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

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Abstract
Human papillomavirus (HPV), especially HPV types 16, 18, and 33, play an important role in the development of cervical cancer.1–3 We,4,5 as well as others,6–9 have shown that DNA from these HPV types immortalize cultured human foreskin keratinocytes (HKc) and human cervical cells. HPV 16-immortalized HKc (HKc/HPV16) represent an attractive in vitro model system to study the process of multistep carcinogenesis in human cells, and for identifying agents that can modify or prevent HPV-induced transformation, since they undergo malignant conversion through four phenotypically defined “steps” or stages. Although immortal, HKc/HPV 16 are not tumorigenic and retain many features characteristic of normal HKc, including a requirement for epidermal growth factor (EGF) and bovine pituitary extract (BPE) for proliferation.4,5 In the next step of this progression model, growth factor independent sublines, HKc/GFI, that no longer require EGF and BPE for proliferation, are selected from HKc/HPV16.5,10 Next, sublines that no longer respond to calcium and serum-induced differentiation, HKc/DR, are selected from HKc/GFI.5 In the final step, malignant conversion of HKc/DR is achieved by transfection with viral Harvey ras (v-Ha-ras) or herpes simplex virus type 2 DNA.11,12 The steps from immortalized HKc/HPV16 to malignant conversion are highly reproducible and at each step the cells progressively acquire phenotypic and biochemical characteristics similar to those found in cell lines established from human tumors.10