Resistance to TNF-induced cytotoxicity correlates with an abnormal cleavage of cytosolic phospholipase A2.

To investigate the mechanism underlying the absence of arachidonic acid (AA) release by TNF in TNF-resistant cells, we first performed comparative analysis of phospholipid pools in both TNF-sensitive (MCF7) and their equivalent resistant cells (C1001). Quantification and incorporation studies of [(3)H]AA indicated that TNF-resistant cells were not depleted in AA. Furthermore, distribution of this fatty acid in different phospholipid pools was similar in both sensitive cells and their resistant counterparts, ruling out a defect in phospholipid pools. Since phospholipase A(2) (PLA(2)) are the main enzymes releasing free AA, we investigated their relative contribution in the acquisition of cell resistance to TNF-induced cell death and AA release. For this purpose, we used two PLA(2) inhibitors, methylarachidonyl fluorophosphate (MAFP) and bromoenol lactone (BEL), which selectively and irreversibly inhibit the cytosolic PLA(2) (cPLA(2)) and the Ca(2+)-independent PLA(2), respectively. Although a significant inhibitory effect of MAFP on both TNF-induced AA release and PLA(2) activity in MCF7 was observed, BEL had no effect. The inhibitory effect of MAFP on cPLA(2) activity correlated with an inhibition of TNF-induced cell death. Western blot analysis revealed that TNF induced a differential cleavage of cPLA(2) in TNF-sensitive vs TNF-resistant cells. Although the p70 (70-kDa) form of cPLA(2) was specifically increased in TNF-sensitive cells, a cleaved form, p50 (50 kDa), was selectively observed in TNF-resistant C1001 cells in the presence or absence of TNF. These findings suggest that the acquisition of cell resistance to this cytokine may involve an abnormal cPLA(2) cleavage.

Overexpression of p53 mRNA in colorectal cancer and its relationship to p53 gene mutation.

We analysed the frequency of p53 mRNA overexpression in a series of 109 primary colorectal carcinomas and its association with p53 gene mutation, which has been correlated with short survival. Sixty-nine of the 109 cases (63%) demonstrated p53 mRNA overexpression, without any correlation with stage or site of disease. Comparison with p53 gene mutation indicated that, besides cases in which p53 gene mutation and p53 mRNA overexpression were either both present (40 cases) or both absent (36 cases), there were also cases in which p53 mRNA was overexpressed in the absence of any mutation (29 cases) and those with a mutant gene in which the mRNA was not overexpressed (four cases). Moreover, the mutant p53 tumours exhibited an increase of p53 mRNA expression, which was significantly higher in tumours expressing the mutated allele alone than in tumours expressing both wild- and mutated-type alleles. These data (1) show that p53 mRNA overexpression is a frequent event in colorectal tumours and is not predictive of the status of the gene, i.e. whether or not a mutation is present; (2) provide further evidence that p53 protein overexpression does not only result from an increase in the half-life of mutated p53 and suggest that inactivation of the p53 function in colorectal cancers involves at least two distinct mechanisms, including p53 overexpression and/or mutation; and (3) suggest that p53 mRNA overexpression is an early event, since it is not correlated with Dukes stage.

Neoplastic progression of human and rat intestinal cell lines after transfer of the ras and polyoma middle T oncogenes.

BACKGROUND: Activation of the p21ras and pp60c-src oncoproteins occurred at high incidence in the early stage of human colorectal carcinogenesis. Our study aimed to investigate the role of these signal-transduction pathways in the process of initiation and promotion of the malignant phenotype in intestinal cells. METHODS: The human Ha-ras and the polyoma middle T (Py-MT) viral oncogenes were transferred into large T oncogene of simian virus 40 immortalized rat intestinal epithelial SLC-44 cells and human colonic adenocarcinoma Caco-2 cells. RESULTS: These transfers conferred the tumorigenic and invasive phenotypes on immortalized SLC-44 cells and potentiated the tumorigenicity of Caco-2 cells and markedly repressed the terminal differentiation of this cell line. In SLC-44T cells, induction of the invasive phenotype by the activated Ha-ras oncogene correlated with weak expression of E-cadherin and reduced accumulation of the transcripts encoding the basement membrane components alpha 1 (IV) collagen, nidogen, and BM40, which might result partly from the inactivation of the transforming growth factor beta signaling pathway. The down-regulation of the alpha 1 (IV) collagen messenger RNA in SLC-44T cells was not due to the protein kinase C-dependent pathways or the secretion of autocrine factor(s). CONCLUSIONS: These results suggest that the activation of the p21ras and Py-MT/pp60c-src oncogenic pathways are critical effectors at different stages of colorectal carcinogenesis and in Caco-2 cells interferes with the program of enterocyte differentiation.