Inhibition of the Jun kinase pathway blocks DNA repair, enhances p53-mediated apoptosis and promotes gene amplification.

We have previously shown, by expression of a nonphosphorylatable dominant inhibitor mutant of c-Jun [cJun(S63A,S73A)], that activation of the NH2-terminal Jun kinase/stress-activated protein kinase by genotoxic damage is required for DNA repair. Here, we examine the consequences of inhibition of DNA repair on p53-induced apoptosis in T98G cells, which are devoid of endogenous wild-type p53. Relative to parental or wild-type c-Jun-expressing control cells, mutant Jun-expressing T98G clones show similar growth rates and plating efficiencies. However, these cells are unable to repair DNA (PCR-stop assays) and exhibit up to an 80-fold increased methotrexate-induced colony formation due to amplification of the dihydrofolate reductase gene. Moreover, the mutant c-Jun clones exhibit increased apoptosis and elevated bax:bcl2 ratios on expression of wild-type p53. These results indicate that inhibition of DNA repair leads to accumulation of DNA damage in tumor cells with unstable genomes and this, in turn, enhances p53mediated apoptosis.

Sensitization of rat glioblastoma multiforme to cisplatin in vivo following restoration of wild-type p53 function.

OBJECT: To study the combined potential of wild-type p53 gene transfer and administration of cisplatin for the treatment of glioblastoma multiforme, the authors used the 9L rat glioblastoma cell line, which expresses a mutant p53. METHODS: Stable expression of wild-type p53 in 9L cells was achieved by transfection of the cells with a wild-type p53-expressing plasmid (pCEP4p53). The resultant cell line, 9LpCEP4p53, was found to be more sensitive to cisplatin treatment in vitro than control (9LpCEP4) cells. The in vitro growth rates of control cells and wild-type p53-modified cells were similar in the absence of cisplatin. Fischer 344 rats were implanted intracerebrally with 9LpCEP4p53 cells and intraperitoneally administered 4 mg/kg cisplatin weekly for 7 weeks. These animals survived significantly longer than animals that were implanted with 9LpCEP4p53 cells but were given no cisplatin treatment. In contrast, concurrent cisplatin treatment provided no benefit for animals implanted with 9LpCEP4 cells. Tumors that developed in animals that had been implanted with 9LpCEP4p53 cells and treated with cisplatin had lost expression of wild-type p53, indicating a correlation between expression of wild-type p53 and cisplatin sensitivity in vivo. CONCLUSIONS: The findings of this study suggest that p53-based gene therapy in combination with cisplatin-based chemotherapy may be superior to single-modality treatment in dealing with glioblastoma multiforme.

Use of wild-type p53 to achieve complete treatment sensitization of tumor cells expressing endogenous mutant p53.

It is known that transfer of the wild-type p53 gene into p53-negative cells from transgenic mice increases their sensitivity to drug and radiation-induced apoptosis. However, unlike many human tumors, these transgenic cells do not express mutant p53, and it is not known from these earlier studies whether wild-type p53 dominates the effects of mutant p53 with respect to drug and radiation sensitivity. We addressed this question in glioblastoma, a disease characterized by an unusually high level of intrinsic resistance to therapy and poor prognosis: mean survival time from diagnosis is only about 1 yr. We introduced the gene for wild-type p53 into human T98G glioblastoma cells, which express endogenous mutant p53 but not wild-type p53. Stable transfectants that co-expressed mutant and wild-type p53 had enhanced sensitivity to cisplatin and gamma radiation, compared with parental cells, control vector-transduced cells, and transduced cells that had lost expression of wild-type p53. Transient wild-type p53 expression after high-efficiency gene transfer by a p53 adenovirus also sensitized the cells to cisplatin and correlated with the induction of apoptosis. The sensitization effect was also observed in p53 adenovirus-infected H23 small cell lung carcinoma cells, which express endogenous mutant p53. Therefore, wild-type p53 gene transfer has dominant effects over mutant p53 in sensitizing tumor cells to therapy, which supports the potential of p53 gene therapy to enhance the efficacy of traditional therapy.

Characterization of a new human glioblastoma cell line that expresses mutant p53 and lacks activation of the PDGF pathway.

We have established and characterized a new glioblastoma cell line, termed GT9, from a biopsy sample of a female adult patient with glioblastoma multiforme. The line has now undergone over 60 passages and has been successfully cultured after cryopreservation. Immunofluorescence analyses with a panel of monoclonal antibodies were positive for glial fibrillary acidic protein and vimentin, and negative for neurofilament, galactocerebroside, and fibronectin, a pattern typical of glial cells. Based on a tetraploid, the composite karyotype of GT9 cells included the loss of chromosome 10, gain of chromosome 7, and the presence of double minute chromosomes, three of the most common karyotypic abnormalities in glioblastoma. Sequence analysis of p53 cDNA revealed a homozygous double mutation at codon 249 (commonly mutated in aflatoxin-associated hepatocellular carcinoma) and codon 250. Moreover, there was a complete absence of wild-type p53. However, unlike the majority of human glioblastomas previously described, the expression of platelet-derived growth factor-B (PDGF-B), a potent mitogenic autocrine factor, was low in GT9 cells. The expression and phosphorylation of c-Jun and Jun-B, downstream mediators of the PDGF pathway, were also low. Thus, deregulation of the PDGF pathway does not appear to be involved in the pathogenesis of the GT9 glioblastoma. Conversely, Jun-D, a negative regulator of cell growth, was also low. In addition, Phosphorylated Egr-1, a recently reported suppressor of PDGF-B/v-sis-transformed cells, was also low, suggesting that the lack of activation of the PDGF pathway was not due to these suppressive mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)