Progressive loss of sensitivity to growth control by retinoic acid and transforming growth factor-beta at late stages of human papillomavirus type 16-initiated transformation of human keratinocytes

Abstract

Retinoids (vitamin A and its natural and synthetic derivatives) have shown potential as chemopreventive agents, and diets poor in vitamin A and/or its precursor β-carotene have been linked to an increased risk of cancer at several sites including the cervix. Human papillomavirus (HPV) plays an important role in the etiology of cervical cancer. We have developed an in vitro model of cancer progression using human keratinocytes (HKc) immortalized by HPV16 DNA (HKc/HPV16). Although immortal, early passage HKc/l HPV16, like normal HKc, require epidermal growth factor (EGF) and bovine pituitary extract (BPE) for proliferation and undergo terminal differentiation in response to serum and calcium. However, following prolonged culture, growth factor independent HKc/HPV16 lines that no longer require EGF and BPE can be selected (HKc/GFI). Further selection of HKc/GFI produces lines that are resistant to serum- and calcium induced terminal differentiation (HKc/DR). HKc/DR, but not early passage HKc/HPV16, are susceptible to malignant conversion following transfection with viral Harvey ras or Herpes simplex virus type II DNA. We have investigated the sensitivity of low to high passage HKc/HPV16 and HKc/GFI to growth control by all-trans- retinoic acid (RA, an active metabolite of vitamin A). Early passage HKc/HPV16 are very sensitive to growth inhibition by RA, and in these cells RA decreases the expression of the HPV16 oncogenes E6 and E7. However, as the cells progress in culture they lose their sensitivity to RA. Growth inhibition by RA may be mediated through the cytokine transforming growth factor-beta (TGF-β), a potent inhibitor of epithelial cell proliferation. RA treatment of HKc/HPV16 and HKc/GFI results in a dose- and time-dependent induction (maximal of 3-fold) in secreted levels of TGF-β. Also, Northern blot analysis of mRNA isolated from HKc/HPV16 demonstrated that RA treatment induced TGF-β1 and TGF-β2 expression about 3- and 50-fold, respectively. We next studied the effect of TGF-β1 and TGF-β2 on the proliferation of early to late passage HKc/HPV16, HKc/GFI and HKc/DR. While early passage HKc/HPV16 were as sensitive as normal HKc to growth inhibition by TGF-β1 and TGF-β2, the cells became increasingly resistant to TGF-β during in vitro progression, with the proliferation of HKc/DR being virtually unaffected by TGF-β1 or TGF-β2 treatment. Overall, loss of growth inhibition by RA parallels loss of TGF-β sensitivity. These results support a mechanism in which RA regulates growth control by enhancing the production of TGF-β, which, then inhibits cellular proliferation in an autocrine or paracrine manner. In addition, the loss of RA and TGF-β sensitivity observed during progression of HKc/HPV16 suggests that retinoids may be most effective in the prevention of cervical cancer and in the treatment of HPV induced lesions at early preneoplastic stages.