SV40 expression in human neoplastic and non-neoplastic tissues: perspectives on diagnosis, prognosis and therapy of human malignant mesothelioma.

We have recently demonstrated the association of SV40 and human pleural malignant mesothelioma. Here, we have investigated whether SV40 viral sequences may be associated with other human tumours or other non-neoplastic pathology and whether SV40 DNA or protein expression may be of diagnostic, prognostic or therapeutic relevance. DNA was extracted from paraffin embedded tissues. SV40, JC and BK viral sequences were detected by the polymerase chain reaction and molecular hybridization with specific probes. The screening with three different sets of SV40-related primers demonstrated that 7/18 (38.8%) mesothelioma specimens were SV40 positive as well as 5/18 (27.7%) tubercular pleural lesions. None of the 18 lung cancers, nor the 20 pleural non-specific inflammatory specimens tested were positive. Twenty-five blood samples and 18 urinary sediments from MM patients were also negative. We have also found that SV40 Tag proteins are present in mesothelioma cells and tumours. Tag proteins may interfere with tumour suppressor gene products, such as p53. Preliminary results suggest that wild type p53 transgene expression, obtained after infection with recombinant adenovirus (AdCMV.p53), inhibited in vitro and in vivo proliferation, inducing apoptosis of mesothelioma cells. Infections with control viruses were ineffective. Thus, SV40 DNA and Tag expression in mesothelioma tumour cells, though probably not relevant for diagnostic or prognostic purposes, may be crucial for innovative gene therapy strategies.

Extracellular adenosine 5' triphosphate involvement in the death of LAK-engaged human tumor cells via P2X-receptor activation.

This study reports that extracellular ATP is a critical factor involved in LAK cell-mediated cytotoxicity. Human colon carcinoma LoVo cells were resistant to LAK cells as well as to ATP, while their multidrug resistant (MDR-1+) derivative, LoVo-Dx cells, were sensitive to both LAK and ATP. LoVo-Dx cells, became resistant to LAK cells and ATP after 48 h pretreatment with Phorbol 12-Myristate-13-Acetate (PMA), while 48 h pretreatment with verapamil in parallel sensitized LoVo cells to LAK cells and to ATP as well. The sensitivity to ATP and LAK cells was not related to the expression of extracellular ecto-ATPase activity on cell targets membranes. Conversely, apyrase, an enzyme with powerful ecto-ATPase activity, abolished the LAK- and ATP-mediated cytotoxicity. Furthermore, ADP-beta-S, an antagonist of ATP, abolished both LAK and ATP-mediated cell killing. Purine binding sites have been detected by radioreceptor assays with ADP-beta[35S] on the cell surface of ATP and LAK-sensitive LoVo-Dx cells. By contrast, no nucleotide receptor was found on the ATP and LAK-resistant cells. Such a putative cytotoxic purinoreceptor has been categorized as P2x purinergic receptor by a panel of synthetic nucleotides. These results demonstrate that extracellular ATP is needed for an efficient LAK cell-mediated killing of tumor cells. We propose that ATP acts as a natural amplifier of physical, or immune cytotoxic damages since it may be released in large amounts from target cells injured by several cytotoxic mediators secreted by LAK effectors.

The Ras suppressor RSU-1 localizes to 10p13 and its expression in the U251 glioblastoma cell line correlates with a decrease in growth rate and tumorigenic potential.

Rsu-1, which was isolated based on its ability to suppress transformation by v-Ras, is a highly conserved gene which shares homology with yeast adenylyl cyclase in the region required for activation by Ras. Genomic DNA clones of human RSU-1 have been isolated and used as a probe for fluorescence in situ hybridization (FISH) to assign RSU-1 to 10p13, confirming the previous results of somatic cell hybrid mapping localizing RSU-1 to chromosome 10. Screening of more than 20 human tumor cell lines for RSU-1 expression revealed that most cell lines contained abundant RSU-1 RNA and protein. However, the p33 RSU-1 protein was undetectable in the U251 glioblastoma cell line and transfection of a rsu-1 expression vector into U251 cells yielded a cell line in which rsu-1 was under the control of a regulatable metallothionein promoter. Addition of Cd2+ to the U251-Rsu-1 transfectant resulted in transcription of rsu-1 RNA and the accumulation of p33 Rsu-1 protein. Appearance of the Rsu-1 protein correlated with a reduction in growth rate of the U251-Rsu-1 transfectant. In addition, reduction in anchorage independent growth and phenotypic alteration in U251-Rsu-1 transfectant agar colonies was observed. Two U251-Rsu-1 transfectant cell lines were non tumorigenic when injected subcutaneously into athymic nude mice. These results, in conjunction with the frequent deletions observed in chromosome 10 in glioblastomas, suggest that RSU-1 loss of function may play a role in the progression of this disease.

Analysis of protein kinase C requirement for exocytosis in permeabilized rat basophilic leukaemia RBL-2H3 cells: a GTP-binding protein(s) as a potential target for protein kinase C.

The role of protein kinase C in calcium-dependent exocytosis was investigated in permeabilized rat basophilic leukaemia cells. When protein kinase C was down-regulated by phorbol myristate acetate (1 microM for 3-6 h) or inhibited by pharmacological agents such as calphostin C (1 microM) or a protein kinase C-specific pseudo-substrate peptide inhibitor (100-200 microM), cells lost the ability to secrete in response to 10 microM free Ca2+. In contrast, a short treatment (15 min) with phorbol myristate acetate, which maximally activates protein kinase C, potentiated the effects of calcium. Biochemical analysis of protein kinase C-deprived cells indicated that loss of the Ca(2+)-induced secretory response correlated with disappearance of protein kinase C-alpha. In addition, at the concentrations effective for exocytosis, calcium caused translocation of protein kinase C-alpha to the membrane fraction and stimulated phospholipase C, suggesting that, in permeabilized cells, protein kinase C can be activated by calcium through generation of the phospholipase C metabolite diacylglycerol. The delta, epsilon and zeta Ca(2+)-independent protein kinase C isoenzymes were insensitive to phorbol myristate acetate-induced down-regulation and did not, as expected, translocate to the particulate fraction in response to calcium. Interestingly, secretory competence was restored in cells depleted of protein kinase C or in which protein kinase C itself was inhibited by non-hydrolysable GTP analogues, but not by GTP, suggesting that protein kinase C might regulate the ability of a G protein(s) directly controlling the exocytotic machinery to be activated by endogenous GTP.