Wild type human p53 is antiproliferative in SV40-transformed hamster cells.

The transformation related protein p53 has been implicated in the process of normal cell proliferation and neoplastic transformation. In this study, the influence of wild type human p53 on cell proliferation was examined. Plasmid constructs encoding the wild type human p53 and various mutant p53 cDNAs, driven by the mouse mammary tumor virus (MMTV) promoter linked to the dominant biochemical selection marker gpt, were used in a colony forming assay employing SV40 transformed HR8 hamster cells. Plasmids encoding wild type p53 drastically reduced the number of gpt+ colonies obtained after transfection, whereas the mutant forms of p53 had no effect. Stable clonal hamster cell lines that constitutively express wild type p53 were isolated and found to have altered growth characteristics (i.e. lower saturation densities, increased doubling times). These findings are consistent with the notion that wild type p53 protein could function as a growth suppressor. The potential role of p53 in the normal cell cycle and in the transformation process is discussed.

Sequence analysis of the human thymidine kinase gene promoter: comparison with the human PCNA promoter.

We report the sequence of 4264 nucleotides of 5' flanking sequence of the human thymidine kinase gene, a gene that is maximally expressed at the G1/S boundary of the cell cycle. The position of nucleotide sequences which can act as binding sites for trans-acting factors, Sp-1, AP-1/jun, AP-2, OTF-1 and CAAT box factors as well as other potential cis-acting sequences have been mapped. The organization of these cis-acting sequences in the promoter of the human PCNA gene (another gene that is maximally expressed at the G1/S boundary) are shown for comparison. The potential role that these sequences may play in the transcriptional regulation of these genes is discussed.

Isolation of a human sequence which complements a mammalian G1-specific temperature-sensitive mutant of the cell cycle.

We have isolated a human genomic sequence which corrects the temperature-sensitive phenotype of the G1-specific cell cycle mutant ts13. Transfection of a thymidine kinase negative (TK-) derivative of ts13 with high molecular weight human DNA from placenta of HeLa cells resulted in the isolation of thermocompetent transformants. These transformants were found to possess discrete Hind III fragments containing human Alu family sequences which were conserved in several independent secondary transformants. A transforming sequence was recovered from a HindIII recombinant bacteriophage library prepared from the DNA of one such secondary transformant. The transforming sequence was isolated as a 12.5 kb Hind III fragment.

Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53.

To investigate the effect that human wild-type p53 (wt-p53) expression has on cell proliferation we constructed a recombinant plasmid, pM47, in which wt-p53 cDNA is under transcriptional control of the hormone-inducible mouse mammary tumor virus promoter linked to the dominant biochemical selection marker gene Eco gpt. The pM47 plasmid was introduced into T98G cells derived from a human glioblastoma multiforme tumor, and a stable clonal cell line, GM47.23, was derived that conditionally expressed wt-p53 following exposure to dexamethasone. We show that induction of wt-p53 expression in exponentially growing cells inhibits cell cycle progression and that the inhibitory effect is reversible upon removal of the inducer or infection with simian virus 40. Moreover, when growth-arrested cells are stimulated to proliferate, induction of wt-p53 expression inhibits G0/G1 progression into S phase and the cells accumulate with a DNA content equivalent to cells arrested in the G0/G1 phase of the cell cycle. Taken together, these studies suggest that wt-p53 may play a negative role in growth regulation.