Resistance of MCF7 human breast carcinoma cells to TNF-induced cell death is associated with loss of p53 function.

We have investigated the relationship between the development of tumor resistance towards the cytotoxic action of tumor necrosis factor-alpha (TNF) and p53 function, using the TNF-sensitive MCF7 human breast adenocarcinoma cell line and two TNF-resistant sublines, MCF7/R-A1 and MCF7/Adr. Use of single-strand conformation polymorphism (SSCP) analysis and DNA sequencing shows that MCF7 has a wild-type p53 gene, whereas both TNF-resistant sublines exhibit mutant p53. This includes a point mutation R280K in MCF7/R-A1 cells, and a point mutation at the splicing acceptor site on the upstream border of exon 5 resulting in a 21 pb deletion in MCF7/Adr cells. These mutations result in loss of p53 capacity to transactivate FASAY (functional assay in yeast). In contrast to what is observed for parental MCF7 cells, treatment of resistant sublines with TNF or gamma-irradiation fails neither to induce the expression of the p53-regulated gene products p21waf1/CIP1 and MDM2, nor to arrest the cells in the G1 phase of the cell cycle. Disruption of p53 wild-type function in MCF7 cells by transfection with human papillomavirus type-16 E6 gene, leads to abrogation of the cytotoxic, but not the cytostatic activity of TNF. Altogether, our results strongly suggest that wild-type p53 is involved in cytotoxic action of TNF, and point out that loss of p53 function contributes to resistance of tumor cell to TNF-induced killing.

High incidence of the t(2;5)(p23;q35) translocation in anaplastic large cell lymphoma and its lack of detection in Hodgkin's disease. Comparison of cytogenetic analysis, reverse transcriptase-polymerase chain reaction, and P-80 immunostaining.

Fifty-six cases of anaplastic large cell lymphoma (ALCL), 23 cases of Hodgkin's disease, and 16 cases of diffuse large cell lymphoma were investigated for the t(2;5)(p23;q35) translocation. The translocation was detected by using cytogenetic analysis, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry with P80 antibody directed against the kinase domain of anaplastic lymphoma kinase (ALK) of the chimeric NPM/ALK protein. In all but three cases of ALCL, we found an agreement between cytogenetic analysis, RT-PCR, and P80 staining. However, in one case, the t(2;5) translocation was detected with cytogenetic analysis, but RT-PCR and P80 staining were found to be negative. Conversely, in another case the karyotype was normal, but the hybrid mRNA and P80 staining were found to be positive. In one case, malignant cells showed a translocation involving chromosomes 1q25 and 2p23 and were strongly positive for P80 staining. Such a result could be expected because P80 antibody detects the kinase domaine of the ALK protein encoded by chromosome 2p23. Overall 73.2% (41 of 56) of cases were found to be positive. However, the highest percentage (23 of 26 cases; 88.5%) of P80 positive cases was found in children compared with 60% (18 of 30 cases) in adult ALCL (P < .05). In Hodgkin's disease, Reed-Sternberg cells were found to be clearly negative by RT-PCR and with P80 antibody. The latter results suggest that Hodgkin's disease and t(2;5)-positive ALCL are distinct biological entities and that the demonstration of the t(2;5) translocation is of diagnostic importance in differentiating these two entities. The results of the present study indicate that immunohistochemistry with P80 antibody is a reliable method for detecting NPM/ALK chimeric protein.