-WT BAK cells with a Chk1 inhibitor impaired BAK dimerization and mutimerization when treated with the DNA damaging agents UV or etoposide. As a result there is a concomitant decrease of cytochrome c release from isolated mitochondria challenged with tBid protein and failure in the activation of caspase3. Interestingly, co-immunoprecipitation studies suggest that Chk1 is required for recruitment of BH3- only protein PUMA to BAK. We also showed that Chk1 is associated with BAK upon DNA damage. CONCLUSION: These findings novelly demonstrate the involvement of a checkpoint kinase Chk1 is required for BAK activation and underscores the importance of involvement of Chk1 in mitochondrial apoptosis upon severe DNA damage.

BAK multimerization for apoptosis, but not bid binding, is inhibited by negatively charged residue in the BAK hydrophobic groove.

BACKGROUND: BCL-2 family proteins BAK and BAX orchestrate outer mitochondrial membrane permeabilization (MOMP) during apoptosis by forming pores in the membrane to release apoptogenic factors that commits a cell to death. BAK and BAX therefore function as a 'point of no return' in the apoptotic cascade. BAK activation is a multi-step process involving conformational changes, mediated by BH3-only proteins or p53, which lead eventually to oligomerization and pore formation. Further, recent reports show that BAK activation is also linked to and dependent upon dephosphorylation of both tyrosine and serine residues. FINDINGS: We hypothesized that phosphorylation of BAK at tyrosine residue 110 (Y110) was functionally important during the BAK activation process. BAK/BAX double knockout HCT116 cells expressing a phosphor-mimetic BAK mutant (BAK Y110E), showed impaired dimerization and multimerization capacity when treated with either UV irradiation or etoposide when compared to cells reconstituted to express wild-type BAK. The Y110E mutant also showed decreased release of cytochrome c from isolated mitochondria challenged with tBid protein, resulting in a failure to activate caspase 3. Interestingly, co-immunoprecipitation experiments suggest that a negative charge at this residue may be important for the recruitment of Bid to BAK, but conversely that this also impairs BAK:BAK interactions. CONCLUSION: These findings implicate dephosphorylation of Y110 as having an important mechanistic role in BAK activation, and underscores how post-translational modifications are intimately linked and coupled to the protein-protein interactions required for BAK activation during apoptosis.

Upregulation of lipocalin-2 in human papillomavirus-positive keratinocytes and cutaneous squamous cell carcinomas.

It has been demonstrated previously that E7 oncogene expression of human papillomavirus (HPV) type 8 in keratinocytes induces cell invasion and accelerated differentiation. Looking for cellular genes deregulated by HPV-8 E7, lipocalin-2 was identified as being upregulated in these cells by cDNA microarray analysis. Lipocalin-2 is known to be overexpressed in many human cancers and is implicated in the regulation of cell proliferation, differentiation and apoptosis. In this study, increased levels of lipocalin-2 were observed in extracts from HPV-8 E7-positive keratinocytes and from keratinocytes expressing E7 of HPV-1, -4, -5, -15, -20 and -38, but not of HPV-16. Similar results were obtained when measuring secreted lipocalin-2 in the supernatants of the cell cultures. Lipocalin-2 expression was associated with cell differentiation in keratinocyte monolayers and in organotypic skin cultures. It was found in the uppermost layers of HPV-5, -8, -15, -16, -20 and 38 E7-expressing but not low-risk HPV-1 and -4 E7-expressing keratinocytes. Immunohistochemical staining of HPV-positive and -negative human skin squamous cell carcinomas (SCCs) revealed lipocalin-2 expression mostly in differentiated, filaggrin-positive areas of 15 out of 17 HPV-positive and three out of nine HPV-negative SCCs. These data indicate that lipocalin-2 expression correlates with HPV positivity of cutaneous SCCs.

The E2 protein of human papillomavirus type 8 increases the expression of matrix metalloproteinase-9 in human keratinocytes and organotypic skin cultures.

Non-melanoma skin cancer (NMSC) is the most frequent human cancer of Caucasian populations. Although the ultraviolet irradiation is a key contributor to the establishment of this keratinocyte malignancy, the infection by some types of human papillomavirus (HPV) has also been implicated in NMSC development. Cancers occur as a result of a complex series of interactions between the cancer cell and its surrounding matrix. The matrix metalloproteinases (MMPs) play a role in degrading the extracellular matrix. MMP9 is an important gelatinase involved in processes such as cell migration, invasion and metastasis. In this report, we demonstrated by EMSA experiments that the MMP9 promoter contains a binding site for the transcriptional regulator E2 of HPV8. Transient reporter gene assays showed that HPV8-E2 activated the MMP9 promoter in a dose-dependent manner in human epidermal keratinocytes. An E2 transactivation-defective mutant (I73L) as well as a DNA-binding deficient mutant (R433K) demonstrated no activation of the MMP9 promoter, suggesting that both an intact transactivation and DNA-binding domain are required for E2 activation of the MMP9-promoter. The functional role of the E2 binding site within the MMP9 promoter was also confirmed by mutating the E2 binding site. In organotypic cultures of human skin, an overexpression of MMP9 was observed in suprabasal layers of the HPV8 E2-expressing epidermis thus confirming the results of the monolayer cultures. These results demonstrate that the early gene E2 of HPV8 is able to increase the expression of MMP9 by direct activation of the MMP9-promoter.

Cutaneous HPV5 E6 causes increased expression of Osteoprotegerin and Interleukin 6 which contribute to evasion of UV-induced apoptosis.

Human papillomaviruses (HPVs) are small DNA viruses, which specifically infect keratinocytes at different body sites. The association between cutaneous squamous cell carcinoma (SCC) formation, ultraviolet (UV) irradiation and infection with a high-risk subset of cutaneous HPVs has been postulated although the underlying molecular mechanisms by which HPV may play a role in SCC development are not yet fully elucidated. Expression of the viral E6 oncoprotein has been shown to interfere with DNA damage responses and inhibit UV-induced apoptosis, suggesting HPV can contribute to early stages in tumorigenesis. However, cutaneous SCCs, in contrast to HPV-associated anogenital cancers do not harbor HPV DNA in every tumor cell. Here, we show that expression of E6 from the prototypic skin cancer-associated HPV type 5 induced the secretion of factors that were able to inhibit UV-induced apoptosis in non-HPV-expressing cell lines and primary human keratinocytes. The anti-apoptotic effect of HPV E6 expression was found to be mediated in part by upregulation of osteoprotegerin (OPG) and interleukin 6 (IL6). Purified OPG and IL6, when added to cells together, but not individually, reduced apoptosis following UV irradiation. We provide evidence that OPG and IL6 inhibit the extrinsic and intrinsic apoptotic pathways, respectively. Furthermore, we show by immunohistochemistry of HPV-typed SCC sections that IL6 protein is upregulated in HPV-positive tumors compared with HPV-negative cancers. These findings support the hypothesis that a small number of HPV-infected cells influence UV-induced apoptosis in the skin and contribute to tumorigenesis.

Human papillomavirus 5 and 8 E6 downregulate interleukin-8 secretion in primary human keratinocytes.

Human papillomaviruses (HPVs) of the genus Betapapillomavirus appear to be involved in the early stages of skin cancer development, since both the prevalence and viral load are higher in precancerous actinic keratoses than in skin cancers. Interleukin-8 (IL-8) is an inflammatory cytokine that serves to alert the surrounding tissue after UV-induced damage. We examined the effects of the E2, E6 and E7 proteins of HPV8 and the E6 proteins of various HPV genotypes on IL-8 secretion from primary keratinocytes. HPV5 and HPV8 E6 showed the highest downregulation of basal IL-8 secretion. HPV8 E6 also negatively modulated IL-8 mRNA expression and protein secretion upon UVB irradiation. The downregulation of IL-8 in actinic keratoses may weaken the response to UV-induced damage and thus favour the accumulation of UVB-induced mutations.

Cutaneous human papillomaviruses down-regulate AKT1, whereas AKT2 up-regulation and activation associates with tumors.

Epithelial tumorigenesis has been linked to AKT up-regulation. Human papillomaviruses (HPV) cause anogenital cancers and anogenital HPV infection up-regulates AKT activity. Mounting evidence points to a role for cutaneous HPVs as etiologic factors in skin tumorigenesis. High-risk cutaneous beta HPVs have been linked to carcinogenesis in immunosuppressed patients, and high-risk cutaneous HPV8 genes enhance tumorigenesis in transgenic mice. We find that, in contrast to anogenital HPVs, cutaneous HPV8 early genes down-regulate epidermal AKT activity by down-regulating AKT1 isoform levels. This down-regulation occurs before papilloma formation or tumorigenesis and leads to cutaneous differentiation changes that may weaken the epidermal squame for viral release. We find that, in viral warts (papillomas) and HPV gene-induced epidermal tumors, AKT activity can be activated focally by up-regulation and phosphorylation of the AKT2 isoform. In squamous cell carcinomas (SCC), AKT1 down-regulation is also common, consistent with a viral influence, whereas AKT2 up-regulation is widespread. Activation of up-regulated AKT2 by serine phosphorylation associates with high-grade tumors. Our data suggest that AKT2 up-regulation is characteristic of SCC and that coincident AKT2 activation through serine phosphorylation correlates with malignancy. These findings highlight differences between the effects of anogenital and cutaneous HPV on epithelial AKT activity and furthermore show that AKT isoforms can behave differently during epidermal tumorigenesis. These findings also suggest AKT2 as a possible therapeutic tumor target in SCC.

Identification of the regions of the HPV 5 E6 protein involved in Bak degradation and inhibition of apoptosis.

UVB induced DNA damage is the major aetiological agent in NMSC development, but mounting evidence suggests a role for human papillomaviruses (HPV) from genus beta, including HPV 5 and HPV 8, in the development of NMSC on sun exposed body sites. We have previously shown that UVB activates Bak, an apoptogenic mitochondrial factor that, following an apoptotic stimulus, undergoes a conformational change that leads to pore formation in the mitochondrial membrane that releases apoptotic factors. The HPV E6 protein effectively inhibits UVB-induced apoptosis and targets Bak for proteolytic degradation. We have now identified the regions of the HPV5 E6 that are required to mediate Bak proteolysis and contribute toward the antiapoptotic activity of the protein. Interestingly, while wild-type HPV5 E6 does not bind or target p53 for proteolysis, we have isolated specific HPV5 E6 mutants that switch target specificity from Bak to p53 in a p53 codon 72 isoform-dependent manner. Furthermore, we demonstrate that the ability of wild-type HPV5 E6 to target Bak or specific E6 mutants to target p53 for proteolysis is not dependent on the E6-AP ubiquitin ligase.

Thiothymidine plus low-dose UVA kills hyperproliferative human skin cells independently of their human papilloma virus status.

The thymidine analogue 4-thiothymidine (S(4)TdR) is a photosensitizer for UVA radiation. The UV absorbance spectrum of S(4)TdR and its incorporation into DNA suggests that it might act synergistically with nonlethal doses of UVA to selectively kill hyperproliferative or cancerous skin cells. We show here that nontoxic concentrations of S(4)TdR combine with nonlethal doses of UVA to kill proliferating cultured skin cells. Established cell lines with a high fraction of proliferating cells were more sensitive than primary keratinocytes or fibroblasts to apoptosis induction by S(4)TdR/UVA. Although S(4)TdR plus UVA treatment induces stabilization of p53, cell death, as measured by apoptosis or clonal survival, occurs to a similar extent in both p53 wild-type and p53-null backgrounds. Furthermore, different types of human papilloma virus E6 proteins, which protect against UVB-induced apoptosis, have little effect on killing by S(4)TdR/UVA. S(4)TdR/UVA offers a possible therapeutic intervention strategy that seems to be applicable to human papilloma virus-associated skin lesions.

The E7 protein of cutaneous human papillomavirus type 8 causes invasion of human keratinocytes into the dermis in organotypic cultures of skin.

Human papillomaviruses (HPV) have been implicated in the development of nonmelanoma skin cancer (NMSC). The molecular mechanisms by which these viruses contribute towards NMSC are poorly understood. We have used an in vitro skin-equivalent model generated by transducing primary adult human epidermal keratinocytes with retroviruses expressing HPV genes to investigate the mechanisms of viral transformation. In this model, keratinocytes expressing HPV genes are seeded onto a mesenchyme composed of deepidermalized human dermis that had been repopulated with primary dermal fibroblasts. Expression of the HPV8 E7 gene caused both an enhancement of terminal differentiation and hyperproliferation, but most strikingly, the acquisition of the ability to migrate and invade through the underlying dermis. The basement membrane integrity was disrupted in a time-dependent manner in areas of invading keratinocytes, as evidenced by immunostaining of its protein components collagen types VII, IV, and laminin 5. This was accompanied by the overexpression of extracellular matrix metalloproteinases MMP-1, MMP-8, and MT-1-MMP. These results suggest that the cutaneous HPV type 8 that is frequently found in NMSC of epidermodysplasia verruciformis patients may actively promote an invasive keratinocyte phenotype. These findings also highlight the importance of epithelial-extracellular matrix-mesenchymal interactions that are required to support cell invasion.

Human papillomavirus type 77 E6 protein selectively inhibits p53-dependent transcription of proapoptotic genes following UV-B irradiation.

DNA damage, such as that elicited by UV-B, can induce either a cell cycle arrest or apoptosis that can be signalled by the p53 protein through the activation of a number of downstream cellular target genes. In contrast to oncogenic anogenital human papillomaviruses (HPVs), which mediate proteolytic degradation of p53, the E6 protein of cutaneous HPVs, such as HPV 77, do not promote p53 degradation. We have previously shown, however, that expression of HPV 77 E6 can effectively block UV-induced apoptosis in cells that have UV-activated p53. Here, we report that expression of the E6 protein from the cutaneous HPV 77 attenuates the UV-induced transactivation of p53-regulated proapoptotic genes Fas, PUMAbeta, Apaf-1, PIG3. This inhibition of p53-activation of proapoptotic genes by HPV77 E6 is exerted selectively, as the increased expression of p53 target genes involved in cell cycle arrest or regulatory functions regulation, such as p21 and Hdm2, is unaffected. Our data suggest that HPV 77 E6 may play an important role in specifically deregulating p53-dependent transactivation of proapoptotic genes upon UV-B irradiation.

Papillomaviruses: death-defying acts in skin cancer.

Ultraviolet (UV) radiation is an environmental agent that has a major impact on humans, and cumulative exposure poses a serious risk in terms of developing skin cancer. Acute doses of UV induce apoptotic cell death in the skin via signalling pathways that are, in part, dependent on the p53 tumour suppressor protein. However, p53-independent mechanisms have also been described. Recent findings show that a high proportion of non-melanoma skin cancers contain human papillomavirus. The viral E6 protein effectively blocks the epidermal apoptotic response to UV and might play a key role in promoting tumour development in cooperation with the mutagenic effects of UV.

Age-associated increase of codon 72 Arginine p53 frequency in gastric cardia and non-cardia adenocarcinoma.

PURPOSE: A common polymorphism of the tumor suppressor gene TP53 at codon 72 has been associated with human cancer susceptibility and prognosis. To examine the role of the polymorphism in the gastric adenocarcinoma, we examined 397 patients with or without the cancer. EXPERIMENTAL DESIGN: DNA samples were extracted from archived gastric tumor tissues and/or normal tissues of gastric adenocarcinoma and noncancer patients. The TP53 codon 72 genotypes were determined by PCR-RFLP. RESULTS: The overall genotype frequencies for Pro/Pro, Arg/Pro, and Arg/Arg were 7.3, 45.1, and 47.6%, respectively. A significant stepwise increased frequency of codon 72 Arg p53 with age was observed in patients with gastric cancer, but not in noncancer patients (P = 0.01). Patients with gastric cardia cancer had a significantly higher frequency of homozygous Arg allele than those with non-cardia tumors (P = 0.03) or than noncancer patients. After adjustment for age and gender, a logistic regression analysis suggested that the risk for a p53 Arg homozygous patient to develop cardia cancer is 3.1 95% confidence interval, 1.4-7.3) times greater than for p53 Pro homozygous and p53 Arg/Pro heterozygous patients. No close relationship was observed among patient gender, tumor histological type, p53 protein expression, and codon 72 genotype distribution. CONCLUSIONS: These findings indicate that codon 72 Arg p53 may be associated with a prolonged survival for patients who have had gastric adenocarcinoma, especially non-cardia adenocarcinoma. It may confer, however, a different role on patients who suffer cardia gastric adenocarcinoma.