Modulation of type M2 pyruvate kinase activity by the human papillomavirus type 16 E7 oncoprotein

We report here that the E7 oncoprotein encoded by the oncogenic human papillomavirus (HPV) type 16 binds to the glycolytic enzyme type M2 pyruvate kinase (M2-PK). M2-PK occurs in a tetrameric form with a high affinity to its substrate phosphoenolpyruvate and a dimeric form with a low affinity to phosphoenolpyruvate, and the transition between both conformations regulates the glycolytic flux in tumor cells. The glycolytic intermediate fructose 1, 6-bisphosphate induces the reassociation of the dimeric to the tetrameric form of M2-PK. The expression of E7 in an experimental cell line shifts the equilibrium to the dimeric state despite a significant increase in the fructose 1,6-bisphosphate levels. Investigations of HPV-16 E7 mutants and the nononcogenic HPV-11 subtype suggest that the interaction of HPV-16 E7 with M2-PK may be linked to the transforming potential of the viral oncoprotein.     

Melittin binds to secretory phospholipase A2 and inhibits its enzymatic activity

Synthetic melittin inhibited the enzymatic activity of secretory phospholipase A2 (PLA2) from various sources, including bee and snake venoms, bovine pancreas, and synovial fluid from rheumatoid arthritis patients, irrespective of substrate (e.g., [14C]-phosphatidylcholine or phosphatidylethanolamine vesicles and [3H]-oleic acid-labeled E.coli). A Lineweaver-Burk analysis showed that melittin was a noncompetitive inhibitor of bee venom PLA2, causing a change in Vmax from 200 to 50 units/min/mg of protein. The Km remained unchanged (0.75 nmole). Melittin inhibited approximately 50% of purified bee venom PLA2 activity in a 30:1 molar ratio (melittin:enzyme). Because the enzyme kinetics indicated a PLA2-melittin interaction, a melittin-sepharose affinity column was used to purify a PLA2 from human serum. Further, an enzyme-linked assay was developed to quantitate PLA2 activity in biological fluids using avidin-peroxidase and ELISA plates coated with biotinylated melittin. These observations may have potential therapeutic significance, as well as provide a convenient basis for the isolation and quantitation of PLA2.     

Immunoexpression of mutant p53 and human papillomavirus related E6 oncoprotein in anal malignancies

Immunohistochemical expression of mutant p53 protein and human papillomavirus (HPV) 16 and 18 related E6 oncoprotein was studied in 36 biopsy proved anal cancers. Mutant p53 was detected in 61.1% cases. HPV 16 and 18 E6 protein was expressed in 22.2% cases, all of which were squamous cell carcinomas. Coexpression of both mutant p53 and E6 protein was found in only 5 cases (13.8%). In HPV 16/18 positive anal tumors, the degradation of p53 is accelerated by viral E6 oncoprotein. In HPV negative tumors, however, other mutagenic factors probably play a role in carcinogenesis.     

The CXXC Zn binding motifs of the human papillomavirus type 16 E7 oncoprotein are not required for its in vitro transforming activity in rodent cells

The conserved region 3 (CR3) of the E7 protein of human papillomaviruses contains two CXXC motifs involved in zinc binding and in the homodimerization of the molecule. Studies have suggested that the intact CXXC motifs in the CR3 of HPV16 and HPV18 E7 are required for the in vitro transforming activity of these proteins. CR3 also contains a low affinity pRb binding site and is involved in the disruption of the E2F/Rb1 complex. E7 is structurally and functionally related to Adenovirus E1A protein, which also has two CXXC motifs in CR3. However, the Ad E1A transforming activity appears to be independent of the presence of such domains. In fact, this viral protein exists in vivo as two different forms of 289 and 243 amino acids. The shorter Ad E1A form (Ad E1A243), where both CXXC motifs are deleted by internal splicing, retains its in vitro transforming activity. We have investigated if the HPV16 E7 CR3 can be functionally replaced by the Ad E1A243 CR3, which lacks both CXXC motifs. A chimeric protein (E7/E1A243) containing the CR1 and CR2 of HPV16 E7 fused to the CR3 of Ad E1A 243 was constructed. The E7/E1A243 while not able to homodimerize in the S. cerevisiae two-hybrid system retains several of the properties of the parental proteins, HPV16 E7 and Ad E1A. It associates with the 'pocket' proteins, induces growth in soft agar of NIH3T3 cells and immortalizes rat embryo fibroblasts. These data suggest that the CXXC motifs in CR3 of E7 do not play a direct role in the transforming properties of this viral protein but probably are important for maintaining the correct protein configuration.     

Human papillomavirus oncoproteins E6 and E7 independently abrogate the mitotic spindle checkpoint

The E6 and E7 genes of the high-risk human papillomavirus (HPV) types encode oncoproteins, and both act by interfering with the activity of cellular tumor suppressor proteins. E7 proteins act by associating with members of the retinoblastoma family, while E6 increases the turnover of p53. p53 has been implicated as a regulator of both the G1/S cell cycle checkpoint and the mitotic spindle checkpoint. When fibroblasts from p53 knockout mice are treated with the spindle inhibitor nocodazole, a rereplication of DNA occurs without transit through mitosis. We investigated whether E6 or E7 could induce a similar loss of mitotic checkpoint activity in human keratinocytes. Recombinant retroviruses expressing high-risk E6 alone, E7 alone, and E6 in combination with E7 were used to infect normal human foreskin keratinocytes (HFKs). Established cell lines were treated with nocodazole, stained with propidium iodide, and analyzed for DNA content by flow cytometry. Cells infected with high-risk E6 were found to continue to replicate DNA and accumulated an octaploid (8N) population. Surprisingly, expression of E7 alone was also able to bypass this checkpoint. Cells expressing E7 alone exhibited increased levels of p53, while those expressing E6 had significantly reduced levels. The p53 present in the E7 cells was active, as increased levels of p21 were observed. This suggested that E7 bypassed the mitotic checkpoint by a p53-independent mechanism. The levels of MDM2, a cellular oncoprotein also implicated in control of the mitotic checkpoint, were significantly elevated in the E7 cells compared to the normal HFKs. In E6-expressing cells, the levels of MDM2 were undetectable. It is possible that abrogation of Rb function by E7 or increased expression of MDM2 contributes to the loss of mitotic spindle checkpoint control in the E7 cells. These findings suggest mechanisms by which both HPV oncoproteins contribute to genomic instability at the mitotic checkpoint.     

Chromosomal instability is correlated with telomere erosion and inactivation of G2 checkpoint function in human fibroblasts expressing human papillomavirus type 16 E6 oncoprotein

Cell cycle checkpoints and tumor suppressor gene functions appear to be required for the maintenance of a stable genome in proliferating cells. In this study chromosomal destabilization was monitored in relation to telomere structure, lifespan control and G2 checkpoint function. Replicative senescence was inactivated in secondary cultures of human skin fibroblasts by expressing the human papillomavirus type 16 (HPV-16) E6 oncoprotein to inactivate p53. Chromosome aberrations were enumerated during in vitro aging of isogenic control (F5neo) and HPV-16E6-expressing (F5E6) fibroblasts. We found that structural and numerical aberrations in chromosomes were significantly increased in F5E6 cells during aging in vitro and fluorescence in situ hybridization (FISH) analysis using chromosome-specific probes demonstrated the occurrence of rearrangements involving chromosome 4 and 6 in genetically unstable F5E6 cells. Flow cytometry and karyotypic analyses revealed increased polyploidy and aneuploidy in F5E6 cells only at passages > 16, although these cells displayed defective mitotic spindle checkpoint function associated with inactivation of p53 at passages 5 and 16. G2 checkpoint function was confirmed to be gradually but progressively inactivated during in vitro aging of E6-expressing cells. Aging of F5neo fibroblasts was documented during in vitro passaging by induction of a senescence-associated marker, pH 6.0 lysosomal beta-galactosidase. F5E6 cells displayed extension of in vitro lifespan and did not induce beta-galactosidase at high passage. Erosion of telomeres during in vitro aging of telomerase-negative F5neo cells was demonstrated by Southern hybridization and by quantitative FISH analysis on an individual cell level. Telomeric signals diminished continuously as F5neo cells aged in vitro being reduced by 80% near the time of replicative senescence. Telomeric signals detected by FISH also decreased continuously during aging of telomerase-negative F5E6 cells, but telomeres appeared to be stabilized at passage 34 when telomerase was expressed. Chromosomal instability in E6-expressing cells was correlated (P < 0.05) with both loss of telomeric signals and inactivation of G2 checkpoint function. The results suggest that chromosomal stability depends upon a complex interaction among the systems of telomere length maintenance and cell cycle checkpoints.     

The bovine papillomavirus E6 protein binds to the LD motif repeats of paxillin and blocks its interaction with vinculin and the focal adhesion kinase

The bovine papillomavirus type 1 (BPV-1) E6 oncoprotein can transform fibroblasts and induce anchorage-independent growth and disassembly of the actin stress fibers. We have previously shown that the E6 protein interacts with the focal adhesion protein, paxillin, suggesting a direct role of E6 in the disruption of the actin cytoskeleton. We have now mapped the E6 binding sites on paxillin to the LD motif repeats region, which has been implicated in mediating paxillin binding to two other focal adhesion proteins, vinculin and the focal adhesion kinase. The five LD motif repeats identified in paxillin do not contribute equally to its interaction with E6. The first LD repeat is most critical for paxillin binding to E6 both in vitro and in vivo. Furthermore, the binding of recombinant wild-type E6 protein to paxillin blocked the interaction of several cellular proteins with paxillin, including vinculin and the focal adhesion kinase. A mutant E6 protein (H105) which does not bind to paxillin had no effect on the binding of these cellular proteins to paxillin. These data suggest that E6 disruption of the actin stress fibers occurs through blocking the interaction of paxillin with its cellular effectors such as vinculin and the focal adhesion kinase.     

Cytotoxic T lymphocyte responses to E6 and E7 proteins of human papillomavirus type 16: relationship to cervical intraepithelial neoplasia

Cytotoxic T lymphocyte (CTL) responses to the human papillomavirus (HPV) type 16 E6 and E7 proteins were measured in 20 women with known HPV and cervical disease status. CTL assays were performed after stimulation with E6 or E7 fusion proteins using autologous B lymphoblastoid cells infected with vaccinia viruses expressing E6 or E7. CTL responses to E6 and E7 were detected in 6 (75%) of 8 and 5 (56%) of 9 HPV-16-positive women without cervical intraepithelial neoplasia (CIN), respectively. Responses to E6 or E7 were each detected in only 2 (29%) of 7 HPV-16-positive women with CIN. Responses to both antigens were found in 63% of women without CIN and 14% of those with CIN. CTL responses to E6 or E7 are more commonly detectable in HPV-16-positive women without CIN than in HPV-16-positive women with CIN, suggesting that CTL response may play a role in disease protection.     

Human papillomavirus type 16 E6 and E7 oncogenes abrogate radiation-induced DNA damage responses in vivo through p53-dependent and p53-independent pathways

E6 and E7 oncoproteins from high risk human papillomaviruses (HPVs) transform cells in tissue culture and induce tumors in vivo. Both E6, which inhibits p53 functions, and E7, which inhibits pRb, can also abrogate growth arrest induced by DNA-damaging agents in cultured cells. In this study, we have used transgenic mice that express HPV-16 E6 or E7 in the epidermis to determine how these two proteins modulate DNA damage responses in vivo. Our results demonstrate that both E6 and E7 abrogate the inhibition of DNA synthesis in the epidermis after treatment with ionizing radiation. Increases in the levels of p53 and p21 proteins after irradiation were suppressed by E6 but not by E7. Through the study of p53-null mice, we found that radiation-induced growth arrest in the epidermis is mediated through both p53-dependent and p53-independent pathways. The abrogation of radiation responses in both E6 and E7 transgenic mice was more complete than was seen in the p53-null epidermis. We conclude that E6 and E7 each have the capacity to modulate p53-dependent as well as p53-independent cellular responses to radiation. Additionally, we found that the conserved region (CR) 1 and CR2 domains in E7 protein, which are involved in the inactivation of pRb function and required for E7's transforming function, were also required for E7 to modulate DNA damage responses in vivo. Thus pRb and/or pRb-like proteins likely mediate both p53-dependent and p53-independent responses to radiation.     

Elevated p16 at senescence and loss of p16 at immortalization in human papillomavirus 16 E6, but not E7, transformed human uroepithelial cells

CDKN2/p16 inhibits the cyclin D/cyclin-dependent kinase complexes that phosphorylate pRb, thus blocking cell cycle progression. We previously reported that p16 levels are low to undetectable in normal human uroepithelial cells (HUCs) and in immortalized uroepithelial cells with functional pRb, whereas p16 levels are markedly elevated in immortal HUCs with altered pRb (T. Yeager et al., Cancer Res., 55: 493-497, 1995). We now report that elevation of p16 levels occurs at senescence in HUCs, including HUCs transformed by human papillomavirus 16 E7 or E6, whose oncoprotein products lead to functional loss of pRb and p53, respectively. We also report that six of six independently immortalized E7 HUCs show high levels of p16 similar to those observed at HUC senescence, whereas p16 is undetectable in five of five immortal E6 HUCs. Four of the five independent E6 HUCs that lost p16 at immortalization showed hemizygous deletion of the 9p21 region. However, no homozygous CDKN2 deletions were detected, and only one CDKN2 mutation was identified. For the first time, these data associate elevated p16 with senescence in human epithelial cells. These data also suggest that a component of immortalization may be abrogation, either by pRb inactivation (as in the E7-transformed HUCs) or by p16 inactivation (as in the E6-transformed HUCs), of a p16-mediated senescence cell cycle block.     

Human papillomavirus infection and its relationship to recent and distant sexual partners

OBJECTIVE: To determine the relation between the detection of genital human papillomavirus (HPV) DNA and the number of new sexual partners in the last year, 1-5 years, and 5-10 years. METHODS: In a cross-sectional study, 298 women collected tampon specimens and completed self-answer questionnaires on the known risk factors for HPV infection, including the number of sexual partners during the last 1, 5, and 10 years. The tampons were analyzed for the presence of HPV DNA by polymerase chain reaction using L1 consensus primers. RESULTS: Ninety-two (30.9%) tampons were positive for HPV DNA. In univariate analysis, the presence of HPV DNA was associated with a younger age, single marital status, a previously abnormal or currently abnormal Papanicolaou smear, and one or more new sexual partners in the last year, 1-5 years, and 5-10 years. The presence of HPV DNA was not associated with education level, past pregnancy, current or past oral contraceptive use, or the age at first intercourse. In multivariate analysis, only the number of sexual partners during the last year and 1-5 years, and a previously abnormal Papanicolaou smear were associated with HPV. CONCLUSION: The presence of HPV DNA is best predicted by the number of new sexual partners in the last 5 years. Transiently detectable HPV DNA is one possible explanation for this observation.     

Dispensability of p53 degradation for tumorigenicity and decreased serum requirement of human papillomavirus type 16 E6

Tonsils and adenoids are secondary lymphoid organs exposed to the environment. The most important classifications of AIDS include the lymphocyte subsets of peripheral blood. We have studied the lymphocyte subsets in peripheral blood and secondary lymphoid organs in a control group of children suffering adenotonsillar pathology and in five children with AIDS and the same adenotonsillar pathology. The antigen surface markers were determined by flow cytometry in lymphocytes isolated from peripheral blood, and from tonsils and adenoids after tonsillectomy and adenoidectomy, in the control group and in children diagnosed with AIDS. The most important findings in tonsils and adenoids were a decrease of the total T lymphocytes, helper T lymphocytes and CD4/CD8 ratio; an increase of cytotoxic T lymphocytes and B lymphocytes, as well as a 200% increase in monocytes of AIDS-affected children. These observations show the value of analyzing the lymphocyte subsets of the tonsils and adenoids of AIDS-affected children, and establishing an earlier relation to clinical symptoms.     

Tumorigenicity by human papillomavirus type 16 E6 and E7 in transgenic mice correlates with alterations in epithelial cell growth and differentiation

The human papillomavirus type 16 (HPV-16) E6 and E7 oncogenes are thought to play a role in the development of most human cervical cancers. These E6 and E7 oncoproteins affect cell growth control at least in part through their association with and inactivation of the cellular tumor suppressor gene products, p53 and Rb. To study the biological activities of the HPV-16 E6 and E7 genes in epithelial cells in vivo, transgenic mice were generated in which expression of E6 and E7 was targeted to the ocular lens. Expression of the transgenes correlated with bilateral microphthalmia and cataracts (100% penetrance) resulting from an efficient impairment of lens fiber cell differentiation and coincident induction of cell proliferation. Lens tumors formed in 40% of adult mice from the mouse lineage with the highest level of E6 and E7 expression. Additionally, when lens cells from neonatal transgenic animals were placed in tissue culture, immortalized cell populations grew out and acquired a tumorigenic phenotype with continuous passage. These observations indicate that genetic changes in addition to the transgenes are likely necessary for tumor formation. These transgenic mice and cell lines provide the basis for further studies into the mechanism of action of E6 and E7 in eliciting the observed pathology and into the genetic alterations required for HPV-16-associated tumor progression.     

Glucocorticoids stimulate growth of human papillomavirus type 16 (HPV16)-immortalized human keratinocytes and support HPV16-mediated immortalization without affecting the levels of HPV16 E6/E7 mRNA

We investigated the effects of the glucocorticoids hydrocortisone and dexamethasone on human papillomavirus type 16 (HPV16)-mediated human cell carcinogenesis using normal human keratinocytes (HKc) and HKc immortalized by transfection with HPV16 DNA (HKc/HPV16). Normal HKc did not require glucocorticoids for proliferation. In contrast, growth of early passage HKc/HPV16 strictly required these hormones, although glucocorticoid dependence became less stringent during in vitro progression. Glucocorticoid dependence was acquired by HKc early after immortalization with HPV16 DNA, and glucocorticoids were required for efficient HKc immortalization. However, treatment of HKc/HPV16 with hydrocortisone or dexamethasone did not increase the steady-state levels of HPV16 E6/E7 mRNA or protein. Firefly luciferase activity expressed under the control of the HPV16 upstream regulatory region and P97 promoter increased by about fourfold following dexamethasone treatment of HeLa, but only twofold in HKc/HPV16, and less than twofold in SiHa. However, all of these cell lines expressed sufficient endogenous glucocorticoid receptors to allow for a dexamethasone response of the mouse mammary tumor virus promoter. These results indicate that mechanisms other than a direct influence by glucocorticoids on HPV16 early gene expression may contribute to the striking biological effects of these steroids on HPV16-mediated human cell carcinogenesis.