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.     

Characterization of events during the late stages of HPV16 infection in vivo using high-affinity synthetic Fabs to E4

HPV late gene expression is initiated as an infected basal cell migrates through the differentiating layers of the epidermis, resulting in the onset of vegetative viral DNA replication and the expression of viral late proteins. We have used a large synthetic immunoglobulin library displayed on phage (diversity 6.5 x 10(10) phage) to isolate three Fabs (TVG405, 406, and 407) which recognize distinct epitopes on the E4 late protein of HPV16. A C-terminal monoclonal (TVG404) was generated by hybridoma technology, and N-terminal polyclonal antiserum was prepared by peptide immunization (alpha N-term). The most potent antibody (TVG405) had an affinity for E4 of approximately 1.0 nM. All antibodies recognized the protein in paraffin-embedded archival material, allowing us to map events in the late stages of virus infection. Expression of E4 in vivo does not coincide with synthesis of the major virus coat protein L1, but precedes it by 1 or 2 cell layers in premalignant lesions caused by HPV16 and by up to 20 cell layers in HPV63-induced warts. In higher grade lesions associated with HPV16, E4 is produced in the absence of L1. By contrast, vegetative viral DNA replication and E4 expression correlate exactly and in some lesions begin as the infected epithelial cell leaves the basal layer. Differentiation markers such as filaggrin, loricrin, and certain keratins are not detectable in E4-positive cells, and nuclear degeneration is delayed. HPV16 E4 has a filamentous distribution in the lower epithelial layers, but associates with solitary perinuclear structures in more differentiated cells. Antibodies to the N-terminus of the protein stained these structures poorly. Our findings are compatible with a role for the HPV16 E4 protein in vegetative DNA replication or in modifying the phenotype of the infected cell to favor virus synthesis or virus release. The Fabs will be of value in the evaluation of model systems for mimicking HPV infection in vitro.     

Transcriptional regulation of the EGF receptor promoter by HPV16 and retinoic acid in human ectocervical epithelial cells

We have previously demonstrated that human papillomavirus 16 (HPV16)-immortalized human ectocervical epithelial cells and cells derived from tumors which express HPV16 oncogenes express high levels of epidermal growth factor receptor (EGFR) compared to normal cervical cells. We have also shown that proliferation of these cells is inhibited by retinoid treatment. We have hypothesized that the retinoid inhibition of cell proliferation may be due to the retinoid-dependent reduction in EGFR level. In this study we examine the regulation of EGFR expression in cervical cells with emphasis on two aspects: (1) the mechanism of retinoid-dependent suppression of EGFR levels in HPV16-positive cells and (2) the mechanism of EGFR upregulation by HPV16. EGFR levels were found to be elevated 5-, 3. 7-, and 1.25-fold in the HPV16-immortalized ECE16-1, ECE16-D1, and ECE16-D2 cells, respectively, compared to normal cervical cells. Treatment of ECE16-1 and ECE16-D1 cells with retinoic acid suppresses proliferation, EGFR level, EGFR mRNA level, and EGFR promoter activity. The reduction in EGFR promoter activity appears to account for the reduction in EGFR protein and mRNA levels. In contrast, retinoic acid does not affect cell growth or EGFR level in ECE16-D2 cells or normal cervical cells. To study the mechanisms regulating EGFR expression in HPV16-positive cells, normal ECE cells were cotransfected with an EGFR promoter reporter plasmid and an expression plasmid encoding the HPV16 E6/E7 open reading frames. In the presence of E6/E7, EGFR promoter activity was increased by 2- to 3-fold, suggesting that the E6/E7 proteins are directly or indirectly responsible for the increased EGFR level and that the EGFR promoter contains the DNA elements necessary to mediate this response. Nevertheless expression of E6/E7 proteins did not confer retinoic acid regulation, as EGFR promoter activity remained elevated in normal cells cotransfected with pHPVE6/E7 and treated with retinoic acid. These results suggest that human papillomavirus and retinoic acid regulate EGFR levels by independent effects on the EGFR promoter.     

G-CSF (filgrastim)-stimulated whole blood kept unprocessed at 4 degrees C does support a BEAM-like regimen in bad-risk lymphoma

In most invasive cervical carcinomas, high-risk human papillomavirus (HPV) DNA is integrated into the host genome, while in pre-invasive cervical lesions the viral genome is typically maintained exclusively as an episome. In contrast, integration of low-risk HPV DNA is rare, as is the association of low-risk HPVs with carcinomas. High-risk HPV integration is associated with a selective growth advantage of affected cells, and hence, integration is likely to be an important genetic alteration contributing to cervical tumor progression. Expression of high-risk, but not low-risk, HPV E6 or E7 proteins disrupts the p53-dependent G1 arrest that cells normally display in response to DNA damage. Absence of this cell cycle checkpoint may predispose cells containing high-risk HPVs to genetic instability and to the accumulation of the genetic alterations that appear to be required for HPV-associated cervical tumor progression. We hypothesized that integration of high-risk HPV DNA into the host cell genome may be facilitated by E6- and/or E7-mediated disruption of the normal DNA damage response pathway. To test this hypothesis, we assessed the integration frequency of a reporter plasmid (pHyGal) in RKO cells expressing individual E6 or E7 genes of either high-risk (HPV16) or low-risk (HPV6, HPV11) type viruses. Cells expressing HPV16 E6 or HPV16 E7 exhibited a significantly increased frequency of pHyGal integration in comparison to RKO control cells or cells expressing low-risk HPV E6 or E7. Thus, expression of high-risk, but not low-risk, E6 and E7 proteins increases the frequency of foreign DNA integration into the host genome. These findings suggest that at least some of the difference in oncogenic potential observed between high-risk and low-risk HPV types may be determined by the increased ability of high-risk HPVs to integrate into host DNA.