A set of growth arrest-specific (gas) genes negatively regulated by serum has been identified. To define the role of gas genes in a model of cell proliferation in vivo we analyzed the expression of one of these genes (gas-6) during liver regeneration after partial hepatectomy (PH). We found that gas-6 mRNA was down-regulated 4 hours after PH, within the G0 to G1 transition. Later on, gas-6 mRNA increased over the level found in normal liver with a peak at 16 hours, before the onset of DNA synthesis. This surge was probably triggered by an inflammatory response caused by the surgical trauma, because an increase of similar extent occurring with the same time course was present in livers of sham-operated and turpentine-treated rats. Comparison of mRNA steady state levels with nuclear transcription rates indicated that gas-6 expression is post-transcriptionally regulated. As we found that down-regulation of gas-6 expression was prevented by treatment with Actinomycin D, a labile protein might be involved in the determination of gas-6 mRNA stability. To investigate the mitogenic signals controlling gas-6 expression during liver regeneration we treated hepatectomized rats with a specific alpha-1-adrenoceptor blocker (prazosin) as well as with drugs which modify intracellular calcium levels. The decrease of gas-6 mRNA 4 hours after PH was prevented by prazosin and by neomycin, an inhibitor of calcium release from endogenous stores. These findings suggest that down-regulation of gas-6 expression during hepatic regeneration is triggered by catecholamines interaction with alpha-1-adrenergic receptors and by subsequent calcium release. In addition we found that the rise of gas-6 gene expression occurring at 16 hours after PH was not affected by prazosin but was inhibited by trifluoperazine. Therefore, we suggest that up-regulation of gas-6 gene expression is mediated by the interaction of calcium with calmodulin, independently of catecholamines.
gas-1 belongs to a family of growth arrest specific genes negatively regulated after growth induction of arrested cells. We report the expression of gas-1 in an in vivo system of cell proliferation. gas-1 mRNA accumulates progressively in the uterus of ovariectomized rats with a peak at three weeks after surgery. After estrogen treatment gas-1 mRNA levels decrease within two hours, at a time when c-myc expression is greatly increased, and return to pretreatment levels at 48 hours. Treatment with cycloheximide does not prevent estrogen-induced down-regulation of gas-1 mRNA levels. The present results show that: i) estrogen affects the uterine growth state by regulating the expression of both positively and negatively acting genes, ii) gas-1 expression is controlled by cellular growth state also in vivo.
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.
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