Regulation of telomerase by human papillomaviruses.

The E6 oncoprotein of human papillomaviruses (HPVs) induces telomerase activity in primary human epithelial cells. This activity is dependent on association of E6 with E6AP, a cellular ubiquitin ligase. E6 activates the transcription of hTERT, the catalytic subunit of telomerase. E boxes near the start of hTERT transcription are required for E6; however, acetylated histones are only present in the E6 cells. We identified two isoforms of NFX1, a new binding partner of E6/E6AP. The NFX1- 91 isoform binds to an X-box motif located adjacent to the proximal E box, binds Sin3A and HDACs, repressing hTERT transcription. It preferentially binds E6/E6AP and is targeted for ubiquitin-mediated degradation. The NFX1-123 isoform has the opposite activity, increasing hTERT transcription or translation. This is the first example of viral oncoproteins disrupting regulation of telomerase, a critical event in tumorigenesis.

Small tumor virus genomes are integrated near nuclear matrix attachment regions in transformed cells.

More than 15% of human cancers have a viral etiology. In benign lesions induced by the small DNA tumor viruses, viral genomes are typically maintained extrachromosomally. Malignant progression is often associated with viral integration into host cell chromatin. To study the role of viral integration in tumorigenesis, we analyzed the positions of integrated viral genomes in tumors and tumor cell lines induced by the small oncogenic viruses, including the high-risk human papillomaviruses, hepatitis B virus, simian virus 40, and human T-cell leukemia virus type 1. We show that viral integrations in tumor cells lie near cellular sequences identified as nuclear matrix attachment regions (MARs), while integrations in nonneoplastic cells show no significant correlation with these regions. In mammalian cells, the nuclear matrix functions in gene expression and DNA replication. MARs play varied but poorly understood roles in eukaryotic gene expression. Our results suggest that integrated tumor virus genomes are subject to MAR-mediated transcriptional regulation, providing insight into mechanisms of viral carcinogenesis. Furthermore, the viral oncoproteins serve as invaluable tools for the study of mechanisms controlling cellular growth. Similarly, our demonstration that integrated viral genomes may be subject to MAR-mediated transcriptional effects should facilitate elucidation of fundamental mechanisms regulating eukaryotic gene expression.

Identification and characterization of functional angiotensin II type 1 receptors on immortalized human fetal aortic vascular smooth muscle cells.

Studies investigating the mechanisms that govern the expression of the human angiotensin II type 1 receptor (hAT(1)R) gene have progressed slowly due to the lack of human cell lines that express the AT(1)R. Recently, however, an immortalized human fetal aortic vascular smooth muscle cell line (FLTR) was generated using an amphotropic recombinant retroviral construct containing the E6/E7 open reading frames of the human papillomavirus type 16. Radioligand binding studies were undertaken to determine whether angiotensin II (Ang II) receptors were expressed on these cells. FLTR cell membranes were shown to express high-affinity Ang II receptors having a B(max) value of 324+/-43 fmol/mg protein and a K(d) of 0.36+/-0.1 nM. In both membranes and intact cells, Ang II, Ang III and the selective AT(1)R antagonist, Losartan, all had a high affinity for the receptor, suggesting that FLTR cells express the AT(1)R subtype. The expression of the hAT(1)R was validated by Northern and Western blot and RT-PCR experiments. In intact FLTR cells, Ang II (100 nM) evoked an increase in intracellular calcium ([Ca(2+)](i)) and induced hyperplasia. Additionally, our results demonstrated that FLTR cells were readily transfected, and hAT(1)R promoter luciferase constructs exhibited robust promoter activity (i.e. approximately 22-fold increase over pGL3-Basic only). Finally, our results demonstrated that the hAT(1)R gene is differentially regulated in FLTR cells vs. H295-R cells, a human adrenocarcinoma cell line that also abundantly expresses the AT(1)R. Taken together, our results suggest that FLTR cells express functional AT(1)Rs and will provide an excellent model system in which to investigate hAT(1)R gene regulation.

Characterization of NR13-related human cell death regulator, Boo/Diva, in normal and cancer tissues.

Mouse Boo/Diva is an ovary-specific member of the Bcl-2 family identified through homology with the avian cell death antagonist NR13. We identified a human orthologue of Boo/Diva, which is highly conserved between mouse and human and related to avian NR13. Human Boo/Diva is also expressed in human liver and kidney in addition to the ovary. We found that green fluorescence protein (EGFP)-tagged Boo/Diva was not exclusively localized to mitochondria before the induction of apoptosis. However, EGFP-Boo/Diva translocated to mitochondria in the process of apoptosis induced by vincristine, a microtubule-interfering agent. Overexpression of human Boo/Diva promoted cell death in HeLa and 293 cells. The cell death antagonist Bcl-XL interacts with Boo, but is unable to protect 293 cells from Boo/Diva-induced cell death. Finally, we mapped human Boo/Diva to chromosome 15q21, a locus known to be related to human cervical cancer. Moreover, we found that genomic DNAs of three of 24 human cervical cancer samples display deletions within their Boo/Diva genes. This result suggests a role for human Boo/Diva in the pathogenesis of cervical cancer.

Expression of truncated FHIT transcripts in cervical cancers and in normal human cells.

To analyse FHIT transcription patterns in cervical cancer, a series of primary cervical tumors and normal control samples were studied using RT-PCR. Full length and truncated FHIT transcripts were detectable in all samples tested. Interestingly, the expression of truncated FHIT transcripts by primary epithelial cells in vitro was associated with confluency. The breakpoints of most transcript deletions coincided with genuine splice site sequences, suggesting that they resulted from alternative splicing. These findings demonstrate that truncated FHIT transcripts are commonly detected in both normal and tumor tissues, and suggest that these altered transcripts are not causally related to tumorigenesis in cervical cancer.

Human papillomavirus infection and survival in oral squamous cell cancer: a population-based study.

OBJECTIVE: To determine whether human papillomavirus (HPV) type 16 affects survival in oral squamous cell carcinoma. STUDY DESIGN: Two hundred fifty-four patients diagnosed with primary oral cancer were studied for survival in relation to tumor HPV type 16 status. Kaplan-Meier analysis and Cox proportional hazard models were used to assess survival and estimate hazard ratios adjusted for potential confounders. RESULTS: HPV type 16 DNA was detected in 15.1% of tumors. HPV 16 positive patients had significantly reduced all-cause mortality (hazard ratio [HR] estimates = 0.34, 95% CI = 0.14, 0.83) and disease-specific mortality (HR = 0.17, 95% CI = 0.04, 0.76) compared with HPV 16 negative patients after adjustment for age, stage, treatment, smoking, alcohol, education, and comorbid disease. CONCLUSIONS: The presence of HPV type 16 DNA is independently associated with a favorable prognosis in patients with oral squamous cell carcinoma. CLINICAL SIGNIFICANCE: Although HPV genotyping is currently not widely available, it may provide important prognostic information.

Human papillomavirus and prognosis of invasive cervical cancer: a population-based study.

PURPOSE: To determine the association between human papillomavirus (HPV) type and prognosis of patients with invasive cervical carcinoma. PATIENTS AND METHODS: Patients diagnosed with International Federation of Gynecology and Obstetrics (FIGO) stage IB to IV cervical cancer between 1986 and 1997 while residents of three Washington State counties were included (n = 399). HPV typing was performed on paraffin-embedded tumor tissue using polymerase chain reaction methods. Patients were observed for a median of 50.8 months. Total mortality (TM) and cervical cancer-specific mortality (CCSM) were determined. Hazards ratios (HR) adjusted for age, stage, and histologic type were estimated using multivariable models. RESULTS: Eighty-six patients had HPV 18-related tumors and 210 patients had HPV 16-related tumors. Cumulative TM among patients with HPV 18-related tumors and among patients with HPV 16-related tumors were 33.7% and 27.6%, respectively; cumulative CCSM in these two groups were 26.7% and 18.1%, respectively. Compared with patients with HPV 16-related cancers, patients with HPV 18-related cancers were at increased risk for TM (HR(TM), 2.2; 95% confidence interval [CI], 1.3 to 3.6) and CCSM (HR(CCSM), 2.5; 95% CI, 1.4 to 4.4). The HPV18 associations were strongest for patients with FIGO stage IB or IIA disease (HR(TM), 3.1; 95% CI, 2.3 to 4.2; and HR(CCSM), 5.8; 95% CI, 3.9 to 8.7), whereas no associations were observed among patients with FIGO stage IIB to IV disease. Virtually identical associations were found in the subset of patients with squamous cell carcinoma (n = 219). CONCLUSION: HPV 18-related cervical carcinomas, particularly those diagnosed at an early stage, are associated with a poor prognosis. Elucidating the mechanism or mechanisms underlying this association could lead to new treatment approaches for patients with invasive cervical carcinoma.

Human papillomavirus and long-term oral contraceptive use increase the risk of adenocarcinoma in situ of the cervix.

We examined United States Surveillance, Epidemiology, and End Results incidence data and conducted a population-based case-control study to examine the role of human papillomavirus (HPV) and oral contraceptive (OC) use in the etiology of adenocarcinoma in situ of the cervix (ACIS). One hundred and fifty women diagnosed with ACIS and 651 randomly selected control women completed in-person interviews. The presence of HPV DNA in archival ACIS specimens was determined by E6 and L1 consensus PCR. Serum samples from case and control subjects were collected at interview, and antibodies to HPV-16 L1 and HPV-18 L1 were detected by virus-like particle capture assays. The overall prevalence of HPV DNA was 86.6%, with 39.0% positive for HPV-16 DNA, 52.4% positive for HPV-18 DNA, and 13.4% positive for more than one HPV type. The age-adjusted relative risk of ACIS associated with HPV-18 seropositivity was 3.3 (95% confidence interval 2.2-4.9). No increased risk was associated with antibodies to HPV-16 L1. Among women born after 1945, the relative risk increased with duration of OC use, with the highest risk for 12 or more years of use (odds ratio, 5.5; 95% confidence interval, 2.1-14.6) relative to nonusers. The detection of HPV DNA in 86.6% of ACIS and the strong association of ACIS with HPV-18 L1 seropositivity underscore the importance of HPV, particularly HPV-18, in the etiology of ACIS. In addition, long-term OC use may contribute to the pathogenesis of these tumors in some women.

Human papillomavirus 16 and 18 L1 serology compared across anogenital cancer sites.

Human papillomavirus (HPV) DNA has been detected in the great majority of cancers of the uterine cervix and anus, whereas the association of HPV DNA with cancer at other anogenital sites has produced less consistent results. This study was designed to compare HPV exposure among anogenital cancer cases and matched controls. Cases (1782) of anogenital cancer diagnosed in the Seattle area from 1978 to 1998 were identified and interviewed. Their responses were compared with those of 2383 age- and sex-matched controls. Blood was drawn at interview from both cases and controls and tested for antibodies to HPV-16 and HPV-18. Tissue blocks were tested for HPV DNA for 649 cases. Serum antibodies to HPV-16 were associated with in situ and invasive cancer at all sites among men and women with the exception of in situ penile cancer. Anti-HPV-18 antibodies were associated with cancers at all sites among women. The increased risk of cancer associated with HPV-16 seropositivity ranged from odds ratio = 1.8 (95% confidence interval, 1.4-2.5) for adenocarcinoma of the cervix to odds ratio = 5.9 (95% confidence interval, 3.4-10.3) for anal cancer in men. Associations between seroprevalence and cancers were stronger when analyses were restricted to HPV-16- or HPV-18 DNA-positive cases. HPV DNA was detected in >80% of cancers from all sites tested. HPV-16 DNA was the type most frequently detected at all sites (range, 40.9-82.2%). HPV-18 DNA was detected in 44.7% of adenocarcinomas of the cervix but detected much less often (2.6-18.1%) at other sites. These findings support an important role for HPV infection in anogenital cancer at all sites. Differences in the proportion of seropositives among HPV-16 DNA-positive cases by site suggest either that the immune response varies by site or that cancer development may lead to changes in antibody responses in a site-specific fashion.

Telomerase activity and cellular immortalization.

Normal human cells undergo a limited number of divisions and eventually undergo senescence. This is accompanied by several changes, including increases in inhibitors of cyclin-dependent kinases and shortening of telomeres. Expression of the catalytic component of telomerase, hTERT, significantly extends the lifespan of human fibroblasts but is not sufficient for the immortalization of human keratinocyte or breast epithelial cells. However, inactivation of the Rb/p16ink4a pathway, or down-regulation of p16ink4a expression in combination with hTERT, is capable of immortalizing human epithelial cells efficiently. Elimination of p53, and of the DNA-damage-induced checkpoint is not required for immortalization, nor is a consistent loss of p19ARF.

"Hit and run" transformation leading to carcinogenesis.

Subgenomic fragments of herpes simplex virus and cytomegalovirus have been shown to transform rodent cells to a neoplastic phenotype in vitro. The transfected DNA does not persist long term in the transformed cells, and viral proteins, although transiently expressed, cannot be detected in the established cell lines. There is evidence that the transforming DNA fragments have mutagenic properties. It has not been established that the effects found in rodent cells can be observed in human cells. Although, the concept of "hit-and-run" transformation has been controversial for many years, it remains the only plausible explanation for the observations of neoplastic transformation, following in vitro transfection of herpesvirus and cytomegalovirus DNAs, which have been made by multiple laboratories over more than two decades.

Genetic and epigenetic changes in human epithelial cells immortalized by telomerase.

Exogenous expression of hTERT, the catalytic component of telomerase, is sufficient for the immortalization of human fibroblasts but insufficient for the immortalization of human foreskin keratinocytes (HFKs) and human mammary epithelial cells (HMECs). These latter cell types can overcome senescence by coexpression of hTERT and human papillomavirus (HPV) E7 or by expression of hTERT and loss of p16(INK4a) expression, indicating that the retinoblastoma (Rb) pathway, along with a telomere maintenance pathway, plays a role in determining the life span of epithelial cells. In this study, we further characterize hTERT-immortalized HFKs and human adenoid epithelial cells (HAKs) for genotypic and phenotypic alterations that are associated with immortalization. Of five hTERT-immortalized HFK and HAK cell lines examined, four exhibited repression of p16(INK4a) expression by promoter methylation or specific large-scale deletion of chromosome 9p, the location of p16(INK4a). Interestingly, one cell line exhibited complete down-regulation of expression of p14(ARF), with only slight down-regulation of expression of p16(INK4a). Yet, all of the immortal cells lines exhibited hyperphosphorylated Rb. Cytogenetic analysis revealed clonal chromosome aberrations in three of the five cell lines. All of the cell lines retained a growth block response with the expression of mutant ras. When grown on organotypic raft cultures, however, the hTERT-immortalized cells exhibited a maturation delay on terminal differentiation. Our results indicate that immortalization of epithelial cells may require both activation of telomerase and other genetic and/or epigenetic alterations that abrogate normal differentiation.

The p53 Arg72Pro polymorphism, human papillomavirus, and invasive squamous cell cervical cancer.

A. Storey et al. [Nature (Lond.), 393: 229-234, 1998)] reported a 7-fold increased risk of cervical cancer associated with having an Arg/Arg polymorphism at codon 72 of p53 compared with the Pro/Arg heterozygotes (odds ratio, 7.4; 95% confidence interval, 2.1-29.4). Complementary in vitro studies suggested that the HPV E6 oncoprotein more readily targets the arginine form, as opposed to the proline form, of p53 for degradation. We investigated the impact of this polymorphism in a population-based case-control study of invasive cervical cancer. Using a PCR assay to detect the p53 codon 72 polymorphism, we tested blood samples from 111 women with invasive squamous cell cancer of the cervix identified by a population-based registry and 164 random-digit telephone-dialed controls. The distribution of the genotype among control women was 38% heterozygous, 7% proline homozygous, and 55% arginine homozygous, and among the cases was 38%, 6%, and 56%, respectively. There was no increased risk of squamous cell invasive cervical cancer associated with homozygosity for the arginine allele (odds ratio, 1.0; 95% confidence interval, 0.6-1.7). Furthermore, there was no modification of this result by human papillomavirus (HPV) DNA status of the tumor, age, or smoking status. Among controls, there was no association between the polymorphism and HPV-16 L1 seropositivity. However, among case subjects, the codon 72 polymorphism may be related to HPV 16L1 seropositivity status.

Genomic changes and HPV type in cervical carcinoma.

To identify chromosomal regions that may include the loci of abnormally expressed cellular genes and may be specifically altered depending on the histological subtype of the tumor, we studied primary cervical carcinoma using CGH and HPV genotyping. Eighty-seven percent of the primary tumors were positive for DNA of a "high-risk" HPV type (e.g., 16 or 18). In the cervical carcinomas, without reference to histologic subtype, overrepresentation of chromosome 3q was the most consistent chromosomal aberration with underrepresentation of chromosome 3p also a frequent finding. Chromosome arms 1q, 5p, 20q, and Xq were overrepresented in many tumors and 3p loss and 5p, 8q, and 16q gain were only associated with squamous cell carcinoma in this series.

Expression of Epstein-Barr virus latent membrane proteins leads to changes in keratinocyte cell adhesion.

Epstein-Barr virus (EBV) has 3 latent membrane proteins (LMPs)--LMP1, LMP2a, and LMP2b--which are expressed in nasopharyngeal carcinoma. Using keratinocyte cell lines expressing LMP2a and LMP2b and coexpressing LMP1/LMP2a, we grew organotypic raft cultures to analyze changes in morphology and expression of the cell adhesion molecule ICAM-1; alpha2, alpha3, alpha5, beta1, and alpha6beta4 integrins; laminin 5; E-cadherin; and desmoplakin. Cells expressing LMP2a or LMP2b were defective in their ability to mature and progress through normal squamous stratification when compared to the parental cell lines. Cells coexpressing LMP1/LMP2a additionally demonstrated "pseudoinvasion" into the raft dermal equivalent. There was a consistent and dramatic up-regulation in the suprabasal expression of laminin 5 and alpha6beta4 and beta1 integrins in the LMP-expressing cell lines. ICAM-1, not expressed in the control cell lines, was up-regulated in the LMP-expressing cell lines. Expression of alpha3 and alpha5 integrins was also up-regulated in the LMP-expressing cell lines, while alpha2 demonstrated a loss of the normal basal layer expression. E-cadherin and desmoplakin expression patterns were essentially unchanged. We conclude that LMP2a and LMP2b singly, and LMP1/LMP2a coexpressed, are capable of altering keratinocyte cell adhesion molecule expression consistent with nasopharyngeal carcinoma.

Generation and characterization of human smooth muscle cell lines derived from atherosclerotic plaque.

The study of atherogenesis in humans has been restricted by the limited availability and brief in vitro life span of plaque smooth muscle cells (SMCs). We describe plaque SMC lines with extended life spans generated by the expression of the human papillomavirus (HPV)-16 E6 and E7 genes, which has been shown to extend the life span of normal adult human aortic SMCs. Resulting cell lines (pdSMC1A and 2) demonstrated at least 10-fold increases in life span; pdSMC1A became immortal. The SMC identity of both pdSMC lines was confirmed by SM22 mRNA expression. pdSMC2 were generally diploid but with various structural and numerical alterations; pdSMC1A demonstrated several chromosomal abnormalities, most commonly -Y, +7, -13, anomalies previously reported in both primary pdSMCs and atherosclerotic tissue. Confluent pdSMC2 appeared grossly similar to HPV-16 E6/E7-expressing normal adult aortic SMCs (AASMCs), exhibiting typical SMC morphology/growth patterns; pdSMC1A displayed irregular cell shape/organization with numerous mitotic figures. Dedifferentiation to a synthetic/proliferative phenotype has been hypothesized as a critical step in atherogenesis, because rat neonatal SMCs and adult intimal SMCs exhibit similar gene expression patterns. To confirm that our pdSMC lines likewise express this apparent plaque phenotype, osteopontin, platelet-derived growth factor B, and elastin mRNA levels were determined in pdSMC1A, pdSMC2, and AASMCs. However, no significant increases in osteopontin or platelet-derived growth factor B expression levels were observed in either pdSMC compared with AASMCs. pdSMC2 alone expressed high levels of elastin mRNA. Lower levels of SM22 mRNA in pdSMC1A suggested greater dedifferentiation and/or additional population doublings in pdSMC1A relative to pdSMC2. Both pdSMC lines (particularly 1A) demonstrated high message levels for matrix Gla protein, previously reported to be highly expressed by human neointimal SMCs in vitro. These results describe 2 novel plaque cell lines exhibiting various features of plaque SMC biology; pdSMC2 may represent an earlier plaque SMC phenotype, whereas pdSMC1A may be representative of cells comprising an advanced atherosclerotic lesion.

Oral cancer risk in relation to sexual history and evidence of human papillomavirus infection.

BACKGROUND: Experimental models and analyses of human tumors suggest that oncogenic, sexually transmittable human papillomaviruses (HPVs) are etiologic factors in the development of oral squamous cell carcinoma (SCC). We conducted a population-based, case-control study to determine whether the risk of this cancer is related to HPV infection and sexual history factors. METHODS: Case subjects (n = 284) were 18-65-year-old residents of three counties in western Washington State who were newly diagnosed with oral SCC from 1990 through 1995. Control subjects (n = 477) similar in age and sex were selected from the general population. Serum samples were tested for HPV type 16 capsid antibodies. Exfoliated oral tissue collected from case and control subjects and tumor tissue from case subjects were tested for HPV DNA. Odds ratios (ORs) were calculated after adjusting for age, sex, cigarette smoking, and alcohol consumption. RESULTS: Among males only, oral SCC risk increased with self-reported decreasing age at first intercourse, increasing number of sex partners, and a history of genital warts. Approximately 26% of the tumors in case subjects contained HPV DNA; 16.5% of the tumors contained HPV type 16 DNA. The prevalence of oncogenic HPV types in exfoliated oral tissue was similar in case and control subjects. The ORs for HPV type 16 capsid seropositivity were 2.3 (95% confidence interval [CI] = 1.6-3.3) for all oral SCCs and 6.8 (95% CI = 3.0-15.2) for oral SCCs containing HPV type 16 DNA. The joint association of cigarette smoking and HPV type 16 capsid seropositivity with oral SCC (OR = 8.5; 95% CI = 5.1-14.4) was stronger than predicted from the sum of individual associations with current smoking (OR = 3.2; 95% CI = 2.0-5.2) and seropositivity (OR = 1.7; 95% CI = 1.1-2.6). CONCLUSIONS: HPV type 16 infection may contribute to the development of a small proportion of oral SCCs in this population, most likely in combination with cigarette smoking.

Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells.

Normal human cells undergo a limited number of divisions in culture and enter a non-dividing state called replicative senescence. Senescence is accompanied by several changes, including an increase in inhibitors of cyclin-dependent kinases and telomere shortening. The mechanisms by which viral oncogenes reverse these processes are not fully understood, although a general requirement for oncoproteins such as human papillomavirus E6 and E7 has suggested that the p53 and Rb pathways are targeted. Expression of the catalytic component of telomerase, hTERT, alone significantly extends the lifespan of human fibroblasts. Here we show that telomerase activity is not sufficient for immortalization of human keratinocyte or mammary epithelial cells: we find that neither addition of hTERT nor induction of telomerase activity by E6, both of which are active in maintaining telomere length, results in immortalization. Inactivation of the Rb/p16 pathway by E7 or downregulation of p16 expression, in combination with telomerase activity, however, is able to immortalize epithelial cells efficiently. Elimination of p53 and of the DNA-damage-induced G1 checkpoint is not necessary for immortalization, neither is elimination of p19ARF.

Telomerase activity and expression of telomerase RNA component and telomerase catalytic subunit gene in cervical cancer.

Telomerase, a ribonucleoprotein complex that includes the telomerase RNA component (hTR) and the telomerase catalytic subunit gene (hTERT) product, has been shown to be activated in the majority of cancer tissues and immortalized cells. To study telomerase activation during the progression of cervical cancer, the expression of hTR and hTERT RNAs in tissues of various stages of cervical cancer was analyzed using the in situ hybridization method and compared with proliferative activity as estimated by Ki-67 immunostaining. To test whether expression of these components is reflected in enzyme activity, we determined the levels of the RNAs in cervical cancer and normal tissues and in primary and immortal keratinocytes by reverse transcription-polymerase chain reaction and RNase protection assays and compared the results to telomerase activities as detected by telomeric repeat amplification protocol assay. In situ hybridization signals of hTR and hTERT were present not only in carcinoma tissues but also in normal epidermal layers. In many adenocarcinoma and fewer squamous cell carcinoma tissues, both signals were focally increased where high proliferative activity was present at the stages of dysplasia/metaplasia, in situ carcinoma, and invasive carcinoma. The level of bTERT, as quantitated by RNase protection assay, was not different between cancer and control tissues or immortal and a subset of primary keratinocytes and did not correlate with telomerase activity. These results indicate that expression of hTR and bTERT is up-regulated in at least a subset of neoplastic cells at an early stage of carcinogenesis and that unidentified factors, such as the modulation or coordination of its protein level with other products, may contribute to the activation of telomerase in cervical cancer.

Human cytomegalovirus IE2 86-kilodalton protein binds p53 but does not abrogate G1 checkpoint function.

Physical interactions between human cytomegalovirus (HCMV) immediate-early (IE) proteins and key cell cycle regulatory proteins have been suggested as a mechanism whereby this herpesvirus modifies cellular control of proliferation. Observed similarities to interactions of other DNA virus proteins (human papillomavirus type 16 E6 and E7, simian virus 40 large T antigen, and adenovirus type 5 E1A and E1B) with cell cycle modulatory proteins such as p53 and Rb have suggested that HCMV IE proteins may likewise alter the G1-to-S phase transition. The IE2 region gene product IE86 has been shown to specifically bind p53, potentially modifying p53 G1 checkpoint function. To examine this possibility, p53-mediated G1 arrest in the presence of IE86 was assessed. Retroviral constructs were created to facilitate the stable expression of IE86 and IE72, another IE protein implicated in HCMV-mediated alteration of cell cycle progression. Western analysis and immunoprecipitation confirmed IE protein expression and binding of IE86 to p53, respectively. Chloramphenicol acetyltransferase assays examining the ability of IE86 to repress activity from the HCMV major IE promoter or activate the HCMV early promoter for the 2.2-kb class of RNAs demonstrated the functional integrity of the IE86 protein. Induction of DNA damage in normal, uninfected fibroblasts (FB) or FB expressing IE86 by actinomycin D (Act D) resulted in increased p53 levels, a predominance of the hypophosphorylated form of Rb, and increased expression of both p21(CIP1/WAF1) and mdm-2. Fluorescence-activated cell sorting revealed that both uninfected and IE86-expressing FB experienced dramatic G1 arrest following exposure to Act D. The clear demonstration of these p53-dependent responses in the presence of IE86 indicates that binding to this viral protein does not compromise the ability of p53 to elicit growth arrest following DNA damage.