Inhibitors of epidermal growth factor receptor kinase and of cyclin-dependent kinase 2 activation induce growth arrest, differentiation, and apoptosis of human papilloma virus 16-immortalized human keratinocytes.

Human papilloma virus 16 (HPV 16) is associated with cervical cancer and is therefore considered a major health risk for women. Immortalization of keratinocytes induced by HPV infection is largely due to the binding of p53 and Rb by the the viral oncoproteins E6 and E7, respectively, and is driven to a large extent by a transforming growth factor alpha/amphiregulin epidermal growth factor receptor autocrine loop. In this study, we show that the growth of HPV 16-immortalized human keratinocytes can be blocked by a selective epidermal growth factor receptor kinase inhibitor, AG 1478, and by AG 555, a blocker of cyclin-dependent kinase 2 (Cdk2) activation. AG 1478 induces a massive increase in the Cdk2 protein inhibitors p27 and p21, whereas AG 555 appears to have a different mechanism of action, inhibiting the activation of Cdk2. Growth arrest induced by AG 1478 and AG 555 is accompanied by up to 20% of cells undergoing apoptosis. Following AG 1478 treatment but not AG 555 treatment, up to 50% of cells undergo terminal keratinocyte differentiation as determined by filaggrin expression and by the decline in the expression of cytokeratin 14. The growth-arresting properties of AG 1478 and AG 555 identifies them as possible lead antipapilloma agents.

Tyrphostins that suppress the growth of human papilloma virus 16-immortalized human keratinocytes.

Human papilloma virus 16 (HPV16) is considered to be the causative agent for cervical cancer, which ranks second to breast cancer in women's malignancies. In an attempt to develop drugs that inhibit the malignant transformation of HPV16-immortalized epithelial cells, we examined the effect of tyrphostins on such cells. We examined the effect of tyrphostins from four different families on the growth of HPV16-immortalized human keratinocytes (HF-1) cells. We found that they alter their cell cycle distribution, their morphology, and induce cell death by apoptosis. The effects of tyrphostins on HF-1 cells are different from their effects on normal keratinocytes. Growth suppression by AG555 and AG1478 is accompanied by 30% apoptosis in HF-1 cells, but this is not observed in normal keratinocytes. Tyrphostin treatment produces distinctive morphological changes in HF-1 cells and in normal keratinocytes; however, the culture organization of normal keratinocytes is less disrupted. These differential effects of the tyrphostins on HPV16-immortalized keratinocytes compared with their effects on normal keratinocytes suggests that these compounds are suitable candidates for the treatment of papilloma. Previous and present results indicate that group 1 tyrphostins, which inhibit Cdk2 activation, and group 2 tyrphostins, represented by AG1478, a potent epidermal growth factor receptor kinase inhibitor, induce cell cycle arrest; and, in the case of HF-1 cells, apoptosis and differentiation. Cells accumulate in the G(1) phase of the cell cycle at the expense of S and G(2) + M. These compounds block the growth of normal keratinocytes without inducing apoptosis or differentiation, causing them to accumulate in G(1). AG17, which belongs to group 4, exerts its antiproliferative effect mainly by increasing the fractions of cells in G(1) with a concomitant decrease in the fraction of cells in S and G(2) + M.