The expression (mRNA level of enzymic activity) of cytosolic and nuclear spermidine acetyltransferases was studied in NIH 3T3 fibroblasts, either (1) serum-starved and stimulated to grow by serum refeeding, or (2) treated with inhibitors of ornithine decarboxylase (ODC) (MDL 72.175) and S-adenosylmethionine decarboxylase (AdoMetDC) (MDL 73.811) and stimulated to grow by spermidine. Expression of the known growth-regulated genes for ODC, AdoMetDC and histone acetyltransferase was also examined. The mRNA for spermidine/spermine N1-acetyltransferase (SAT) accumulated after serum refeeding (between 6 and 16 h) and even more after spermidine addition (16 h). Histone acetyltransferase activity increased after both growth stimuli, whereas spermidine N8-acetyltransferase activity remained unchanged. After serum stimulation, the ODC mRNA level and activity rose between 6 and 16 h, whereas AdoMetDC mRNA accumulation occurred later (16 h) than the increase in enzyme activity (6 h). Stimulation of ODC and AdoMetDC activities was suppressed by the inhibitors added alone or in combination with spermidine, whereas mRNA accumulation was down-regulated by spermidine. These results indicate that the expression of SAT was growth-controlled and that SAT mRNA level was regulated by polyamines.
Summary A set of growth arrest-specific (gas) genes negatively regulated by serum has been identified. We report the analysis of the expression of one of them (gas-i) in transformed cells. We found a down regulation of gas-I expression in NIH 3T3 cells transfected in vitro with an activated Ha-ras oncogene. In five chemically-induced mouse tumours grown in vivo the amounts of gas-I mRNA were largely different but not related to the proliferating activity (evaluated by both H3 histone expression and 3H-thymidine incorporation into DNA). The amount of gas-I mRNA in the tumours was in general higher than in normal tissues. Expression of c-myc was also evaluated and found to be high in tumours which exhibited low gas-I expression. Two fibrosarcomas, CA-2 and CB-20, with similar phenotype, similar growth rate, different expression of c-myc and 100-fold difference in gas-I expression were further investigated and gas-I expression was found to be correlated with the expression of a differentiated function (as judged from collagen expression). Cell lines derived from CA-2 and CB-20 and maintained under different culture conditions showed that the cell cycle regulation and serum response of gas-I expression were lost in CA-2. The higher steady state level of gas-I mRNA in spite of a shorter mRNA half life suggests that in CB-20 cells the gas-I gene is transcribed faster than in CA-2 cells indicating that transcriptional regulation is the major determinant of gas-I gene expression in tumour cells. The finding of gas-I expression in tumour cells suggests that its expression is not sufficient to maintain cells into quiescence, however, as a marker specific for the Go phase, it could be useful, in conjunction with other growth related genes, to define the cell cycle distribution of a cell population.
Osteoqectin is one of the major non-collagenous proteins of bone. However, its transcript has been found in many soft, extracellular matrix-producing tissues; an osteonectin-related protein was detected in tumor basement membrane. We have investigated the expression of osteonectin gene in fresh BALB/c fibroblasts transformed by v-Ki-ras. Transformed cells exhibited lower levels of RNA as compared with normal fibroblasts. The transformed cells were cloned after in vivo tumorigenic assay, and 4 clones were analyzed for osteonectin expression by Northern blots. Two of them were selected for high or low osteonectin expression and tested in vivo in spontaneous and artificial metastasis assays. High osteonectin expression was correlated with high lung colonization. When 10s cells were injected i.v., median colony value was 55 and 20 in higher expressor vs. lower expressor respective (p < 0.005). Spontaneous metastasis indicates a possible reverse correlation. Our data align osteonectin with ot R er matrix-components and adhesion molecules in affecting potential metastatic spreading of transformed cefls.
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