Abstract
Interleukin 1alpha (IL-1alpha), Interleukin 6 (IL-6) and epidermal growth factor (EGF) were tested for their ability to regulate epithelial cervical cell cytokine production and secretion and to induce proliferation of human normal and neoplastic epithelial cervical cells. IL-1alpha, and IL-6 enhanced tumour and normal cell growth by 20-120%. The interleukins efficacy was similar to that of EGF for some cell lines but not for normal esocervical cells. The stimulatory effects of the interleukins were observed in both human papilloma virus (HPV)-infected and HPV-non-infected cervical cells. Normal cells constitutively expressed IL-1alpha, IL-6 and EGF mRNA. All cell lines except C33A expressed IL-1alpha mRNA. CaSki, C-4II and HT-3 expressed mRNA for IL-6. IL-1alpha induced or increased IL-6 mRNA levels in the Me-180 and HT-3 lines and in normal cervical cells. IL-6 induced: (1) the expression of its own mRNA only in Me-180 cells that constitutively lacked IL-6 mRNA; (2) the expression of IL-1alpha mRNA in C-33A and increased IL-1alpha mRNA level in the case of Me180 cells. Increased amounts of IL-6 mRNA were found in normal cells when treated with IL-1alpha. In spite of the pattern of mRNA expression, only HT-3 and normal cervical cells constitutively secreted IL-6, and only normal cells were able to produce IL-1alpha protein. A significant IL-1alpha-dependent increase of IL-6 secretion was found in Me-1 80, HT-3 and normal cells. IL-1alpha- and IL-6-driven cell proliferations were almost completely inhibited by the addition of neutralizing anti-IL-6 antibodies. Taken together, these data suggest that interleukins play a role in cervical carcinogenesis as autocrine and/or paracrine stimuli.
Highlights
All neoplastic and normal cervical cells were sensitive to the stimulatory effect of epidermal growth factor (EGF)
The magnitude of this effect was similar to that of IL-la and Interleukin 6 (IL-6) in the case of ME- 180 and CaSki cell lines, whereas it was significantly higher in the remaining lines and in normal cervical cells (Student's t-test; P < 0.05 for C-33A and P < 0.005 for the other cells)
This lack of effect may be due to the absence of insulin and transferrin in the culture medium as we have found that the stimulatory effect of both these interleukins and EGF is insulin and transferrin dependent
Summary
Cervical cell culturesNormal epithelial cervical cells were isolated using a two-step enzymatic digestion with dispase and trypsin, as described by Pirisi et al (1988). Tissues from hysterectomies for endometriosis or fibroids were incubated overnight at 4°C in phosphate-buffered saline (PBS) containing 25 U ml-l of dispase (Gibco-Life Technologies, Grand Island, NY, USA). The ectocervical epithelium was lifted from the submucosa, recovered as an intact sheet, and further incubated at 37°C for 30 min in the presence of 0.13% trypsin (Gibco Life Technologies) with frequent agitation. Dickinson), 0.2 ng ml-1 human recombinant EGF (Becton Dickinson), 5 ,ug ml insulin, 5 ,ug ml' transferrin, 2 mm glutamine, 0.1 mm phosphoethanolamine and 0.1 mm ethanolamine (Sigma). The purity of these preparations was checked by immunostaining with anti-cytokeratin 14 and anti-cytokeratin 18 antibodies (Woodworth et al, 1993). Cervical carcinoma cell lines (ME-180, C-411, CaSki, HT-3 and C-33A) were obtained from ATCC (Rockville, MD, USA) and maintained in culture as Primer
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