Although rodent cells have been immortalized following transfection with a mutant p53 gene, the role of p53 in the immortalization of human cells is unknown. Therefore, human epithelial cell lines were examined for p53 mutations in exons 4-9 which include the evolutionarily conserved regions. A spontaneously immortalized skin keratinocyte cell line, HaCat, and three ras-transfected clones, have a p53 mutational spectrum that is typical of ultraviolet light induced mutations. A normal finite lifespan cell strain (184) and two benzo[a]pyrene immortalized mammary epithelial cell lines derived from 184 (184A1 and 184B5) contain wild type p53 sequences in exons 4-9, although elevated levels of nuclear p53 indicate an alteration in the stability of the normally transient protein. Wild type p53 was found in human bronchial, esophageal and hepatic epithelial cells immortalized by SV40 T antigen gene and human renal epithelial cells immortalized by adenovirus 5. BEAS-2B, an SV40 T antigen immortalized bronchial epithelial cell line and two subclones, have a germline polymorphism at codon 47. Inactivation of p53 by mechanisms such as mutation or complexing with proteins of DNA tumor viruses appears to be important in the immortalization of human epithelial cells.

Estrogen-induced progression through G 1 phase of the cell cycle is preceded by increased expression of the G 1 -phase regulatory proteins c-Myc and cyclin D1. To investigate the potential contribution of these proteins to estrogen action, we derived clonal MCF-7 breast cancer cell lines in which c-Myc or cyclin D1 was expressed under the control of the metal-inducible metallothionein promoter. Inducible expression of either c-Myc or cyclin D1 was sufficient for S-phase entry in cells previously arrested in G 1 phase by pretreatment with ICI 182780, a potent estrogen antagonist. c-Myc expression was not accompanied by increased cyclin D1 expression or Cdk4 activation, nor was cyclin D1 induction accompanied by increases in c-Myc. Expression of c-Myc or cyclin D1 was sufficient to activate cyclin E-Cdk2 by promoting the formation of high-molecular-weight complexes lacking the cyclin-dependent kinase inhibitor p21, as has been described, following estrogen treatment. Interestingly, this was accompanied by an association between active cyclin E-Cdk2 complexes and hyperphosphorylated p130, identifying a previously undefined role for p130 in estrogen action. These data provide evidence for distinct c-Myc and cyclin D1 pathways in estrogen-induced mitogenesis which converge on or prior to the formation of active cyclin E-Cdk2-p130 complexes and loss of inactive cyclin E-Cdk2-p21 complexes, indicating a physiologically relevant role for the cyclin E binding motifs shared by p130 and p21.Estrogenic steroids elicit mitogenic responses in a variety of cell types, particularly those of female reproductive tissues, including uterus and mammary gland tissues. In addition, estrogens have well-described mitogenic actions on neoplastic breast epithelial cells both in vivo (55) and in vitro (25), and this effect has been linked to the established role of estrogens in the development and progression of the majority of human breast cancers (16). Estrogenic steroids, e.g., 17␤-estradiol (E 2 ), stimulate resting (G 0 -phase) cells to enter the cell cycle and accelerate G 1 -S-phase progression (23,58). Advances in the understanding of molecular mechanisms controlling cell cycle progression (31,50,51,66) have identified cyclin-dependent kinases (CDKs) as potential targets of E 2 -induced mitogenesis (2,12,39,42).Sensitivity to mitogenic stimulation is limited to G 1 phase of the cell cycle, transit through which is regulated by the activities of Cdk4, Cdk6, and Cdk2. These CDKs are activated by cyclin binding: Cdk4 and Cdk6 by D-type cyclins (50) and Cdk2 by cyclin E (22). Additional control of cyclin-CDK activity is achieved by phosphorylation/dephosphorylation of specific residues conserved among CDKs and by interaction with two families of CDK inhibitors: the INK4 family, of which p16 INK4A is prototypic, and the p21 WAF1,CIP1,SDI1 /p27 KIP1 /p57 KIP2 family (reviewed in references 31 and 51). Other factors, such as the activity of Cdc25 phosphatases that catalyze the removal of inhibitory phosphates on CDKs (31), identify a furt...

INK4 expression and a novel mechanism for the elongation of telomeres.

No abstract