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Induction of Cell-Cycle Arrest in Cervical Cancer Cells by the Human Immunodeficiency Virus Type 1 Viral Protein R

From the Departments of Obstetrics and Gynecology, Microbiology and Immunology, Biochemistry, and Medicine, University of Rochester Cancer Center, Rochester, New York

Address reprint requests to: Vicente Planelles, PhD, Departments of Medicine and Microbiology and Immunology, University of Rochester Cancer Center, 601 Elmwood Avenue, Box #704, Rochester, NY 14642, E-mail: vicente_planelles{at}urmc.rochester.edu

	Abstract

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Objective: To determine the ability of the human immunodeficiency virus type 1 (HIV-1) gene vpr to induce cell-cycle arrest in cervical cancer cells with or without human papillomavirus (HPV) type 16 E6 or E7 expression.

Methods: High- and low-level expression vectors for vpr (designated pVPR_HIGH and pVPR_LOW, respectively) were used in conjunction with HPV-16 E6 or E7 vectors to transfect HPV-negative C33A cervical cancer cells. Vpr expression vectors encode a cell surface marker gene, murine Thy-1, for specific detection of transfected cells. Dual staining for the surface molecule Thy-1 and DNA content was used to determine cell-cycle profile and G₂-phase arrest.

Results: C33A cells not expressing HPV-16 E6 showed some but not maximal G₂-phase arrest when transfected with pVPR_HIGH alone (43.2% of cells in the phase). Addition of HPV-16 E6 or E6 plus E7 to pVPR_HIGH substantially increased the percentages of cells in the G2 phase (51.3% and 53.0%, respectively). Cotransfection with pVPR_HIGH and HPV-16 E7 did not increase significantly the

percentage of cells in the G₂ phase compared with pVPR_HIGH alone (40.6% versus 43.2%). In transfections involving pVPR_LOW, a slight degree of G₂-phase arrest was observed when Vpr was expressed alone (29.0% of cells in the G₂ phase) or in cotransfection with HPV-16 E7 (33.2% of cells), and G₂-phase arrest was augmented with the addition of HPV-16 E6 (41.7%) or E6 plus E7 (45.7%).

Conclusion: Cervical cancer cells are susceptible to cell-cycle arrest induced by HIV-1 vpr. This effect is exacerbated by coexpression of HPV-16 E6, although E6 alone is incapable of inducing any detectable G₂-phase arrest, suggesting that E6 and VPR share links in cell-cycle signaling pathways.

Infection with high-risk human papillomavirus (HPV) subtypes has been implicated as a risk factor for cervical dysplasia and neoplasia. Human papillomavirus–associated viral oncoproteins E6 and E7 bind to tumor suppressor proteins p53 and retinoblastoma protein, respectively, causing inactivation and subsequent loss of cell-cycle regulation. Human papillomavirus DNA is found in more than 80% of specimens obtained from women with invasive cervical carcinoma, mostly of high-risk subtypes.¹

Investigation of potential anti-HPV therapies has been hindered by difficulty in propagating the virus in vitro.² The focus of much work with HPV has been on the relationship of particular viral genes and their gene products with the cell cycle on the molecular level. Production of an HPV vaccine has been at the forefront of microbiologic research,^3–5 and therapies that target particular oncoproteins have shown promise.⁶ Blocking of E6 and E7 gene expression in established cervical carcinoma cell lines and primary cell cultures has inhibited tumor cell growth effectively.⁶ Selective targeting of HPV gene products is a strategy for dealing with sequelae of HPV infections.

Human immunodeficiency virus type 1 (HIV-1) is the etiologic agent associated with AIDS. The HIV-1 genome contains three structural and six accessory genes.⁷ One of the accessory genes, vpr, encodes a 96–amino acid product (Vpr) that induces cell-cycle arrest in the G₂ phase.^8–11 Vpr disrupts the checkpoint in the progression from the G₂ phase to the M phase and subsequently induces apoptosis.¹² The vpr gene is highly conserved in evolution and has sequence and functional homology with homologous genes in other Lentiviruses, such as simian immunodeficiency virus and HIV type 2.⁸ Vpr is capable of inducing G₂-phase arrest in many transformed cells, and this ability is independent of p53 and retinoblastoma protein status.^13,14

It was shown that Vpr can suppress the growth of several different tumor cell lines, including leukemia-lymphoma, osteosarcoma, colon carcinoma, and cervical carcinoma cell lines. Therefore, it was suggested that Vpr or a derivative might be useful as an antiproliferative agent for the treatment of human tumors.¹³ Before Vpr-based therapies can be designed for treating HPV infection, the mechanism of Vpr action needs to be understood on a molecular level. Human papillomavirus type 16 encodes two genes, E6 and E7, that are well known to affect the function of regulators of cellular proliferation. The objective of the present study was to examine the cell cycle–inhibiting effect of HIV-1 Vpr on cells expressing HPV-16 E6, E7, or both.

	Materials and Methods

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Expression vectors encoding HIV-1 vpr and a surface marker gene, thy-1, enabled specific detection of transfected and untransfected cells. The high-level expression vector, pVPR_HIGH, has been described^8,9 and was referred to previously as BSVprThy. The low-level vpr expression vector, pVPR_LOW, was constructed by inserting an internal ribosome entry site¹⁵ between the vpr and thy-1 open reading frames, to allow expression of both genes with use of a single promoter. Vpr expression from pVPR_LOW was lower than that from pVPR_HIGH and resulted in lower levels of cells in the G₂ phase under conditions in which equal proportions of cells expressed the reporter molecule Thy-1. Human papillomavirus type 16 vectors were described previously¹⁶ and were constructed with use of E6, E7, and E6-E7 coding sequences for HPV-16 cloned into the Bam HI restriction endonuclease site of the simian virus 40–based expression vector pSG5 (Stratagene, La Jolla, CA). Those HPV vectors were used in conjunction with pVPR_HIGH or pVPR_LOW vectors. The vector pVPR_NULL was constructed by deleting vpr from pVPR_HIGH by first synthesizing a linker by annealing two oligonucleotides of the following sequences: 5'-TCGAGATATCGATATAAGCTTCAGGCGCGCCTTAGGCCTGGACGCGTCCAGATCTCCT-3' and 5'-CTAGAGGAGATCTGGACGCGTCCAGGCCTAAGGCGCGCCTGAAGCTTATATCGATATC-3'. The linker then was cloned into pVPR_HIGH between the Xho I and Xba I restriction endonuclease sites, which resulted in excision of the vpr open reading frame.

The HPV-negative cervical carcinoma cell line, C33A,¹⁷ was obtained from the American Type Culture Collection (Manassas, VA) and cultured in Dulbecco’s modified Eagle’s medium (Gibco, Gaithersburg, MD) with 10% fetal calf serum (Omega Scientific, Bedford, OH). Those cells are known to have a mutated p53 phenotype and are retinoblastoma protein–positive.

C33A cells were plated in six-well plates at a density of 2.5 x 10⁵ cells per well and were cultured overnight. Plasmids (2 µg) were mixed with 500 µL of serum-free medium (total volume 0.6 mL) with a 5:1 molar excess of HPV vector. A separate aliquot of 12 µL of lipofectin (GIBCO BRL, Gaithersburg, MD) in 100 µL of serum-free medium was prepared. The DNA and lipofectin solutions were allowed to stand separately at room temperature for 30 minutes and then were mixed, with subsequent incubation at room temperature for 10–15 minutes. C33A cells were washed with 2 mL of serum-free medium per well in preparation for transfection. An additional 0.8 mL of serum-free medium was added to each DNA-lipofectin complex before the complex was transferred to C33A cells. C33A cells were cultured at 37C for 18–24 hours, after which the DNA-lipofectin solution was removed and replaced with normal medium. C33A cells were harvested 48 hours after transfection, for staining and flow cytometry.

Simultaneous staining for surface molecules and DNA content was done as previously described.¹⁸ Briefly, 10⁶ cells were harvested 48 hours after transfection, resuspended in 100 µL of anti–Thy-1 monoclonal antibody conjugated to fluorescein isothiocyanate (Caltag Laboratories, San Francisco, CA), diluted to 1:200 in flow cytometry buffer (phosphate-buffered saline with 2% fetal calf serum and 0.01% sodium azide), and incubated for 20 minutes on ice. Cells were washed once with flow cytometry buffer, fixed in phosphate-buffered saline with 0.3% paraformaldehyde for 1 hour, washed again, and permeabilized in 0.2% Triton X-100 (Sigma Chemical Co., St. Louis, MO) in phosphate-buffered saline for 15 minutes. Cells were washed once more with phosphate-buffered saline, resuspended in flow cytometry buffer containing propidium iodide (10 µg/mL) and DNase-free ribonuclease A (11.25 Kunitz units), and kept on ice until analysis by flow cytometry. At least 5000 events (cells) were collected for flow cytometry.

Flow cytometric analysis was done at the Flow Cytometry Facility, University of Rochester, in an Epics Elite ESP (Coulter Corp., Hialeah, FL). Cell-cycle analysis was done with the use of Multicycle AV software (Phoenix Flow Systems, San Diego, CA). Cell-cycle profiles were generated, with each sample being at least 5000 cells. Statistical significance was tested with ² analysis. ² values were no more than 5.0 for all experiments shown.

	Results

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To determine the effect of Vpr on C33A cervical carcinoma cells, we used pVPR_HIGH to provide optimal expression of vpr. The pVPR_HIGH vector (Figure 1) was used alone to transfect C33A cells then in conjunction with pSG5-E6, pSG5-E7, or pSG5-E6/E7 (Figure 1) to permit study of potential effects of coexpression on cell-cycle profile. Staining for DNA content enabled generation of cell-cycle analyses. Thy-1–positive cells showed successfully transfected cells expressing Vpr or HPV proteins and were gated for cell-cycle analysis.

View larger version (35K): [in this window] [in a new window]   Figure 1. Transcriptional elements of the human immunodeficiency virus type 1 vpr expression vectors (pVPRHIGH, pVPRNULL, and pVPRLOW) are shown. Two separate copies of the human cytomega-lovirus immediate-early promoter (CMV IE) allow for optimal expression of vpr and thy-1. The pVPRLOW vector directs production of a dicistronic messenger RNA by the encephalomyocarditis virus internal ribosome entry site (EMCV-IRES). Expression of HPV genes was achieved with the expression vector pSG5, containing indicated segments of the human papillomavirus type 16 genome. SV40 p(A) and SV40 E are simian virus 40 polyadenylation and early promoter sequences, respectively.

View larger version (34K): [in this window] [in a new window]   Figure 2. Cell-cycle profiles from C33A cells (2.5 x 105 cells) transfected with human immunodeficiency virus type 1 vpr expression vector pVPRHIGH and indicated human papillomavirus genes, after staining and flow cytometry. For simplicity, only data for the genes expressed are shown. DNA contents of nontransfected (Thy-1–negative) and transfected (Thy-1–positive) cells are shown in the lower and upper panels, respectively. % G2 = percentage of cells in the G2 phase.

To determine the effect of HPV-16 proteins on G₂-phase arrest, we used pVPR_NULL alone or with HPV-16 E7, E6, or E6 plus E7. Expression of the thy-1 gene by the pVPR_NULL vector allowed for similar electronic gating of cells into THY-1–positive (transfected) and Thy-1–negative (nontransfected) populations.

Cell-cycle profiles were generated for each of the four cell groups (Figure 3). The pVPR_NULL vector alone was associated with no G₂-phase arrest (26.9% of cells in the G₂ phase). With the addition of HPV-16 E7, the proportion of cells in the G₂ phase decreased (16.0%). A similar cell-cycle profile was generated with the pVPR_NULL and HPV-16 E6 cotransfection (13.5% of cells in the G₂ phase). The final vector combination of pVPR_NULL and HPV-16 E6 plus E7 also was associated with no G₂-phase arrest (21.4% of cells in the G₂ phase).

View larger version (32K): [in this window] [in a new window]   Figure 3. Cell-cycle profiles from C33A cells (2.5 x 105 cells) transfected with human immunodeficiency virus type 1 vpr expression vector pVPRNULL with or without vectors expressing human papillomavirus genes, after staining and flow cytometry. DNA contents of nontransfected (Thy-1–negative) and transfected (Thy-1–positive) cells are shown in the lower and upper panels, respectively. % G2 = percentage of cells in the G2 phase.

	Discussion

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Human immunodeficiency virus type 1 vpr–induced cell-cycle arrest in the G₂ phase has been well characterized in T lymphocytes, and a cervical carcinoma model offers an easily accessible alternative for investigating the growth-arrest potential of vpr. Advances in prevention and treatment of cervical dysplasia and neoplasia most likely will occur within the context of HPV infection. All HPV subtypes express E6 and E7 proteins, which are responsible for immortalization and transformation of infected cells.^19,20 Selective targeting and arrest of E6-expressing cells could prevent transformation of an otherwise benign proliferating cell. We provide evidence that the ability of VPR to arrest cells in the G₂ phase is augmented by expression of HPV-16 E6. Therefore, expression of HPV-16 E6 in cells from cervical dysplasia or neoplasia might render those cells highly susceptible to the cytostatic activity of HIV-1 vpr. Genotoxic agents, agonists of Vpr, might be effective against cervical tumors.

High-level expression of Vpr induces cell-cycle arrest at detectable levels in the G₂ phase. The proportion of cells that are arrested in the G₂ phase increases significantly with the addition of E6. Vpr with HPV-16 E7 has little or no effect on G₂-phase arrest.

Any G₂-phase arrest induced by HPV-16 E6, E7, or E6 plus E7 was measured by cotransfection with a control vector, pVPR_NULL, that does not encode vpr. We conclude that no detectable G2-phase arrest can be attributed solely to HPV genes in the experimental system we describe here. Therefore, the exacerbation of G₂-phase arrest seen with vpr expression vectors in combination with HPV E6 is not due simply to an additive effect.

Low-level vpr expression induces a small degree of G₂-phase arrest, but that effect is enhanced by coexpression of HPV-16 E6. Expression of E7 has little or no effect on the cell-cycle profile of cells that express low levels of Vpr.

The data presented here suggest that vpr-induced cell-cycle arrest might involve cell-cycle regulatory pathways, which might be used therapeutically to interfere with the growth of HPV-infected cells. A low-level vpr expression vector might allow cells that express HPV-16 E6 to arrest in the G₂ phase, with cells not expressing HPV-16 (ie, phenotypically normal cells) remaining unaffected. Therefore, vpr gene delivery could be used to treat HPV-infected dysplastic or neoplastic cells, arresting them in the ^G2 phase, with, perhaps, subsequent apoptosis of those cells.¹² Because normal or HPV-negative cells are spared, successful gene delivery of vpr to all HPV-infected cells could result in eradication of the virus and preclude potential infection sequelae, preventing and treating dysplasia associated with HPV infection.

E6 is known to cause ubiquitin-dependent degradation of p53, which contributes to release of G1/S checkpoint.²¹ The p53 protein in C33A cells is inactive, so any link between Vpr and E6 would be through a p53-independent pathway. That also excludes p53-dependent G₂-phase arrest by 14-3-3 sigma, another recently elucidated cell-cycle inhibitor.²²

Human immunodeficiency virus type 1 vpr and HPV-16 E6 might share a link in the cell cycle that has yet to be fully determined. The simplest concept would be that the two bind to a common protein that regulates cell-cycle progression. The implication of this relationship is that identification of that protein or group of proteins could offer more specific anti-HPV therapy through increased or uninhibited expression of it within the cell-cycle scheme. Identification of the putative protein that interacts with E6 and Vpr will provide further insight into the mechanism of T cell immunosuppression with HIV infection. One could target that protein for anti–HIV therapy and hinder Vpr’s ability to cause G₂-phase arrest in T cells.

Patients infected with HIV have elevated risks of cancer. The pathogenesis of that increase in tumor prevalence appears to be related to the persistence of several herpesviruses and HPV.²³ The relationship between HPV infection and female cervical intraepithelial neoplasia (CIN) has been established. Several studies have found an increased prevalence of cervical HPV infection and CIN among HIV-positive women compared with HIV-negative women. A high recurrence rate of CIN after standard treatment was noted in HIV-infected women, and the severity of those lesions seems to be inversely correlated to immune function.²³ Human immunodeficiency virus infection may influence the pathogenesis of HPV-associated cervical disease by molecular interaction between HIV and HPV genes. Upregulated HPV E6 and E7 genes expressed by HIV proteins (such as Tat) have been shown in vitro.^24,25

Potential molecular interactions between HIV Tat and HPV genes did not affect the experiments we presented here because we focused on the vpr gene. By transfection methods, we have been able to introduce vpr plus E6 or E7 and study their effects on the cell cycle. The effects of coinfection with HIV and HPV on the cell cycle might be different than with isolated genes, and this may warrant further experimentation with viruses instead of expression plasmids.

It is not clear whether HIV and HPV in vivo can infect the same cell type. It appears that target cells for infection by HPV, mucosal and epithelial cells,²⁶ might not overlap with those infected by HIV, monocyte-derived macrophages, including Langerhans’ cells in various epithelia and microglial cells in the brain, and CD4 cells.²⁷ Whether in cervical carcinoma cells or T lymphocytes, cell growth and cell-cycle progression are important foci for gene delivery and manipulation of molecular biology. Patients with AIDS are known to be at a higher risk development and progression of cervical dysplasia, yet vpr is able to arrest cells, including cervical cells, and to inhibit their cell cycles. Also, HPV-16 E6 causes loss of cell-cycle inhibition by inactivating p53 yet seems to act with vpr synergistically to enhance cell-cycle arrest.

View larger version (34K): [in this window] [in a new window]   Figure 4. Cell-cycle profiles from C33A cells (2.5 x 105 cells) transfected with the low-level vpr expression pVPRLOW vector with or without vectors expressing human papillomavirus genes. DNA contents of nontransfected (Thy-1–negative) and transfected (Thy-1–positive) cells are shown in the lower and upper panels, respectively. % G2 = percentage of cells in the G2 phase.

	Footnotes

This work was supported by National Institutes of Health research grants R29-AI41407 (National Institute of Allergy and Infectious Diseases) (VP) and AI01146 (LR-R).

PII S0029-7844(99)00464-0

Received April 7, 1999. Received in revised form June 8, 1999. Accepted June 24, 1999.

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