Effect of Glucocorticoid Hormones on Viral Gene Expression, Growth, and Dysplastic Differentiation in HPV16-Immortalized Ectocervical Cells

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

232, 353–360 (1997)

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Effect of Glucocorticoid Hormones on Viral Gene Expression, Growth, and Dysplastic Differentiation in HPV16-Immortalized Ectocervical Cells Suvarnalatha Khare, Mary M. Pater,1 Shou-Ching Tang, and Alan Pater2 Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John’s, NF Canada A1B 3V6

Steroid hormones are proposed to act as cofactors with human papillomaviruses (HPVs) in the etiology of cervical cancer. We and others reported that progesterone and glucocorticoid hormones induce the expression of HPV16 via three glucocorticoid response elements (GREs) in the viral regulatory region. Consensus GREs (GREcs) are useful in other systems for examining the effect of hormones after enhancing the response with mutated GREc constructs. Therefore, this study used human ectocervical cells (HEC) and HPV type 16 containing three GREcs to establish immortalized cells (HEC-16GREc). Northern blot assays showed that the level of viral E6–E7 oncogene RNA was increased by hormones substantially more in HEC-16GREc than in wild-type HPV16-immortalized human ectocervical cells (HEC-16). The saturation density and the hormone response of the growth rate were significantly higher for HEC-16GREc and the doubling was faster in the presence of hormone than for HEC-16. Although both were nontumorigenic, only HEC-16GREc showed anchorage-independent growth, which was dependent on hormone. Also, HEC-16GREc were more abnormal in their epithelium differentiation pattern in organotypic (raft) cultures. Furthermore, hormone-treated HEC-16GREc rafts showed more dysplastic features than hormone-treated HEC16 rafts. These results suggest new features of the role of hormones: that enhanced expression of viral oncogenes in response to hormones apparently confers a greater risk for cervical cells containing HPV16. Further, HEC-16GREc could be ideal for studying hormone-dependent and -independent malignant transformation. q 1997 Academic Press

INTRODUCTION

The association of certain human papillomaviruses (HPVs) with cancer of the uterine cervix is unequivocally established (zur Hausen and de Villiers, 1994). In addition, prolonged latency between the onset of HPV 1

Mary Pater passed away on November 2, 1994. To whom correspondence and reprint requests should be addressed. Fax: (709) 737-7010; E-mail: [email protected]. 2

infection and malignant conversion implicates cofactors in the malignant transformation of benign lesions. Specifically, the glucocorticoid and progesterone steroid hormones are implicated as an important cofactor with HPV in oncogenesis (Stern et al., 1977; Brinton et al., 1993; reviewed by Khare et al., 1995). Oral contraceptives, which contain progestin and estrogen steroid hormones as active ingredients, are epidemiologically associated with an increased risk for cervical cancer (Negrini et al., 1990; Brinton, 1991; Brinton et al., 1993). In addition, the increased level of progesterone during pregnancy is a significant risk factor for malignant conversion (Ferenczy, 1989; Bokhman and Urmancheyeva, 1989). HPV16 is the most frequent HPV in cancers and the most extensively studied for the effect of hormones. Dexamethasone (dex) glucocorticoid hormone and progesterone induced transformation by HPV16 in in vitro studies. Hormones from oral contraceptives facilitated transformation by HPV16, and transformation by dex was specifically abolished by the RU486 steroid antagonist (Pater et al., 1990; Pater and Pater, 1991). Also, glucocorticoid hormone or progesterone were necessary for the efficient transformation by HPV16 DNA of rodent epithelial cells (Pater et al., 1988; Crook et al., 1988) and human cells (Durst et al., 1989). For the mechanism of action, the regulatory region of HPV16 harbors a glucocorticoid response element (GRE), which mediated induction of expression by hormones (Gloss et al., 1989; Chan et al., 1989). Two additional HPV16 GREs also functioned in hormone-enhanced transformation of rodent epithelial cells (Mittal et al., 1993b). Further, all three GREs probably mediate hormone-induced expression of the HPV E6 and E7 oncogenes from the HPV16 promoter in immortalized cervical cells. A few studies reported the effect of glucocorticoid hormones on HPV16-containing cells. Each of the three GREs in the HPV16 regulatory region was functional in hormone-induced expression in normal human ectocervical cells (HEC) (Mittal et al., 1993a). In SiHa cervical carcinoma cells, the expression of the integrated HPV16 E6 and E7 transforming genes was increased by dex (Chan et al., 1989). Hydrocortisone increased the transformation of keratinocytes by HPV16 (Durst

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et al., 1989). Additional studies suggested that glucocorticoids have a significant role in the pathogenesis of HPV16-infected cells (Jee et al., 1993; Zheng and Vaheri, 1995). However, glucocorticoids also have a significant role, which does not involve HPVs, in the physiology of cervical cells (Reagan and Fu, 1983). In this report, we investigated the direct HPV16 GRE-mediated role of hormones in the growth, dysplastic differentiation, and viral expression of cervical cells. HPV16 GREs were replaced with GRE consensus sequences (GREcs) for examining in immortalized ectocervical cells the role of a high hormone response in cervical oncogenesis. MATERIALS AND METHODS Plasmids, cell culture, and immortalization. GREs were mutated into GREcs by site-directed mutagenesis using the HPV16 nt 6150– 863 sequences as template and the method described previously (Mittal et al., 1993a). The mutations were confirmed by sequencing. To produce pHPV3 1 GREc, the sequences containing the GREc mutations were used to replace the GREs of the wild-type HPV16 plasmid (pHPV[WT]). GRE sequences of both plasmids are shown on the top of Fig. 1. Primary HEC were cultured from cervical specimens that were histologically free of cervical intraepithelial neoplasia (CIN). HEC were initiated, as described previously (Boyce and Ham, 1985). Cells immortalized by HPV16 containing the three GREcs (HEC-16GREc) were established from secondary HEC by transfection with pHPV3 1 GREc (Fig. 1), using previously described methods (Felgner et al., 1987). HEC-16 were established previously (Tsutsumi et al., 1992). Cultures were grown in keratinocyte growth medium (KGM) (Clonetics, San Diego, CA) or in Dulbecco’s modified Eagle’s medium (DMEM) for different assays. When used, the concentration of dex was 1006 M. Cell growth assays. HEC-16GREc and HEC-16 were assayed for growth rate using plates on every second day for 8 days. Doubling time was then determined as the time required for cells to undergo one cell division. To measure cell growth rate, triplicate aliquots of 104 cells grown to 70% confluence were replated onto three 60-mm dishes. Saturation density was measured by growing cells in KGM in triplicate 60-mm dishes to confluence and counting. Cell numbers were counted with a hemocytometer. Southern, spot, and Northern blot assays. HEC-16 and HEC16GREc were grown in KGM. Analyses were of high molecular weight DNA by Southern blot assays, undigested high molecular weight DNA by spot blot assays, and total RNA by Northern blot assays. Whole HPV16 DNA probe was used and methods were as described previously (Sambrook et al., 1989). Organotypic (raft) culture. The protocol was described previously (McCance et al., 1988; Shindoh et al., 1995). In brief, 3 1 105 cervical cells grown in KGM were seeded on collagen gels impregnated with 3T3 J2 fibroblasts and grown in E-medium for 2 days. The confluent monolayer was then rafted at the air/liquid interface to allow epithelial stratification. The medium was changed to fresh medium containing 0 or 1006 M dex on Day 0 and on every second day until Day 12. For histology, Day 12 raft specimens were fixed, paraffinembedded, sectioned, and stained with hematoxylin and eosine (Sun et al., 1992). Soft agar and tumorigenicity assays. Soft agar growth assays for 105 cells per dish in DMEM were as described previously (Tsutsumi et al., 1994). Colony formation was examined for 5–6 weeks. For tumorigenicity assays, HEC-16GREc and HEC-16 grown in DMEM were washed with PBS and 107 cells were injected into each of three

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1-month female nude mice subcutaneously at three sites. These mice were monitored weekly for tumor formation over 8 months.

RESULTS

Immortalization of Ectocervical Cells by HPV16 Containing Triple Consensus GREs Previous studies showed that glucocorticoid hormone and progesterone induced the expression of HPV16 from the HPV16 enhancer-promoter harboring wildtype and triple GREc mutations in HEC cultured from the human ectocervix (Mittal et al., 1993a; Khare et al., 1996). These HEC transfected with HPV DNA resemble the infected lesions containing replicating viral episomal DNA in women. Here, we studied the role of hormones in a model for malignant lesions. Thus, HEC were transfected with the triple GREc-containing whole HPV16 construct, pHPV3 1 GREc (Fig. 1). The untransfected and the control vector-transfected HEC underwent senescence after passage 6. However, the HEC transfected with pHPV3 1 GREc continued to grow until passage 18, at which time they underwent crisis for 4 weeks. However, growth was reinitiated and these cells have been in culture for more than 80 passages. These cells were designated HEC-16GREc. HEC-16 were previously established by immortalization of HEC by HPV16-containing wild-type GREs using pHPV(WT) (Tsutsumi et al., 1992). Effect of Enhanced Hormone Response from GREcs on Morphology in Monolayer Culture and HPV16 DNA Level of Immortalized Cells The morphology in monolayers of cervical cells is an identified indicator of their oncogenicity (Vooijs et al., 1991). The three types of ectocervical cells, normal HEC, HEC-16, and HEC-16GREc, were compared for their morphology in monolayer culture at confluence (Fig. 1A). HEC were composed of keratinocyte-like polygonal cells forming a typical cobblestone monolayer for primary (Fig. 1A(a)) and secondary (Fig. 1A(b)) cultures, as described previously (Tsutsumi et al., 1992). HEC-16 (Fig. 1A(c)) were moderately less pleomorphic than HEC-16GREc (Fig. 1A(d)). Also, most HEC16GREc were larger and flatter than primary and secondary HEC. To examine whether HPV16 DNA was present and to quantify the DNA, spot blot analysis was used (Fig. 1B). HEC-16GREc contained HPV16 DNA and the level was similar to that for HEC-16. Also, both levels were intermediate between those of SiHa, containing one copy of HPV16, and CaSki, containing 600 copies per cell. Based on the results for SiHa, CaSki, and HPV16 DNA spot blots, HEC16GREc contained approximately 100 copies per cell. Effect of Enhanced Hormone Response on HPV16 DNA Status and RNA Expression in Immortalized Cells To reexamine the presence and to examine the physical state of HPV16 DNA, high molecular weight DNA

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FIG. 1. Effect of enhanced hormone response construct on morphology in vitro and HPV16 DNA content of immortalized cells in monolayer cultures. On top are shown the wild-type (WT) and triple consensus GRE sequences in pHPV3 1 GREc in the two respective plasmids. The first nucleotides of each GRE are numbered. The wild-type sequences are shown in capital letters and the consensus mutations created by site-directed mutagenesis are in lower case letters, and the other wild-type nucleotides are shown by dashes. The flanking sequences of the oligonucleotides used for nt 7385 and 7474 GREs are in lower-case italics. These sequences are in whole HPV16 plasmids. (A) Monolayer cultures. The panels are for primary culture of HEC (a), secondary culture of HEC (b), HEC-16 (c), and HEC-16GREc (d). The bar for primary HEC represents 10 mm for all panels. (B) Spot blot analysis of HPV16 DNA. HPV16 DNA (100 pg) and total cellular DNA from the indicated cells (5 mg) was examined, as indicated and described in the text.

was analyzed by Southern blot. HEC-16GREc contained integrated HPV16 DNA, as shown for immortalized cell DNA that was undigested or digested with HPV16-noncleaving XbaI (Fig. 2a, lanes UD, and XbaI, lane 1, respectively). The pattern for multiple site-

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cleaving BamHI and PstI showed that the whole HPV16 genome was present. Further, the presence of a 1.8-kb BamHI and PstI fragment indicated that the E6–E7 region and the regulatory region (Durst et al., 1987) were retained intact in HEC-16GREc and HEC-

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FIG. 2. Effect of enhanced hormone response on HPV16 status in triple GREc construct-immortalized cells. High molecular weight DNA from the cells was analyzed by Southern blot assays. Molecular weights are shown on the left in kb. The HPV16 genomic DNA (16) digested with the indicated enzymes was used as marker. (a) The lanes are: UD, undigested HEC-16GREc; C, C33A as a negative control; 1, HEC-16GREc; 2, CaSki as a positive control. (b) DNA was from HEC-16 cells and cleaved with: BamHI and PstI (B/P), BamHI (B), and XbaI (X).

16 (Fig. 2a, BamHI / PstI, lane 1; Fig. 2b, lane B/P, respectively). HEC-16GREc DNA for single site-cleaving BamHI revealed multiple sites of HPV16 integration in a complex pattern compared with the single intact 7.8-kb band for HEC-16 (Fig. 2a, BamHI, lane 1; Fig. 2b, lane B, respectively). Apparently, the multiple copies of whole HPV16 DNA were integrated into more sites and were less tandemly repeated in HEC-16GREc than in HEC-16. Previously, an association was seen between the expression level of HPV16 and cell growth in cysts and tumors (Durst et al., 1991). Since the GREcs were designed to directly enhance the expression of the integrated pHPV3 1 GREc HPV16 DNA, total RNA from HEC-16GREc and HEC-16 was compared in Northern blots. Viral DNA was expressed predominantly as two transcripts of 4.5 and 2.3 kb (Fig. 3). Both contain most of the E6–E7 oncogenes (Durst et al., 1987). Compared with the actin internal control, the viral RNA levels were substantially higher in hydrocortisone-treated HEC-16GREc (Fig. 3, lane /H) or dex-treated HEC16GREc (Fig. 3, lane /D) than in untreated HEC16GREc (Fig. 3, lane 0). The same RNA transcripts were seen before and after induction of expression by either hormone. Also, the induction was specific, since the RU486 glucocorticoid antagonist abolished the induction (Fig. 3, lane D / R). For HEC-16, a marginal induction of expression was observed in the presence of dex. Previously, histopathological and in situ hybridization assays revealed the absence of HPV expression in untransfected HEC (Khare et al., data not shown).

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Effect of Enhanced Hormone Response on Growth Properties of Immortalized Ectocervical Cells HPV16 was expressed at elevated levels in HEC16GREc and HEC-16, as evaluated by in situ hybridization assays (Khare et al.) and Northern blot assays

FIG. 3. Effect of enhanced hormone response construct on expression of HPV16 DNA in immortalized cells. The Northern blot assays used total RNA isolated from the indicated cells. Molecular weight markers are shown on the right in kb. The same blots were probed with g-actin DNA as an internal control. Lanes were for cells cultured in medium containing no added hormone (0) or 1 mg/ml hydrocortisone (/H), dexamethasone (/D), and dexamethasone plus RU486 in an equimolar ratio (D / R).

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TABLE 1 Effect of Enhanced Hormone Response on Growth Properties of HEC-16GREc and HEC-16 Growth property

HEC-16

Soft agar growth efficiency Early passagea 0dex /dex Late passagea 0dex /dex Tumorigenicityb Doubling timec KGM DMEM Saturation densityd Induction of growth ratee

HEC-16GREc

0 0

0 0

0 0 0/3

0 5 1 1005 0/3

37 39 16 1.2

{ { { {

4 2 4 0.1

25 31 29 2.4

{ { { {

3 4 3 0.3

a Early passage was passage 30 and less. Late passage was passage 77 and more. b The tumor formation/mice injected at three sites each is shown. c The doubling time in medium with dex is given as hours { standard error and based on the average number of duplicate counts of triplicate 60-mm dishes. d The number of cells { standard error 1 1005 is presented as the average number of triplicate counts of three dishes. e The ratio of the growth rate in KGM with and without dex is given as the average { the standard error for the cumulative numbers of cells grown during 8 days for triplicate experiments.

(Fig. 3). Therefore, the effect of hormones on the growth potential of HEC-16GREc was of special interest. Primary cultures of HEC grew more slowly in medium with dex (data not shown). HEC-16 had a 1.2-fold increase in growth rate, whereas HEC-16GREc exhibited a 2.4-fold higher growth rate (Table 1). The doubling time for HEC-16GREc was 1.5- and 1.2-fold faster than for HEC-16 in dex-containing KGM and DMEM, respectively. Next, the cells were assayed for anchorageindependent (soft agar) growth. Early passage and late passage HEC-16 failed to form colonies in soft agar assays. Although HEC-16GREc also failed to form colonies for assays of early passage cells, late passage cells dex-independently produced colonies at an efficiency of 5 1 1005. Also showing the enhanced growth potential of HEC-16GREc, the saturation density was 1.8-fold higher. To test the tumorigenicity, nude mice were injected with a total each of 3 1 107 HEC-16GREc and HEC-16, but no tumors were produced (Table 1). Effect of Enhanced Hormone Response on Differentiation of Ectocervical Cells in Organotypic (raft) Culture Rafts mimic the differentiating cervical epithelium and are used to characterize the ability of cultured cells to form normal or dysplastic stratified lesions (McCance et al., 1988). Therefore, rafts from the three types of ectocervical cells were examined. HEC recon-

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structed rafts of normal epithelium with a single layer of basal cells and multiple superficial layers of well differentiated cells (Fig. 4), similar to previous studies (McCance et al., 1988). In contrast, HEC-16 and HEC16GREc rafts resembled low-grade dysplasia in organotypic cultures reconstructed without dex. The dysplastic features included modest parabasal cell crowding, a decreased cytoplasmic/nuclear ratio, and mitotic cells with large nuclei (Fig. 4). In addition, HEC-16GREc had an altered differentiation pattern, with abundant koilocytes containing abnormal nuclei and vacuolated cytoplasms (Fig. 4, arrowheads). The presence of dex reduced the number of stratified layers reconstructed from HEC and HEC-16, especially the basal layer of dividing cells. Thus, these rafts showed premature differentiation. Interestingly, the stratification of dividing and differentiating HEC-16GREc was almost unaffected by dex. Only the HEC-16GREc raft showed the enhanced growth characteristic of a more dysplastic phenotype (Fig. 4). DISCUSSION

The association of hormones and HPVs with cervical cancer was clearly established (reviewed in Khare et al., 1995; zur Hausen and de Villiers, 1994). Physiologically, hormones are intricately associated with the growth and differentiation of the cervical epithelium (Reagan and Fu, 1983). Therefore, studying the effect of glucocorticoid hormones on HPV16-containing cells is important for understanding cervical carcinogenesis. Further, GREcs in the regulatory region of cellular genes were useful to study the action of glucocorticoids (Rozansky et al., 1994). Thus, immortalized HEC-16GREc were established using HPV16 containing three GREcs. Also used, was the organotypic (raft) system. Raft culture reconstructs the in vivo-like stratification and differentiation of an epithelium. The physiology or pathology of the raft epithelium indicate the cell phenotypes of cultured epithelial cells. Also, the effects of exogenous agents, such as hormones, can be tested in vitro (McCance et al., 1988; Meyers et al., 1992). HEC-16GREc rafts clearly showed the dysplastic property of greater cell growth. Previously, some differential effects of glucocorticoid hormone on growth and viral gene expression in HPV-containing cells were found (von Knebel Doeberitz et al., 1991). HPV-independent antiproliferative and differentiation-promoting effects of glucocorticoids were also identified (Denis et al., 1992; Gregoire et al., 1991). Consistently, hormone reduced the growth of our HEC-16 raft and led to premature differentiation. However, the HEC-16GREc raft showed little change. Possibly, the hormone-induced higher level of the HPV E6-E7 oncoproteins in HEC-16GREc counteracted the HPV-independent antiproliferative action of hormone. HEC-16GREc viral DNA was integrated into the host

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FIG. 4. Effect of enhanced hormone response on morphological differentiation of epithelia from immortalized cells in organotypic (raft) culture. Cell differentiation is toward the top of each panel and the basal layer is toward the bottom. The bar in the upper left panel represents 10 mm for all panels.

genome. A previous study observed that extended life span, HPV-transfected cells containing episomal DNA, and immortalized cells containing integrated HPV resembled the cervical lesions that often progress to neoplasia (Yokoyama et al., 1995; Daniel et al., 1995). Cervical lesions with integrated viral DNA shared with HEC-16GREc two more conditions that apparently lead to carcinomas, enhanced cell growth, and enhanced viral oncogene expression (Jeon et al., 1995). The enhanced expression of E6–E7 may directly cause the enhanced growth of HEC-16GREc. This suggests that an enhanced response to hormones confers a greater oncogenic risk for cervical cells containing HPV16. Cervical carcinoma cells in which HPV18 E6– E7 expression was up-regulated or down-regulated from a heterologous promoter showed that the level of expression is important (von Knebel Doeberitz et al., 1988). However, HEC-16GREc regulated expression from the homologous HPV16 promoter and represent a system resembling early HPV16-containing lesions. They have not undergone the tumorigenic changes of cervical carcinoma cells. Thus, HEC-16GREc were useful to study the importance of E6–E7 levels in the initial events of oncogenesis.

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E6 – E7 expression is necessary to maintain the altered growth phenotype of immortalized cells (Munger et al., 1989). Apparently, the expression of the E6 – E7 oncogenes in HEC-16GREc was substantially higher than that in HEC-16 due to the three GREcs in HEC-16GREc. In situ hybridization assays revealed a significant induction by dex of HPV expression in HEC transiently transfected with the pHPV3 1 GREc plasmid that is integrated in HEC16GREc (Khare et. al., 1996). The glucocorticoid-mediated expression of integrated HPV16 in some carcinoma cell lines was disrupted, but was restored after excising the DNA from the flanking cell DNA (von Knebel Doeberitz et al., 1991). Consistently, episomal HPV16 DNA is responsive to dex and progesterone (Mittal et al., 1993a; Mittal et al., 1993b). We have investigated the role of hormone and HPV in epithelial morphology and differentiation in an in vitro model, using the raft system. HEC-16GREc were more resistant to dex-induced differentiation signals than HEC and HEC-16. Dex had a significant role in different stages of oncogenesis through the effects of the hormone on oncogene expression, growth, and differentiation of cervical cells. We suggest that the

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potentially variable level of expression of viral oncogenes in response to hormones leads to a corresponding risk for cervical cells containing integrated HPV16 DNA. This suggestion supports the previous findings (von Knebel Doeberitz et al., 1988, 1991). Further, the response to hormone was retained following immortalization for HEC-16 and HEC16GREc. Taken together, the results suggest that the loss of hormone response is a late event in the transfection, infection, immortalization, and carcinoma formation of cervical cells. Potential late-acting factors in oncogenesis act with or following the action of hormones. EGF, like hormones, is implicated in early events (Pim et al., 1992). Dex inhibited the proliferation of cells synergistically with retinoic acid (Song and Cheng, 1993; Song, 1994). Further, loss of sensitivity to retinoic acid during HPV16-associated tumorigenesis was a late event (Sarma et al., 1996). Smoke is another exogenous agent that was experimentally implicated as a late-acting factor in HPV-associated cervical cancer (Yang et al., 1996; Nakao et al., 1996). In addition, only late passages of HEC-16GREc showed the oncogenic phenotype in the presence of dex in anchorage independence assays and no growth was seen without dex. Loss of response to dex after transformation of rodent cells by HPV16 was associated with changes in cell factor-mediated viral gene expression (Pater et al., 1993). Similarly, dex-independent HEC-16GREc featuring altered patterns of growth, differentiation, and viral gene expression may emerge during a late event. Therefore, we are selecting HEC-16GREc that are hormone-independent to develop a model for cancers in which late events confer resistance to hormone therapy (Darbre and King, 1987). We thank Ms. G. Jin for excellent technical assistance, Mr. E. Evelly for assistance in the histological studies, Dr. M. Pirai of the Grace General Hospital for analysis and samples of cervical tissues, and Ms. J. Petten for typing the manuscript. This work was supported in part by the National Cancer Institute of Canada, with funds from the Canadian Cancer Society, and by the Medical Research Council of Canada.

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Received September 9, 1996 Revised version received January 14, 1997

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