Analyses of five wild-type p53 containing cell lines revealed lineage specific differences in phosphorylation of Thr18 after treatment with ionizing (IR) or ultraviolet (UV) radiation. Importantly, Thr18 phosphorylation correlated with induction of the p53 downstream targets p21 Waf1/Cip1 (p21) and Mdm-2, suggesting a transactivation enhancing role. Thr18 phosphorylation has been shown to abolish side-chain hydrogen bonding between Thr18 and Asp21, an interaction necessary for stabilizing alpha -helical conformation within the transactivation domain. Mutagenesisderived hydrogen bond disruption attenuated the interaction of p53 with the transactivation repressor Mdm-2 but had no direct effect on the interaction of p53 with the basal transcription factor TAF II 31. However, prior incubation of p53 mutants with Mdm-2 modulated TAF II 31 interaction with p53, suggesting Mdm-2 blocks the accessibility of p53 to TAF II 31. Consistently, p53-null cells transfected with hydrogen bond disrupting p53 mutants demonstrated enhanced endogenous p21 expression, whereas p53/Mdm-2-double null cells exhibited no discernible differences in p21 expression. We conclude disruption of intramolecular hydrogen bonding between Thr18 and Asp21 enhances p53 transactivation by modulating Mdm-2 binding, facilitating TAF II 31 recruitment.

We investigated the induction and physiological role of Ser20 phosphorylation of p53 in response to DNA damage caused by ionizing radiation (IR) or ultraviolet radiation (UV). A polyclonal antibody that speci®cally recognizes a p53 peptide containing phosphorylated Ser20 was generated and used to detect p53 phosphorylation at Ser20. Western blot analyses of p53 in four cell lines with this antibody revealed that the p53 protein was phosphorylated at Ser20 to a dierent extent after treatment with IR or UV. The phosphorylation of Ser20 of wild-type p53 correlated with enhanced induction of the p53 downstream target genes p21 WAF1/Cip1 (p21) and mdm-2. These results suggest that DNA damage-induced phosphorylation of p53 at Ser20 enhances the transactivation function of p53 for p21 and mdm-2 in vivo.

In this study, we examined the effects of radiation and ara-C on induction of apoptosis and on the apoptosis-promoting genes p53, Bax and Fas/APO-1, in BV173 human leukemia cells, which harbor the wild-type p53 gene. It has been reported that p53 upregulates Fas/APO-1 and Bax expression. Both irradiation and ara-C treatment resulted in apoptosis and induction of p53 proteins within hours. The Bax gene was activated in irradiated and ara-C-treated BV173 cells, but Fas/APO-1 was induced only in irradiated BV173 cells. Radiation and ara-C treatment did not induce Bax or Fas/APO-1 protein expression in p53-null HL60 cells. Radiation weakly induced Fas/APO-1 expression in KBM-7 cells, which harbor a partially defective p53 gene. Both HL60 and KBM-7 cells are more resistant to radiation-and ara-Cinduced apoptosis than BV173 cells. These results suggest that functional p53 is necessary for the activation of Bax and Fas/APO-1 expression. However, elevated p53 protein is not sufficient to activate Fas/APO-1 gene expression in ara-Ctreated cells. Using two-dimensional gel electrophoresis, we found that the p53 proteins in irradiated and ara-C-treated BV173 cells have different isoelectric points; they converged to a single isoelectric point after in vitro treatment with phosphatase. These results suggest that different genotoxic treatments cause different phosphorylations of p53, which may account for the different levels of activation of Fas/APO-1 expression.

Tumor cell lines are an indispensable tool for cancer research. However, among cell lines of the same pathological group, heterogeneity has been detected in gene expression, gene mutation, and cellular response to various treatments. In this study, we systematically investigated the extent of heterogeneity of gene expression in three glioblastoma cell lines using cDNA array technology in which the expression of 588 cellular genes is studied simultaneously. Comparison of the expression pro®les revealed substantial qualitative and quantitative heterogeneity. Among the 588 genes, 197 genes were expressed in all three lines and 56 genes were not expressed in any of the three lines; total of 222 genes were expressed in only two of the three cell lines, and 113 genes were expressed in only one of the three cell lines. These results provide molecular evidence that cell lines of the same pathological origin can be highly heterogeneous.

Previous studies have demonstrated the irradiation-induced phosphorylation of p53 at Thr18 and Ser20, residues integral within an a-helical segment of the transactivation domain. Importantly, phosphorylation at either site has been correlated with decreased binding to the inhibitory partner Mdm-2 and enhanced transactivation of p53 target genes. In this study, we investigated the impact of Asp substitution at Thr18 and Ser20 (p53T18D/S20D) on the functional regulation of p53. Asp substitution is commonly accepted as a means of mimicking phosphorylation due to the introduction of negative charge within the functional group. p53T18D/S20D was refractory to in vitro digestion by calpain, a protease recognizing a-helical structure within the transactivation domain. In addition, transfected p53T18D/S20D poorly bound GST-Mdm-2 in vitro, enhanced the endogenous expression of the p53 transactivation targets p21 Waf1/Cip1 and fas/APO-1, and significantly curtailed cell proliferation relative to wild-type p53 transfected cells. Thus, Asp substitution at Thr18 and Ser20 within the a-helical segment of the transactivation domain reduced Mdm-2 interaction, upregulating transactivation of cell-cycle and apoptotic regulatory targets, curtailing cellular proliferation.