Casein kinase II phosphorylation of the human papillomavirus-18 E7 protein is critical for promoting S-phase entry.

The human papillomavirus type 18 E7 protein subverts the pRb/E2F pathway to promote S-phase reentry by postmitotic, differentiated primary human keratinocytes in support of viral DNA amplification. We prepared a panel of HPV-18 E7 mutations in pRb binding or in casein kinase II (CKII) phosphorylation. Our results showed that the ability of E7 binding to pRb correlated with the activation of DNA polymerase alpha or cyclin E to various extents in differentiated keratinocytes of organotypic cultures but was insufficient to induce the proliferating cell nuclear antigen. Proteins mutated in the CKII recognition sequence or in one or both serine substrates (S32 and S34) bound pRb in vitro, but only those with negative charges at these two residues induced proliferating cell nuclear antigen effectively. Nevertheless, unscheduled cellular DNA synthesis occurred very inefficiently relative to the wild-type E7, if at all. Thus, both pRb binding and CKII phosphorylation of E7 are critical for activating cellular genes essential for S-phase entry.

Placebo-controlled trial of indole-3-carbinol in the treatment of CIN.

OBJECTIVE: Most precancerous lesions of the cervix are treated with surgery or ablative therapy. Chemoprevention, using natural and synthetic compounds, may intervene in the early precancerous stages of carcinogenesis and prevent the development of invasive disease. Our trial used indole-3-carbinol (I-3-C) administered orally to treat women with CIN as a therapeutic for cervical CIN. METHODS: Thirty patients with biopsy proven CIN II-III were randomized to receive placebo or 200, or 400 mg/day I-3-C administered orally for 12 weeks. If persistent CIN was diagnosed by cervical biopsy at the end of the trial, loop electrocautery excision procedure of the transformation zone was performed. HPV status was assessed in all patients. RESULTS: None (0 of 10) of the patients in the placebo group had complete regression of CIN. In contrast 4 of 8 patients in the 200 mg/day arm and 4 of 9 patients in the 400 mg/day arm had complete regression based on their 12-week biopsy. This protective effect of I-3-C is shown by a relative risk (RR) of 0.50 ((95% CI, 0. 25 to 0.99) P = 0.023) for the 200 mg/day group and a RR of 0.55 ((95% CI, 0.31 to 0.99) P = 0.032) for the 400 mg/day group. HPV was detected in 7 of 10 placebo patients, in 7 of 8 in the 200 mg/day group, and in 8 of 9 in the 400 mg/day group. CONCLUSIONS: There was a statistically significant regression of CIN in patients treated with I-3-C orally compared with placebo. The 2/16 alpha-hydroxyestrone ratio changed in a dose-dependent fashion.

Presence of a 5-HT7 receptor positively coupled to adenylate cyclase activation in human granulosa-lutein cells.

Although serotonin (5-HT) has been shown to stimulate progesterone production by human granulosa-lutein cells (hGLC), the receptor type and associated signaling pathway remain uncharacterized. We report here that 5-HT receptors in these cells are positively coupled to adenylate cyclase activity. Formation of cAMP was stimulated by 5-HT and its agonists in a dose- and time-dependent manner. Mianserin, amoxapine, and loxapine were equipotent in antagonizing 5-HT-induced cAMP formation. For both cAMP formation in cells and adenylate cyclase assay using membrane fractions, the rank order of potency for agonists of 5-HT were: 5-carboxy-aminotryptamine >5-HT> or =5-methoxytryptamine, consistent with a typical pharmacological profile of human 5-ht7 (h5-ht7) receptor. Sequence data of amplified complementary DNA fragments reverse transcribed from hGLC RNA revealed complete identity with published sequence of h5-ht7 receptor complementary DNA. Northern analysis showed the presence of 2.8-kb h5-ht7 transcripts in hGLC. The three variants h5-ht7A, h5-ht7B, and h5-ht7D were also detected in hGLC. Preincubation of hGLC with 5-HT (10(-8)-10(-6) M) resulted in a marked reduction in the cAMP response when the cells were subsequently stimulated with gonadotropin, and this heterologous desensitization could be reversed by 5-ht7 receptor antagonist clozapine. These data demonstrate that h5-ht7 receptor is present and stimulate cAMP formation in hGLC. In addition, the h5-ht7 receptor seems to be implicated in the heterologous down-regulation hCG-stimulated cAMP response in hGLC, with a possible ramification for luteal insufficiency.

HIV+ patients have increased lymphocyte infiltrates in CIN lesions.

OBJECTIVE: The objective of our study was to analyze immunocyte infiltrates in CIN lesions from HIV+ patients to assess whether local immunosuppression, defined as a decrease in T cell infiltrates, could explain the aggressive nature of CIN in HIV-infected patients. MATERIALS AND METHODS: Cervical tissue was obtained from 6 HIV+ CIN patients, 6 HIV- CIN patients, who underwent LLETZ (large loop excision of the transformation zone) for CIN, and 17 normal patients who underwent hysterectomy for benign indications. The following cell surface markers were analyzed: CD20 (B cells), CD4 (T helper cells), and CD8 (T suppressor/cytotoxic cells). Each tissue section was visualized with a Leica microscope at 400x and the image was captured for analysis by Harmony Group image analysis software. RESULTS: A significantly higher number of lymphocytes (both B and T cells) was detected in the stroma of HIV+/CIN tissue compared to either HIV-/CIN or normal tissue. There was also a significant increase in CD8+ cells in the HIV+/CIN group compared to HIV-/CIN or normal tissue. There was a trend toward a decreased CD4+/CD8+ ratio in the HIV+/CIN compared to the other two groups; however, this did not reach statistical significance. CONCLUSIONS: This study indicates that HIV+/CIN cervical tissue has a greater number of tissue lymphocytes recruited to the neoplastic site compared to HIV- individuals. In addition, HIV+ patients may have a decreased CD4/CD8 ratio in locally infiltrating immunocytes in CIN lesions. The local immunomodulatory effects of HIV may be detectable early in infection and therefore may explain the aggressive nature of CIN in the HIV+ patient.

Post-transcriptional induction of p21cip1 protein by human papillomavirus E7 inhibits unscheduled DNA synthesis reactivated in differentiated keratinocytes.

Productive infection by human papillomaviruses (HPVs) occurs only in differentiated squamous epithelial cells in papillomas, condylomata, and low grade intraepithelial neoplasias. Host DNA replication is reactivated in a fraction of terminally differentiated keratinocytes in benign human lesions and in organotypic raft cultures of primary human keratinocytes (PHKs) transduced with retroviruses expressing HPV-18 E7 oncogene from its native upstream regulatory region (URR). Thus the natural function of E7 protein, which inactivates pRB family proteins, is to induce host genes essential to support viral DNA replication in post-mitotic cells. Using this raft culture model system, we show that HPV-18 URR-E7 induces the universal cyclin-dependent kinase inhibitor p21cip1 protein in a fraction of differentiated PHKs. Induction is mediated by posttranscriptional mechanisms independent of p53. Double immunofluorescence studies demonstrate that, in raft cultures and in laryngeal papillomas, p21cip1 induction and reactivated host DNA synthesis take place in a mutually exclusive manner in PCNA-positive, differentiated keratinocytes. We suggest that p21cip1 induction effectively blocks unscheduled DNA synthesis reactivated by E7. These results begin to explain the inverse relationship between p21cip1 induction and HPV activities previously observed in a spectrum of benign lesions regardless of HPV types present.

Post-transcriptional induction of p21cip1 protein in condylomata and dysplasias is inversely related to human papillomavirus activities.

Infections of the genital and oral epithelia by human papillomaviruses cause condylomata, papillomas, and squamous intraepithelial neoplasms, some of which can progress to invasive cancers. We describe an induction of p21cip1/WAF1/sdi1 protein in a fraction of the spinous cells in benign lesions and in cervical intraepithelial neoplasia grades I and II. The induction appears to be post-transcriptional and independent of p53. p21cip1 antigen-positive cells were sporadic in cervical intraepithelial neoplasia III and rare and focal in carcinomas. In contrast, p21cip1 protein was below or at the threshold of detection in the differentiated cells of normal squamous epithelia from different body sites despite an up-regulation of p21cip1 RNA. In cervical intraepithelial neoplasias from patients who were also positive for the human immunodeficiency virus, there was an additional increase in p21cip1 RNA in the upper spinous cells without concomitant p21cip1 protein induction. A consistent inverse relationship was observed between the p21cip1 protein induction and abundant human papillomavirus DNA and RNAs. We propose that p21cip1 protein induction is a novel host response that inhibits viral DNA replication and thus prevents elevated viral transcription. This hypothesis can partly account for the heterogeneity and the differentiation-dependent viral activities commonly observed in benign human papillomavirus lesions.

A cervical teratoma with invasive squamous cell carcinoma in an HIV-infected patient: a case report.

HIV infection among women is increasing in the United States. Since January 1993, cervical cancer has been considered an AIDS-defining illness. We report a rare case of squamous cell carcinoma arising in a teratoma of the uterine cervix in an HIV-infected patient. This patient was treated with a radical hysterectomy, para-aortic and pelvic lymphadenectomy. She is currently 1 year from treatment and is without evidence of recurrence.

Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes.

mRNA transcription, DNA amplification, and progeny production of human papillomaviruses (HPVs) are closely linked to squamous epithelial differentiation in patient papillomas. Because suprabasal, differentiated keratinocytes have exited the cell cycle for days or weeks and because viral DNA synthesis requires the host DNA replication machinery, HPVs must have a mechanism to reactivate the essential host genes. In this study, we show via acute recombinant retrovirus infection that an intact E7 gene of either high-risk or of low-risk HPV genotypes, under the control of its respective native enhancer-promoter, induced proliferating cell nuclear antigen (PCNAs) expression in the suprabasal cells of epithelial raft cultures of primary human foreskin keratinocytes (PHK). The cellular differentiation program was unaltered by the viral oncoprotein; it was essential for high HPV promoter activity. Furthermore, extensive host chromosomal DNA replication took place in differentiated cells of HPV-18 E7-expressing raft cultures and of patient laryngeal papillomas caused by HPV-6. These results indicate that the main function of the E7 protein is to reactivate host DNA replication machinery to support viral replication in differentiated, noncycling cells.