Proto-oncogene Transcription Research Articles

Possible role of c- fos, c-N- ras and c- mos proto-oncogenes in muscular development

Time course analyses of various proto-oncogene transcripts compared with cytoskeleton-specific and muscle-specific messenger RNAs (mRNAs) were carried out during growth and differentiation of a clonal line of rat myoblasts that retain the capacity to form non-contractile fibres in vitro. Throughout their growth phase, these cells express consistent levels of c- fos, c- myc, c-Ki- ras and c-N- ras RNA and no c- mos RNA. When the cultures approach confluency the level of c- fos RNA rises sharply 3–4-fold, peaks, and rapidly declines when muscle-specific transcripts start accumulating, to become negligible in myotube-forming cells. These changes occur whatever the concentration in seric factors. By contrast, the level of c-N- ras RNA rises up to 3-fold and both c- myc and c-Ki- ras RNAs are slowly eliminated during the myogenic process, whereas no c- mos RNA is detectable. However, skeletal muscles from prenatal fetuses and adult animals were reproducibly found to contain both low and high levels of c- mos RNA respectively. These data and the demonstration that inactivation of the c- fos gene correlates with the loss of myogenic capability in six lines of neoplastic myoblasts, including four lines transformed by the v- fos oncogene, suggest a physiological function for this proto-oncogene during early stages of myogenesis and for the c-N- ras and c- mos genes in later stages of muscular development.

Oncogene expression in reticuloendotheliosis virus-transformed lymphoid cell lines and avian tissues.

The genome of reticuloendotheliosis virus (REV-T) contains a unique oncogene, designated v-rel, which is inserted into the env region. Employing a cloned rel DNA probe, a single 2.9- to 3.0-kilobase v-rel mRNA was identified in poly(A)+ RNA from REV-T-transformed lymphoid cell lines. A 4.0-kilobase rel-specific transcript corresponding to the cellular homolog of the v-rel oncogene was identified in MSB-1 cells, a herpesvirus-transformed lymphoid cell line. Cytodot hybridization was used to quantitate the levels of rel, c-rel, c-myc, c-myb, c-abl, c-fms, c-Ha-ras, c-Ki-ras, c-src, c-yes, c-mos, and c-sis mRNA in REV-T-transformed cells. The levels of rel transcription in REV-T-transformed cells were elevated only two to eightfold over levels found in the transformed immature avian lymphoid cell line MSB-1. The relatively modest levels of rel transcription in REV-T-transformed cells and the significant differences between the lengths of the v-rel and c-rel mRNA suggest that REV-T transformation is the result of the production of an altered rel protein. The c-rel proto-oncogene is expressed in all avian hematopoietic tissues but is not expressed at significant levels in brain and muscle. The transcription of other proto-oncogenes is not enhanced in REV-T-transformed lymphoid cell lines.

Modulation of c-fos and c-myc mRNA levels in normal human lymphocytes by calcium ionophore A23187 and phorbol ester.

A transient expression of the proto-oncogenes c-fos and c-myc is rapidly induced by growth factors or mitogens in different cell types including lectin-stimulated lymphocytes. To elucidate the early events of lymphocyte activation, we determined (by Northern blot analysis) the steady-state levels of c-fos and c-myc mRNA in normal human peripheral blood lymphocytes (PBL) stimulated with the Ca2+ ionophore A23187 and/or 12-O-tetradecanoylphorbol-13-acetate (TPA), whose biological activities are well defined. We report that ionophore A23187 (0.5 microM) or, to a significantly lesser extent, TPA (0.5 ng/ml), neither of which is mitogenic alone at these concentrations, nevertheless can induce a transient accumulation of the proto-oncogene transcripts. These results indicate that a significant accumulation of c-fos and c-myc mRNA can occur without subsequent lymphocyte proliferation. The combination of these two mitogens increases the induced levels of both types of c-onc mRNAs. The level of mRNA accumulation correlates with the degree of proliferation induced by mitogenic combinations of ionophore A23187 and TPA, as measured by [3H]thymidine incorporation. Thus, these compounds act synergistically both with respect to c-fos and c-myc mRNA accumulation and to mitogenicity in human PBL. We also studied the level of c-fos transcripts following stimulation of the T lymphocyte T3/Ti receptor complex, using monoclonal anti-T3 antibodies. We observed that mitogenic doses of anti-T3 also induce an accumulation of c-fos mRNA, whose induction also is synergized by TPA. These results suggest that transient accumulation of c-fos; as well as c-myc, mRNA is a normal event of the immune response. They confirm that Ca2+ ionophore combined with TPA can mimic some molecular features of the early steps of normal human PBL activation.

Regulation of C-myc expression during growth and differentiation of normal and leukemic human myeloid progenitor cells.

C-myc proto-oncogene transcripts from serially harvested, colony-stimulating activity (CSA)-stimulated, normal progenitor-enriched human bone marrow cells were compared to those of the promyelocytic leukemia cell line HL-60 and to those of freshly obtained human myeloid leukemic cells. During the early culture period both normal and leukemic cells expressed the c-myc oncogene. In normal cells maximal expression occurred after 24 h of culture and did not occur in the absence of CSA. At this time, progranulocytes predominated in the cultured cells. Although cellular proliferation occurred for 96 h in vitro, c-myc expression ceased after 24-36 h. Terminally differentiated cells predominated in these cultures by 120 h. In contrast, although leukemic cells also expressed c-myc in vitro, transcription persisted throughout the culture period and, in the case of HL-60 cells, occurred in the absence of exogenous CSA. We also noted that normal cells with only one diploid gene copy exhibited, after 24 h of culture, only twofold fewer transcripts than did HL-60 cells in which there were 16 myc copies. Furthermore, c-myc mRNA degradation rates were similar in normal cells and in HL-60 cells. We conclude that c-myc transcription is a normal event in granulopoiesis linked to proliferative activity as well as to primitive developmental stage. Furthermore, the most consistent abnormality in leukemic cells in vitro is their failure to suppress transcriptional activity of this gene. We suggest that c-myc transcription in HL-60 cells may be appropriate for cells arrested at that developmental stage and that the amplified genes in HL-60 cells are quiescent relative to c-myc in normal cells at the same differentiation stage. The techniques described herein may be of value in identifying mechanisms by which normal hematopoietic cells suppress c-myc expression and aberrancies of these mechanisms in leukemic cells.

Nerve growth factor and epidermal growth factor induce rapid transient changes in proto-oncogene transcription in PC12 cells.

Nerve growth factor (NGF) promotes neuronal differentiation of PC12 pheochromocytoma cells. We show here that within 5 min after its addition, NGF transiently stimulates c-fos proto-oncogene and actin transcription by greater than 100-fold in nonsynchronized PC12 cells. c-myc and ornithine decarboxylase transcription are also transiently activated, but more slowly. The corresponding mRNAs are induced as well. Two weeks' exposure to NGF causes no significant changes in the transcription of these and a variety of other genes analyzed; however, c-fos mRNA levels are increased severalfold under these conditions. Neuronally differentiated PC12 cells retain the capacity for rapid transcriptional responses. Removal of NGF from such cells for several hours followed by its readdition results in rapid induction of c-fos and actin transcription. These NGF-promoted transcriptional changes in PC12 cells are similar to those previously observed in quiescent fibroblasts stimulated by platelet-derived growth factor (Greenberg, M.E., and Ziff, E.B. (1984) Nature 311, 433-438). This rapid transcriptional activation in PC12 cells could be necessary for neuronal differentiation, but is apparently not sufficient since diverse agents without differentiating activity such as epidermal growth factor, insulin, dibutyryl cAMP, phorbol ester, and elevated K+ were also found to induce transcription. These results suggest that c-fos, c-myc, and actin induction may be general nuclear responses to growth or differentiation factors in a variety of different cell types.

Expression of c-Ki-ras, c-Ha-ras, and c-myc in specific cell types during hepatocarcinogenesis.

We examined the expression of six proto-oncogenes in (i) whole rat liver and isolated liver cell populations during the course of hepatocarcinogenesis induced by a choline-deficient diet containing 0.1% ethionine and (ii) fetal rat liver at different stages of development. The abundance of c-Ki-ras, c-Ha-ras, and c-myc transcripts in polysomal polyadenylated RNA from liver cells increased by 2 weeks after the start of the carcinogenic diet. c-Ki-ras and c-myc expression remained elevated during the 35 weeks of the diet, whereas c-Ha-ras transcripts increased transiently. A primary tumor sampled at 35 weeks after the carcinogenic diet was started contained high levels of both c-Ki-ras and c-myc RNA. The abundance of c-src transcripts was unchanged throughout carcinogenesis; c-abl and c-mos transcripts were not detected in either preneoplastic or neoplastic livers. To determine which cell types within the liver contained proto-oncogene transcripts, we isolated hepatocytes, oval cells, and bile duct cells from normal and preneoplastic livers. The results indicate that proto-oncogenes are expressed differentially in these cell types during hepatocarcinogenesis and that the expression of c-Ki-ras and c-myc is high in oval cells throughout carcinogenesis. In developing livers, c-Ki-ras, c-Ha-ras, and c-myc transcript levels were high at 17 days of gestation but reached the low values characteristic of adult rat livers between 20 days of gestation and 3 days after birth.

Expression of c-Ki-ras, c-Ha-ras, and c-myc in specific cell types during hepatocarcinogenesis.

We examined the expression of six proto-oncogenes in (i) whole rat liver and isolated liver cell populations during the course of hepatocarcinogenesis induced by a choline-deficient diet containing 0.1% ethionine and (ii) fetal rat liver at different stages of development. The abundance of c-Ki-ras, c-Ha-ras, and c-myc transcripts in polysomal polyadenylated RNA from liver cells increased by 2 weeks after the start of the carcinogenic diet. c-Ki-ras and c-myc expression remained elevated during the 35 weeks of the diet, whereas c-Ha-ras transcripts increased transiently. A primary tumor sampled at 35 weeks after the carcinogenic diet was started contained high levels of both c-Ki-ras and c-myc RNA. The abundance of c-src transcripts was unchanged throughout carcinogenesis; c-abl and c-mos transcripts were not detected in either preneoplastic or neoplastic livers. To determine which cell types within the liver contained proto-oncogene transcripts, we isolated hepatocytes, oval cells, and bile duct cells from normal and preneoplastic livers. The results indicate that proto-oncogenes are expressed differentially in these cell types during hepatocarcinogenesis and that the expression of c-Ki-ras and c-myc is high in oval cells throughout carcinogenesis. In developing livers, c-Ki-ras, c-Ha-ras, and c-myc transcript levels were high at 17 days of gestation but reached the low values characteristic of adult rat livers between 20 days of gestation and 3 days after birth.