Complexity of the Early Genetic Response to Growth Factors in Mouse Fibroblasts

Abstract

Genes whose expression is growth factor regulated are likely to be important components in the mechanisms controlling cell proliferation and differentiation. With the aim of identifying some of those genes, a lambda cDNA library was prepared with poly(A)+ RNA from quiescent NIH 3T3 cells stimulated with serum for 4 h in the presence of cycloheximide. Differential screening of approximately 200,000 recombinant phage plaques revealed 2,540 clones that cross hybridized preferentially with [32P]cDNA derived from RNA of stimulated cells rather than with cDNA derived from nonstimulated cells. Cross hybridization of these clones identified 82 independent sequences, including c-fos and c-myc. Seventy-one clones were further studied. Analysis of the changes in transcription and mRNA levels after serum stimulation demonstrated that the kinetics and extent of the induction vary dramatically between the different genes. Cycloheximide in all cases superinduced the mRNA levels by two mechanisms, inhibiting the shutoff of transcription and prolonging the half-lives of the mRNAs. Our results showed that induction of proliferation is accompanied by the onset of a complex genetic program.

Growth Factor-Induced Delayed Early Response Genes

“…Cells were grown to confluence and made quiescent by changing the medium to MEM-0.5 for 3 days. For each time point, four 15-cmdiameter dishes were stimulated with MEM-20, PDGF (50 ng/ml), or FGF (50 ng/ml), and at the indicated time, cells were rinsed three times with ice-cold phosphate-buffered saline and nuclei were isolated as described previously (1). Labelled RNA was prepared and isolated as previously described (15,16), except that the base hydrolysis step was omitted.…”

“…We and others have been analyzing a prototypic genetic program induced by extracellular signaling agents, namely the program induced in the mouse fibroblastic 3T3 cell line by serum or purified growth factors. When nondividing mouse 3T3 cells are stimulated by serum, platelet-derived growth factor (PDGF), or fibroblast growth factor (FGF), a set of specific genes is activated within minutes, even in the presence of an inhibitor of protein synthesis (1,9,25). These primary response genes have been called immediate-early genes, analogous to the immediate-early genes of viruses.…”

Structure, Chromosome Location, and Expression of the Mouse Zinc Finger Gene Krox-20: Multiple Gene Products and Coregulation with the Proto-Oncogene c-fos

“…Therefore, it appeared likely that intron 1 was subject to alternative splicing but that the Northern blotting procedure did not allow distinction between the two RNA molecules generated. The intron probe was then used to screen the original lambda NM1149 cDNA library from which the AC16 DNA had been isolated (1,10). Nine clones among approximately 300,000 screened plaques hybridized to the probe on duplicate filters.…”

“…Serum stimulation of quiescent fibroblasts leads to rapid activation of a number of so-called immediate-early response genes, including the proto-oncogenes c-fos and c-myc, whose expression has been demonstrated to be essential to reach DNA synthesis (33,35,52,60). More than 80 unrelated cDNAs corresponding to immediate-early genes have been isolated in different laboratories (1,14,38,39,43). The recent observation that some of these genes encode potential transcription factors (10,15,37,42,56,61,62, 66; M. Zerial et al, unpublished data) is of particular interest since they might therefore represent members of the hierarchy of regulatory genes controlling cell proliferation.…”

“…Indeed, cycloheximide leads to superinduction of the genes, * Corresponding author. due to both stabilization of the mRNA and prolongation of the period of transcription (1,10,42). Both Krox-20 and Krox-24 encode proteins containing three zinc finger domains (10,42,48,66).…”