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ORIGINAL RESEARCH |
Effects on Cell Growth and Differentiation in Cervical Carcinoma Cell Lines
From the Department of Obstetrics and Gynecology, Saga Medical School, Saga, Japan; and the Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada.
Address reprint requests to: Masatoshi Yokoyama, MD, Department of Obstetrics and Gynecology, Saga Medical School, Saga 849-8501, Japan; E-mail: yokoyam1{at}post.saga-med.ac.jp.
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ABSTRACT |
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(IFN-) on premalignant and malignant models of cervical cancer.
METHODS: Cell growth rate was examined after treatment for 4, 7, and 10 days with RA and/or IFN- of human papillomavirus type 18 (HPV 18)-immortalized endo- and ectocervical cells, nontransformed serum-adapted cells, transformed cells, three adenocarcinoma, and three squamous cell carcinoma cell lines. The effect on epithelial differentiation by RA and IFN- was examined in organotypic culture. Induction of apoptosis was examined by modified terminal transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) and DNA fragmentation.
RESULTS: Cell growth rate was inhibited by RA, 84–96% in HPV 18-immortalized endocervical cells, SiHa, and ME180, 0% in OMC-4, and 18–62% in other cell lines; and by IFN- about 75% in SiHa and ME180 and 14–40% in the other cell lines. Combining RA and IFN- increased the antiproliferative effect in premalignant cell lines and some cancer cell lines except OMC-4, SiHa, and HT-3. In rafts, RA treatment reversed human endocervical cell metaplasia and HPV 18-immortalized endo- and ectocervical cell dysplastic epithelial differentiation. Interferon-, not RA, treatment of HPV 18-immortalized endo- and ectocervical cells induced apoptosis.
CONCLUSION: Cell growth inhibition by treatment with RA, IFN-, and their combination differentially depends on treatment type and time, cell origin, cell line, and oncogenic state. In a premalignant model of cervical carcinoma, RA reduces dysplastic differentiation and IFN- induces apoptosis. These data confirm that these treatments may be effective for preventing or treating premalignant cervical lesions.
Cervical cancer is the second most common female cancer in the world. Cervical cancer develops through a multistep process in which increasing severely premalignant dysplastic lesions called low-grade squamous intraepithelial lesion (SIL), high-grade SIL, and carcinoma in situ progress to invasive cancer.1 Previously, we reported that human papillomavirus (HPV) type 18-immortalized cells are useful as a model for studying progressing premalignant cervical lesions.2
Retinoids play a key role in epithelial differentiation and growth.3 Retinoic acid (RA), the direct acting vitamin A metabolite, regulates epithelial development, reversing it in some dermatologic conditions and precancerous cervical lesions.4 Retinoic acid and several other retinoids were shown to have an antineoplastic effect on cervical cells.5–9 Retinoids have been used in several clinical trials to prevent cervical cancer.10 A phase II trial of all-trans RA for cervical intraepithelial neoplasia (CIN) resulted in a 50% complete clinical response.11 A randomized phase III trial of patients with CIN II all-trans RA increased the complete regression rate from 27% in the placebo group to 43% in the treated group.12 Interferon- (IFN-) has antiproliferative activity on many normal and transformed cells, including cervical cancer cells.13 Some clinical trials suggest that IFN- can induce regression of premalignant lesions of the cervix.14,15 Furthermore, two clinical trials combining cis-RA and IFN- for the therapy of advanced cervical cancer showed promising clinical results.16,17 However, little is known about potential cellular targets of this activity in cervical oncogenesis.
This study used an in vitro cervical oncogenesis model composed of various HPV 18-immortalized ectocervical, endocervical, and cancerous cervical cell lines, to compare the effect of RA and/or IFN- on cell proliferation in monolayers and metaplastic differentiation, CIN, or carcinoma in situ formation and apoptosis in rafts.
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MATERIALS AND METHODS |
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on cervical cell growth rate and premalignant lesion formation, keratinocyte serum-free growth medium (K-SFM, GIBCO, Grand Island, NY) was used for the growth of primary human endo- and ectocervical cells,18 HPV 18-immortalized human endocervical cells,2 and HPV 18-immortalized human ectocervical cells.2 Non-transformed, serum-adapted HPV 18-immortalized human endocervical cells and transformed HPV 18-immortalized human ectocervical cells induced by the treatment with cigarette smoke condensate19 were cultured in Dulbecco’s modified Eagle’s medium plus 10% fetal calf serum. Cervical cancer cell lines are summarized in Table 1
. OMC-4, CAC-1, and TMCC-1 were kindly obtained from T. Yamada (Osaka Medical College, Osaka, Japan), O. Hayakawa (Sapporo Medical College, Sapporo, Japan), and M. Sakamoto (Tokyo Medical College, Tokyo, Japan), respectively. SiHa, ME180, and HT-3 were maintained at 37C in Dulbecco’s modified Eagle’s medium plus 10% fetal calf serum (GIBCO).
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(Takeda Drug Corporation, Osaka, Japan), which was changed every second day. Cells were counted with a Coulter counter. Growth inhibition was expressed with the following formula:
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Morphology of raft epithelia formed from cells was examined using histopathology. Rafts were prepared as described previously.20 Briefly, cells were seeded on a collagen matrix support. When the cells reached confluence, the gel was raised to the air-liquid interface. The rafts were incubated with or without RA and/or IFN-, and the reconstructed epithelia were recovered after 12 days. The rafts were embedded in paraffin and stained with hematoxilineosin for histopathology and alcian blue for mucin.
Apoptotic DNA fragmentation was detected by modified terminal transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL). Paraffin-embedded blocks were deparaffinized, immersed in 0.3% H2O2 to block endogenous peroxidase, and treated with 2 µg/mL proteinase K for 15 minutes. They were then incubated at 37C for 1 hour in a solution including TdT and digoxigenin-labeled dUTP, washed, treated with peroxidase-labeled antidigoxigenin antibody for 30 minutes, stained by 3,3'-diaminobenzidine in 0.01% H2O2 and couterstained with hematoxylin. The normal thymus of a 20-week fetus was a positive control. For confirmation DNA fragmentation analysis, high molecular weight DNA was extracted, resolved by 1.5% agarose gel electrophoresis, and stained using an Apoptosis Ladder Detection kit (Wako, Osaka, Japan).
Assays of growth were performed in replicates of three. The mean and standard deviation of all samples were calculated and compared with untreated controls. For statistical analysis, all results were compared with two-tailed Student t test. All experiments were repeated, and the results were reproducible.
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RESULTS |
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) for 4, 7, and 10 days. Retinoic acid inhibited growth 84–96% in HPV 18-immortalized human endocervical cells, SiHa, ME180, and HT-3, and 18–62% in serum-adapted HPV 18-immortalized human endocervical cells, HPV 18-immortalized human ectocervical cells, transformed HPV 18-immortalized human ectocervical cells, CAC-1, and TMCC-1, whereas growth was unaffected in OMC-4 (Tables 2 and 3). Interferon- inhibited growth rate 75% for SiHa and ME180 and 14–40% for the remaining eight cell lines. The inhibitory effect of IFN- was less than that of RA (Tables 2 and 3). The cells varied in their sensitivity to the inhibitory effect of RA or IFN-, but dose-dependent reductions in cell growth were observed (data not shown). Combination of IFN- and RA treatment inhibited cell growth synergistically in HPV 18-immortalized human endocervical cells, HPV 18-immortalized human ectocervical cells at 7 or 10 days, and ME180 cells and additively in serum-adapted HPV 18-immortalized human endocervical cells, CAC-1, TMCC-1, and transformed HPV 18-immortalized human ectocervical cells. Growth was unaffected by combination in OMC-4, SiHa, and HT-3.
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treatment on morphology was examined using organotypic culture (rafts). Raft epithelial development from untreated HPV 18-immortalized human ectocervical cells displayed low-grade SIL containing stratified and well-formed cornified layer. Generally, the raft epithelia retained the parental cell differentiation feature, such as keratinization. Retinoic acid blocked keratinization in the superficial layer, and IFN- had no effect (Figure 1d–f). In contrast, HPV 18-immortalized human endocervical cell morphology resembled high-grade SIL/carcinoma in situ. The entire epithelium was composed of highly atypical cells with prominent polymorphism. The rafts also showed sporadic cells with vacuolated cytoplasm, suggesting glandular cell differentiation. Alcian blue specifically stained these cells, confirming their mucin content and corroborating their characteristics as colmunar epithelium (data not shown). However, RA blocked this glandular differentiation, and INF- had no effect (Figure 1a–c). Human endocervical cells, parental cells of HPV 18-immortalized human endocervical cells, had the features resembling metaplasia in control medium and RA treatment induced glandular alcian blue mucin staining of the raft epithelium (Figure 2A–C).
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treatment on apoptosis was examined by TUNEL assay of rafts and DNA fragmentation assays of monolayer cells. Apoptosis in HPV 18-immortalized human ectocervical cells was not induced by RA (Figure 3A), but was by IFN- (Figure 3B). Similar results were observed for HPV 18-immortalized human endocervical cells using RA (Figure 3C) and IFN- (Figure 3D). Furthermore, IFN- treatment of HPV 18-immortalized human endocervical cells and HPV 18-immortalized human ectocervical cells cultures resulted in internucleosomal DNA fragmentation, evidenced by the formation of a DNA ladder in agarose gel (Figure 4). These results suggest that IFN- induces apoptosis in precursor lesions of cervical carcinoma.
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DISCUSSION |
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The relative susceptibility of endocervical glandular compared with squamous lesions to treatment with different drugs remains unclear. Retinoic acid, the main active vitamin A metabolite, represents such a drug. Here, it inhibited cell growth for cell lines derived from the endocervix and adenocarcinoma (about 35%, and 96% by HPV 18-immortalized human endocervical cells) generally less than those derived from squamous counterparts (about 75%). Human papillomavirus 18-immortalized endocervical cells were much more sensitive to RA before than after serum adaptation (which confers greater cervical cell growth potential and higher grade CIN19) and all three adenocarcinoma-derived cell lines. We have previously consistently observed greater RA sensitivity of two nontumorigenic HPV 16-immortalized endocervical lines than the corresponding tumorigenic cells.9 These results support the hypothesis that the sensitivity to RA in HPV-positive endocervical cell lines decreases according to the progression of carcinogenic process.9
Mice fed vitamin A developed normal endocervical glandular epithelium.27 Progressing from our previous study,18 we now show that human endocervical cells treated with RA differentiated into mucin-positive, glandular epithelium from squamous metaplasia in organotypic culture (rafts). These results suggest that human endocervical cells have the bipotential to differentiate to metaplasia and glandular structure. Retinoic acid might block the differentiation to squamous metaplasia in human endocervical cells. We previously found that RA reverses the histology and differentiation markers of carcinoma in situ of HPV 16-immortalized endocervical cells,20 and RA was found to block terminal squamous differentiation in HPV-immortalized and SiHa squamous cell lines.8 Analogously, we now show that RA treatment blocks keratinization of HPV 18-immortalized ectocervical cells in rafts. It also leads to loss of the glandular phenotype in HPV 18-immortalized endocervical cells. The results reveal that RA blocks the terminal differentiation of epithelial cells from both squamous and columnar origin.
Interferon- but not RA was shown to inhibit HPV 16 oncogene expression in HPV-positive immortalized and cancer cells,28 but we found no change was induced in HPV 18 transcripts by either agent (data not shown). However, it, but not RA, induced apoptosis in this study in monolayers and rafts. Thus, INF- may induce apoptosis via inhibiting HPV 16 E6 or through other mechanisms for HPV 18. It is reported that INF- or RA treatment in SiHa and ME180 cell induced the cell detachment.29,30 The cells in the supernatants by INF- treatment represented apoptosis and secondary necrosis.30 When the cells started to detach from the plate, they lost the capacity to proliferate.29 In contrast, adhering cells remained viable.30 Although the possibility of cell lysis and clumping in preparation for cell counting cannot be completely ruled out, growth rate in the present study represents viable and proliferating cell number. Previous studies have suggested that RA acts by inducing cervical carcinoma cell apoptosis, but only in one of eight cervical carcinoma cell lines.6 The differential effects of INF- and RA on cervical cell growth, differentiation, and apoptosis in culture may explain the synergism of these two agents in some cervical lesions.
Several clinical trials have produced favorable results for combination IFN-/RA cervical carcinoma therapy.16,17,31 This report demonstrates that RA, IFN-, and their combination inhibited the cell growth rate in monolayer and reversed metaplastic differentiation of CIN in rafts of cell lines derived from the cervix. Retinoic acid and/or IFN- were more effective on average in cell lines of ectocervix/squamous carcinoma compared with endocervix/adenocarcinoma origin (Tables 2 and 3), suggesting ectocervical cells have greater sensitivity. However, adenocarcinoma cell lines have the tendency to be more sensitive to IFN- than to RA. A recent report also described that the growth of RA-treated cervical adenocarcinoma cell line, HeLa, was less effectively suppressed than that of IFN-treated ones.32 Only when combined were IFN- and RA able to increase the expression of the 2-5A synthetase gene and HLA-A2 gene that regulate responses to IFN- or RA.29 The association of the effects of IFN and RA with the cyclin-dependent kinase inhibitor p21 was also reported.33 Recently Um et al described that combined RA and IFN-
treatment caused a marked increase in the level of IRF-1, which codes for the transcriptional factor that regulates the IFN gene.32 A combination of RA and IFN- leads to an additive antiproliferative effect on cell growth.32 It is also reported that the combination of IFN- and RA increases the expression of IRF-1 and enhances the IFN--induced apoptosis.29 The antiproliferative effects of the combination of IFN- and RA in immortalized cervical cell lines and tumorigenic lines in vitro and in vivo might be coupled with the different mechanism of these agents and caused by an increased expression of these genes. In premalignant cells, the antiproliferative effect was synergistic. Hence, the combination use of RA and IFN would be effective to prevent premalignant cervical lesions from progressing into cervical carcinoma. Our results might help explain the possible effects that the combination of IFN and RA has given in clinical studies.
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Footnotes |
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Received January 9, 2001. Received in revised form April 16, 2001. Accepted April 19, 2001.
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REFERENCES |
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