Expression of growth-regulated genes in tsJT60 cells, a temperature-sensitive mutant of the cell cycle

We have investigated the expression of growth-regulated genes in tsJT60 cells, a temperature-sensitive (ts) mutant of Fischer rat cells, which, on the basis of its kinetic behavior, can be classified as a G0 mutant. It grows normally at 34 degrees C and also at 39.5 degrees C if shifted to the higher temperature during exponential growth. However, if the cell population is first made quiescent by serum deprivation, subsequent stimulation by serum induces the cells to enter S phase at 34 degrees C but not at 39.5 degrees C. A panel of growth-regulated genes was used that included three protooncogenes (c-fos, c-myc, and p53), several genes that are induced in G0 cells stimulated by growth factors (beta-actin, 2A9, 2F1, vimentin, JE-3, KC-1, and ornithine decarboxylase), and an S-phase gene (histone H3). The expression of these growth-regulated genes was studied in both tsJT60 cells and its parental cell line, rat 3Y1 cells. All the genes tested, except histone H3, are similarly induced when quiescent tsJT60 cells are stimulated by serum at either permissive or restrictive temperatures. These results raise intriguing questions on the nature of quiescence and the relationship between G0 and G1 in cells in culture.     

Prostaglandin endoperoxide synthase (cyclooxygenase) mRNA and protein production in mouse myoblasts and a differentiation-defective variant.

Northern blot analysis revealed that a differentiation-defective variant (DD-1) of MM14 mouse myoblasts has seven times the prostaglandin endoperoxide synthase mRNA than the parental MM14 myoblasts. There was an even greater increase in the level of prostaglandin endoperoxide synthase protein in the DD-1 cells as compared to that in the MM14 myoblasts. In fact, prostaglandin endoperoxide synthase was not detectable by Western blot analysis of extracts from MM14 myoblasts. Since prostaglandin endoperoxide synthase has been reported to be a gene whose expression is induced transiently, i.e., growth-regulated, upon mitogen stimulation of quiescent cells, the RNA abundance of other growth-regulated genes was examined including: KC, JE, c-myc, 1B6, and vimentin. Northern blot analysis revealed that the mRNA abundance of JE, KC, and c-myc is 12-, 17-, and 2-fold higher, respectively, in growing DD-1 cells than in growing MM14 myoblasts. In contrast, there was little difference in the mRNA abundance of 1B6 and vimentin. These results are consistent with the hypothesis that increases in the levels of expression of prostaglandin endoperoxide synthase and some growth-regulated genes are integral to the expression of the differentiation-defective phenotype and may in fact contribute to this phenotype.     

The growth-regulated gene 1B6 is identified as the heavy chain of calpactin I

The expression of 1B6, a growth-regulated sequence isolated from a Syrian hamster fibroblast cDNA library, was studied in BALB/c 3T3 cells. The level of cytoplasmic 1B6 mRNA (1600 bases) was low in quiescent cells and plateaued in mid/late G1 after the cells were stimulated with 15% fetal calf serum (FCS). Protein synthesis was not required for the induction of 1B6 mRNA; therefore, the expression of 1B6 is a primary response to serum stimulation. The induction of 1B6 mRNA was also observed after stimulation with insulin, epidermal growth factor, and fibroblast growth factor but not with platelet-derived growth factor. When quiescent cells were serum-stimulated, the percentage of cells that became committed to enter DNA synthesis was proportional to the length of their incubation with serum. To determine if 1B6 expression was also correlated with the time of exposure to serum, quiescent cells were stimulated with a pulse of 15% FCS and the abundance level of 1B6 induced by that pulse was determined. The amount of 1B6 mRNA increased with increasing time of exposure to serum and paralleled the increase in the percentage of nuclei that were induced into DNA synthesis by the serum pulse. Comparison of the nucleotide sequence of the p1B6 cDNA to the GenBank database revealed a striking identity of 1B6 to the 3' end of p36, the heavy chain of calpactin I. The previous characterization of p36 as a substrate for tyrosine kinases suggests a possible role for 1B6/p36 in cell proliferation.     

Expression of c-myc and induction of DNA synthesis by platelet-poor plasma in human diploid fibroblasts

When WI-38 human diploid fibroblasts become confluent, they stop synthesizing DNA and dividing. Addition of serum causes the quiescent cell to reenter the cell cycle. Prolonged quiescence after confluence decreases and delays the response to serum. For a few days after reaching confluence, WI-38 cells also respond to platelet-poor plasma. During this period, although not cycling, WI-38 cells still express c-myc and other growth-regulated genes, as measured by steady-state RNA levels. If the quiescence is prolonged further, c-myc expression (and that of two other growth-regulated genes) is no longer detectable, and its disappearance coincides with a loss of response to platelet-poor plasma. These results suggest that, also under physiological conditions, the expression of c-myc and other growth-regulated genes can cooperate with platelet-poor plasma in inducing cellular DNA synthesis in human diploid fibroblasts.