E2F as a Regulator of Keratinocyte Proliferation: Implications for Skin Tumor Development

Abstract

E2F and DP family members are established regulators of the cell cycle. In this study, we examined their activity/expression during keratinocyte growth arrest. Treating human epidermal keratinocytes with the growth inhibitors TPA or IFN-gamma or allowing the cells to reach confluence resulted in 90% inhibition of DNA synthesis, whereas a keratinocyte-derived squamous carcinoma cell line (SCC25) was resistant to growth inhibitors. Gel shift analysis of keratinocytes using an E2F response element indicated that growth arrest was associated with a decrease in all E2F binding complexes. This indicates that growth inhibition is not due to negative regulation by pocket proteins. Conversely, gel shift analysis of growth inhibitor-resistant SCC25 cells showed no decrease in E2F binding. If deregulated E2F expression/activity is involved in tumor development, then the deliberate deregulation of E2F activity may make keratinocytes resistant to growth inhibitors in much the same way as the SCC cells. The HPV16 E7 protein is known to activate E2F. Retroviral infection of keratinocytes with E7-expressing constructs resulted in growth inhibitor resistance, whereas infection with E6 constructs did not. E2F is a heterodimeric complex consisting of E2F family members (1-5) and DP proteins (1-3). Examination of the expression levels for E2F genes and other genes associated with the cell cycle indicated that E2F1 was profoundly decreased in growth-arrested keratinocytes (90%), whereas E2F3, E2F5, and DP1 were not. E2F2 and E2F4 were increased in IFN-gamma-treated keratinocytes but not in TPA-treated or confluent keratinocytes. In contrast, SCC25 cells did not undergo growth arrest and did not downregulate E2F1 mRNA expression in response to growth inhibitors. Our results indicate that E2F DNA binding and in particular E2F1 mRNA expression are associated with keratinocyte proliferation. Our results with the SCC25 cells and the E7-infected cells are consistent with the proposition that deregulated E2F expression/activity (in particular E2F1) may be involved in the unregulated proliferation of skin tumor cells.

E2F-1 induces proliferation-specific genes and suppresses squamous differentiation-specific genes in human epidermal keratinocytes

“…Interestingly, other E2F family members (E2F-2, 3, 4 or 5) did not appear to be associated with keratinocyte proliferation or di erentiation status (Jones et al, 1997b).…”

“…Squamous di erentiation of keratinocytes requires a change in intermediate ®lament (keratin) expression and the commitment of non-proliferative keratinocytes to form a corni®ed cross-linked envelope. This di erentiation programme is a tightly regulated process, characterized by the irreversible growth arrest of keratinocytes and transcriptional downregulation of growth related genes such as E2F-1, p53, keratin 14 and cdk-1 (Lane et al, 1985;Woodworth et al, 1993;Saunders and Jetten, 1994;Jones et al, 1997b;Dahler et al, 1998). This is followed by the induction of squamous di erentiation-speci®c genes such as transglutaminase type 1 (TG I), keratin 10 and corni®n (Rieger and Franke, 1988;Marvin et al, 1992;Saunders et al, 1993b;Saunders and Jetten, 1994).…”

“…Squamous tumours of the skin frequently demonstrate defective control of the di erentiation programme (Lymboussaki et al, 1996;Moll et al, 1997;Hauser et al, 1998), such as insensitivity to di erentiation inducing stimuli (Jones et al, 1997b) and inappropriate expression of di erentiation-speci®c mRNA in proliferating cells (Dicker et al submitted). This deregulation of transcriptional processes may be causally associated with the development of squamous carcinomas, since deliberate activation of E2F in normal keratinocytes leads to growth-inhibitor insensitivity (Jones et al, 1997b).…”

“…This deregulation of transcriptional processes may be causally associated with the development of squamous carcinomas, since deliberate activation of E2F in normal keratinocytes leads to growth-inhibitor insensitivity (Jones et al, 1997b). A better understanding of normal keratinocyte di erentiation may lead to alternate skin tumour therapies that manipulate the deregulated control of di erentiation that is characteristic of squamous tumours.…”

“…A number of studies have suggested an important role for cell cycle proteins, and in particular the pRb pathway, in regulation of keratinocyte di erentiation (Kikuchi et al, 1992;Betz and Pelling, 1995;Lymboussaki, et al, 1996;Harvat et al, 1998), and both E2F-1 mRNA expression and E2F DNA binding activity have been shown to closely correlate with the di erentiation status of normal keratinocytes (Jones, et al, 1997b). Interestingly, other E2F family members (E2F-2, 3, 4 or 5) did not appear to be associated with keratinocyte proliferation or di erentiation status (Jones et al, 1997b).…”