Crude Pedersen fetuin, derived from fetal bovine serum, contains adipogenic activity. Biochemical characterization was undertaken by following the differentiation of the 1246 adipogenic cell line. The present paper provides evidence that crude fetuin contains distinct proteins with adipogenic activity. By molecular sieve fractionation using Sephacryl S-300, the majority of adipogenic activity eluted in two distinct peaks, FI (molecular weight greater than 669 kDa) and FII (molecular weight ranging from 445 and 232 kDa). In addition a minor activity was found in a third peak, FIII (molecular weight around 69 kDa). Partial purification and biochemical characterization indicate that FI and FII are two distinct factors. FI has a PI higher than 9.4, is destroyed by alkaline treatment, and is stable when treated with acid. FII has a PI lower than 4.0, is alkali stable, but is destroyed completely by treatment with acid. Moreover, our data show that adipogenic factors are distinct from another protein alpha 2 macroglobulin known to be found in crude Pedersen fetuin. These results suggest that serum contains two large molecular weight proteins bearing adipogenic activity which could play an important role in the control of the adipose differentiation process.

Four temperature-sensitive (ts) mutants of rat 3Y1 cells (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) are arrested at 39.8 degrees C mainly with a 2N DNA content (temperature-arrested cells). The states of these cells were compared with findings in case of cells arrested at 33.8 degrees C at saturation density (density-arrested cells), with regard to the ability to enter S phase after release from arrest or after serum stimulation at 39.8 degrees C. With the 3Y1tsD123, the ts defect is an event which seems essential for the initiation of S phase and occurs after mitosis but not after release from the density arrest. The defect in 3Y1tsF121 related to the efficiency of utilization of serum component(s). In case of 3Y1tsG125, the state of temperature arrest appeared to locate between the state of density arrest and the beginning of S phase. There was no significant difference between the density- and the temperature-arrested cells, in case of 3Y1tsH203.

Alterations in cellular characteristics associated with transformation were compared among various agents, including simian virus 40, mouse polyoma virus, adenovirus type 12, the ElA region of adenovirus type 12, Rous sarcoma virus, v-Hras oncogene, and N-methyl-N'-nitro-N-nitrosoguanidine. To avoid the difficulty in interpretation due to differences in the original cellular characteristics, all transformed lines (three to four independent lines for each agent) were derived from the same parental line, i.e. rat clonal diploid fibroblasts 3Y1. Doubling time, saturation density, serum requirement, efficiency of colony formation, Ca dependency, anchorage dependency, cell volume, density-dependent inhibition of proliferation, and alteration of morphology were examined. In the lines transformed either by DNA virus, RNA virus, or transforming oncogene, the characteristics were similar among the lines induced by the same transforming agent but distinguishable between the lines induced by different agents. In such cases, a transformed line displayed phenotypes specific to each transforming agent. In contrast, nitrosoguanidine-transformed lines showed different properties. Only one characteristic was shared by all the transformed lines: the ability to attain a higher saturation density than that of the untransformed parent. The fact that (cellular) densitydependent inhibition of DNA synthesis became weak may be responsible for this characteristic.

We compared proliferation and survival of various syngeneic transformed cell lines under conditions of depletion of 15 amino acids in Dulbecco-Eagle's medium. We used a normal fibroblast line 3Y1 and 22 transformed sublines of 3Y1 which had been induced by one of seven transforming agents--simian virus 40, mouse polyomavirus, adenovirus type 12, E1A gene of adenovirus type 12, cDNA of Harvey murine sarcoma virus, Rous sarcoma virus, or N-methyl-N'-nitro-N-nitrosoguanidine. Unlike other untransformed cells examined (mouse BALB/c-3T3 line, mouse NIH-3T3 line, and primary Fischer rat embryo fibroblasts), 3Y1 ceased to proliferate and accumulated in a viable state with a G1-phase DNA content under 14 singular deprivations of amino acid. None of the transformed 3Y1 lines completely arrested in the G1 phase of the cell cycle and each showed different levels of survival, depending on each transforming agent. As for transformed 3Y1 cells induced by a given virus or a given transforming gene, any one of the three sublines shared the same trend with respect to proliferation and survival. Transformed derivatives induced by N-methyl-N'-nitro-N-nitrosoguanidine showed almost the same trend in proliferation, but the patterns of survival were not uniform. Our observations suggest that the unique responses of 3Y1 to amino acid depletion are differently modified by different transforming agents.

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