Interaction with fibronectin regulates cytokine gene expression in human melanoma cells.

Our study was aimed at investigating whether interaction of human melanoma cells with the extracellular matrix (ECM) protein fibronectin (FN) could regulate lymphokine gene expression. Serum-deprived cells (quiescent condition) of a metastatic melanoma cloned line were cultured either on uncoated or on FN- or BSA-coated surfaces. By means of reverse transcriptase- polymerase chain reaction (RT-PCR), we analyzed mRNA expression of 4 cytokines interleukin (IL)-1alpha, IL, 1beta, IL-6 and IL-8-and 9 growth factors-endothelial cell growth factor (ECGF), basic fibroblast growth factor (bFGF), fibroblast growth factor (FGF)-5, HST, keratinocyte growth factor (KGF), transforming growth factor (TGF)-alpha TGF-beta1, TGF-beta2 and TGF-beta3. When cultured on FN, melanoma cells expressed IL-1beta and IL-6 transcripts in addition to IL-1beta, IL-8, ECGF, TGF-beta1, TGF-beta2 and TGF-beta3, already present in quiescent cells. Amplification parameters to achieve semi-quantitative RT-PCR were then determined for each detectable factor, thus allowing us to measure a selective enhancement of mRNA levels for IL-1alpha, IL-6, IL-8 and TGF-beta2 upon interaction with FN by quiescent melanoma cells. This augmented expression was inhibited by an anti-integrin beta1 chain monoclonal antibody (MAb). Moreover, the amounts of IL-6, IL-8 and IL-beta produced in the supernatants, as assessed by ELISA, correlated with the corresponding mRNA expression. Extension of this analysis to the other 5 human primary and metastatic melanoma lines confirmed the ability of FN to selectively up-regulate only IL-6 and IL-8 secretion. Our data indicate that FN is able to modulate expression and secretion of a defined subset of lymphokines in human melanoma.

Expression of interleukin 1 alpha, interleukin 6, and tumor necrosis factor alpha genes in human melanoma clones is associated with that of mutated N-RAS oncogene.

To assess whether RAS oncogenes may affect the expression of cytokines in tumor cells, the presence of interleukins (IL) 1 alpha, 1 beta, 4, 6, 7, and 8, tumor necrosis factor (TNF) alpha and interferon gamma mRNA has been analyzed by reverse transcriptase-polymerase chain reaction in 19 melanoma clones derived from the metastatic cell line 665/2 and previously characterized for RAS mutation and expression. Five of these clones and the parental cell line showed a mutation at codon 61 of N-RAS that resulted in Gln-->Arg substitution (N-RAS/61+), while in the remaining 14, only the wild-type allele for N-RAS was present (N-RAS/61-). With the exception of interferon gamma and IL-4, all the cytokines tested were expressed by the parental 665/2 cell line, whereas IL-1 alpha, IL-6, and TNF-alpha were coordinately transcribed only in the subset of the clones bearing the mutated N-RAS gene. The other cytokine genes studied (IL-1 beta, IL-4, IL-7, and IL-8) displayed a variable degree of expression, and such an heterogeneity was not correlated to the N-RAS phenotype of the clones. The association between N-RAS oncogene and IL-1 alpha, IL-6, and TNF-alpha expression was also found in a 665/2 subline (665/2/5) in which loss of mutated N-RAS genes simultaneously occurred with the loss of IL-1 alpha, IL-6, and TNF-alpha expression. Direct evidence that N-RAS oncogene could influence the pattern of cytokine expression was provided by the coordinate induction of IL-1 alpha, IL-6, and TNF-alpha messenger RNA achieved in N-RAS/61+ transfectants of the N-RAS wild-type melanoma clone 2/21. Furthermore, IL-1 alpha, IL-6, and TNF-alpha could be detected by enzyme-linked immunosorbent assay in the culture medium obtained from N-RAS/61+ melanoma clones as well as from positive transfectants, indicating that lymphokine mRNA expression triggered by the activated N-RAS oncogene lead to a secreted protein. In an N-RAS/61+ melanoma clone, by adding specific antibodies against each cytokine, it was found that soluble IL-1 alpha exerted a positive control on IL-6 mRNA and a negative one on its own expression. In addition, IL-1 alpha and IL-6 were negatively regulated by soluble IL-6 and TNF-alpha.

Involvement of somatically acquired ecotropic viruses in the immunogenicity of nude-transplanted NIH/3T3 transformed cell lines.

Immunogenic tumor variants were previously derived after transplantation in vivo into nude mice of NIH/3T3-transformed cell lines. Nude-passaged cell lines were rejected by immunocompetent H-2q NIH mice, were recognized by specific CTL clones, and expressed new retroviral Ag. The aim of the present work was to investigate whether somatically acquired proviral sequences were present in the genome of nude-passaged cells and to test directly for a causative relationship between murine leukemia virus (MuLV) expression and immunogenicity. Southern blot analysis of PstI-digested DNA indicated that in contrast to the parental NIH/3T3 transformed cell lines (pT, T12N/5a, NS-1) all the nude-passaged immunogenic variants (pT-nude, T12N/5a-nude, NS-1-nude) contained newly acquired ecotropic-related proviruses. Immediately after in vitro establishment, these tumors displayed multiple integration sites as assessed by analysis of 3' proviral-cellular junctions. Long term in vitro culture of one of the cell lines (pT-nude) resulted in a cell line (pT-nude/vitro) that was clonal or oligo-clonal with respect to viral integration. Northern blot analysis established that the new proviruses were actively transcribed in all the immunogenic variants. To assess whether the somatically acquired ecotropic proviral sequences encode for target structures recognized by specific CTL, obtained after immunization of NIH mice with pT-nude, the parental cell line pT was transfected with plasmids containing the entire AKV MuLV genome, the cloned AKV gag or env genes. Screening of transfectants for their ability to stimulate the production of TNF by anti-pT-nude effectors indicated that cells transfected with the entire ecotropic virus or with MuLV-env gene products could be recognized by an NIH anti-pT-nude CTL line and NIH anti-pT-nude Kq-restricted CTL clones as well as the immunizing target pT-nude.

Mitochondrial enzymatic adaptation of skeletal muscle to endurance training.

Some mitochondrial enzymatic activities (succinate dehydrogenase, NADH cytochrome reductase, cytochrome oxidase) were studied in the gastrocnemius and soleus muscle of the rat. The modifications of the enzyme activity, induced by endurance training, were found to be functions of 1) daily work load and 2) total training time. The treatment with an effective dose of vasodilating substances (papaverine, nicergoline, dipyridamole, and bamethan) showed that 1) nicergoline, bamethan, and dipyridamole were differently able to shorten the time of appearance of the increase in the enzymatic activities; 2) however, long-term treatments with these drugs did not prove able to modify the plateau level of the enzymatic activity increase, for a given amount of endurance training; 3) the pharmacodynamic effect on enzymatic activities was in no way related to the vasodilating effect of these drugs, since the effect was not observed with papaverine. The transition from a given level of endurance training to a lower one led to a proportional decrease of the mitochondrial enzymatic activities, thus pointing out the relation between amount of training and enzymatic activity. The drugs studied were unable to modify the decrease of enzymatic activity induced by lower work load.