Cell cycle arrest at the G1 checkpoint allows completion of critical macromolecular events prior to S phase. G1 and G2 checkpoints (4, 5). Phosphorylation of the RB protein by Cdk and the release of RB-associated proteins-e.g., the transcription factor E2F-is correlated with the transition across the G, checkpoint (6-10). The free E2F is then available to transcriptionally activate genes encoding proteins critical for S-phase function, including deoxynucleotide biosynthesis (11).

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A failure of normal apoptosis, often due to mutant p53, may contribute to the formation of a cancer and to its resistance to therapy. Mesothelioma, an asbestos-induced tumor, is highly resistant to therapy but generally expresses wild-type p53. We asked whether mesothelioma was resistant to apoptosis and whether resistance was associated with altered expression of the antiapoptotic protein Bcl-2 or proapoptotic protein Bax. We found that three mesothelioma cell lines (1 with wild-type p53) were highly resistant to apoptosis induced by oxidant stimuli (asbestos, H2O2) or nonoxidant stimuli (calcium ionophore) compared with primary cultured mesothelial cells. By immunostaining, one of these three lines expressed Bcl-2 but only during mitosis. By immunoblotting, 3 of 14 additional mesothelioma lines (9 of 14 with wild type p53) expressed Bcl-2 but all 14 of 14 expressed the proapoptotic Bax, giving a low ratio of Bcl-2 to Bax. We conclude that mesothelioma cell lines are resistant to apoptosis and that the failure in apoptosis is not explained by Bcl-2 but by other mechanisms that counteract the proapoptotic effect of Bax.

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Loss of normal functions and gain of oncogenic functions when the p53 tumor suppressor gene is mutated are considered critical events in the development of the majority of human cancers. Human bronchial epithelial cells provide an in vitro model system to study growth, differentiation, and neoplastic transformation of progenitor cells of lung carcinoma. When wild-type (WT) or mutant (MT; codon 143va-A) human p53 cDNA was transfected into nontumorigenic BEAS-2B cells, we observed that (i) transfected WT p53 suppresses and MT p53 enhances the colony-forming efficiency of these cells, (it) MT p53 increases resistance to transforming growth factor (31, and (iii) clones of MT p53 transfected BEAS-2B cells are tumorigenic when inoculated into athymic nude mice. These results are consistent with the hypothesis that certain mutations in p53 may function in multistage lung carcinogenesis by reducing the responsiveness of bronchial epithelial cells to negative growth factors.Mutation of the p53 tumor suppressor gene has been shown to be a frequent event in many types of human cancer (1). Studies of the actions of p53 that contribute to growth regulation, including those showing that the wild-type (WT) gene product is a nuclear phosphoprotein that blocks tumor cell proliferation through arrest at the G1/S boundary, have been recently reviewed (2, 3). In contrast, although not all mutations in p53 have identical activities (4), most mutations provide cells with a growth advantage (2, 3). Recent studies show that different mutations in the p53 gene lead to proteins that vary in their DNA-binding specificity and affinity (4)(5)(6)(7) and activity in transcriptional transactivation assays (8-10). One study of transactivation in different human lung tumor cell lines indicates that the cell line, as well as the kind of mutation, has a major influence on the degree of transactivation (58). Therefore, both the cell and the precise mutation will play a critical role in determining the consequences for a specific cell type.Normal human bronchial epithelial (NHBE) cells represent the progenitor cells for human bronchogenic carcinoma. This tumor results from exposure to such exogenous genotoxic carcinogens as tobacco smoke (11) and radon (12). To study the pathogenesis of this disease, we have utilized the simian virus 40 (SV40) large T "immortalized" cell line BEAS-2B derived from NHBE cells (13). BEAS-2B cells produce SV40 T antigen, are nontumorigenic, and respond to treatment with serum or transforming growth factor /,3 (TGF-,81) by ceasing cell division and initiating terminal squamous differentiation (14,15 MATERIALS AND METHODSTransfection and Cloning. BEAS-2B cells (passage 67) were transfected by strontium phosphate precipitation (22) with 10 ,g of DNA from pC53-SN, pCS3-CX3, or CMV-neo (16). After 14 days of selection in LHC-9 medium with 125 jig/ml of G418 (Geneticin, GIBCO/BRL) and 3% chemically denatured serum (Upstate BioTechnologies, Lake Placid, NY) (23), mass cultures were established and expanded. Mass cult...

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Monolayer cultures of human mesothelial cells made quiescent by serum deprivation are induced to undergo one round of DNA synthesis by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor type beta 1 (TGF-beta 1). This one-time stimulation is independent of other serum components. The kinetics for induction of DNA synthesis observed for PDGF, EGF, and TGF-beta 1 are all similar to one another, with a peak of DNA synthesis occurring 24-36 h after the addition of the growth factors. Repetitive rounds of DNA synthesis and cell division do not ensue after addition of PDGF, EGF, or TGF-beta 1 alone or in combination; however, in media supplemented with chemically denatured serum, each of these factors is capable of sustaining continuous replication of mesothelial cells. Stimulation of growth by PDGF and TGF-beta 1 is unusual for an epithelial cell type, and indicates that mesothelial cells have growth regulatory properties similar to connective tissue cells.