c-myc expression is down-regulated by cell-cell and cell-extracellular matrix contacts in normal hepatocytes, but not in hepatoma cells.

Primary culture of adult rat hepatocytes resulted in marked increase of c-myc expression within a few hours. The high level of c-myc mRNA was maintained throughout culture on collagen-coated dishes, but decreased greatly with time during culture on collagen-gel or matrigel. Expression of c-myc was also down-regulated at high cell density. The decrease in its expression appeared closely related to inhibitions of DNA synthesis and cell spreading. In contrast, hepatoma H4TG cells showed a high level of c-myc expression which was not affected by culture on any extracellular matrices examined or by the cell density. These results suggest that up-regulation of expression of the c-myc gene is linked to G0 to G1 transition during cell cycle progression, which in normal hepatocytes is strictly regulated by cell-cell and cell-extracellular matrix interactions, but that this control mechanism is defective in malignant hepatic tumor cells.     

Cellular density regulation of plasminogen gene expression in mouse hepatocytes

The liver produces a variety of proteins including plasminogen. Plasminogen is pro-enzyme that is converted into plasmin by plasminogen activator. Plasmin has a broad substrate spectrum and participates in several biological processes, such as fibrinolysis, tissue remodeling, cell migration, angiogenesis and embryogenesis. In the present study, the regulation of plasminogen expression in mouse hepatocytes was investigated in the primary culture system. Expression level of plasminogen mRNA in the culture at the low cell density condition (0.2 x 10(5) cells / cm(2)) was compared with that at the high cell density condition (1.0 x 10 (5) cells / cm(2)). In the low cell density culture, the expression level of plasminogen mRNA decreased by a time-dependent manner. However, mRNAs for albumin and alpha(2)-antiplasmin were not influenced by the low cell density culture. On the other hand, in the high cell density culture, plasminongen mRNA expressed constantly as well as albumin and alpha(2)-antiplasmin mRNAs. Thus, the decrease in plasminogen mRNA expression could specifically occur when the density of hepatocytes was low. The down-regulation of plasminogen mRNA in the low cell density culture is not observed in the presence of cycloheximide, suggesting that the de novo protein synthesis is required for the regulatory mechanism. These findings indicate that the expression of plasminogen mRNA from hepatocyte is dependent on the cell density and the stimulation by cell-cell contact may be an important factor for the constitutive expression of plasminogen gene in hepatocytes.     

Altered regulation of c-myc expression in adenovirus-transformed cells

Expression of the oncogenes c-myc, c-raski, and p53 is studied in normal primary mouse cultures and in two adenovirus-transformed mouse cell lines. In all cases oncogene expression is measured in cells arrested in G1 (or G0 for primary cells) by serum starvation and at different times after cell cycle traverse is stimulated by addition of high serum. For primary mouse cells, c-myc mRNA levels are found to increase four- to six-fold within 1 h of serum addition and then decline by 4 h to nearly the level observed in serum-starved cells. This level is maintained throughout the remainder of the cell cycle. The early induction of c-myc is dependent on serum concentration and is independent of cell density. These results confirm and extend previous observations for primary cells. By contrast, expression of c-raski does not vary at all through the cell cycle and p53 increases with time after mitogenic stimulation. In the adenovirus-transformed cell lines, the regulation of expression of c-myc with respect to the cell cycle is altered. There is an increase in c-myc in S phase cells which is dependent on cell density and the early induction in response to serum addition as seen in primary cells is absent. Expressions of c-raski and p53 are found to show similar profiles to those observed for primary cells.     

Enhanced growth-dependent expression of TGF beta 1 and hsp70 genes in aortic smooth muscle cells from spontaneously hypertensive rats.

Enhanced proliferation of vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) as compared with Wistar-Kyoto rats (WKY) persists in long-term culture and is characterized by an accelerated entry of these cells into the synthetic S phase of the cell cycle and a higher specific growth rate, particularly evident at high cell density. In the present study, we investigated by Northern blot experiments the expression of genes putatively involved in the regulation of VSMC growth. One of them is the transforming growth factor beta 1 gene (TGF beta 1), a bifunctional modulator of cell growth whose action is dependent on cell density. The accumulation of TGF beta 1 mRNA was enhanced in growing SHR cells at every density studied as early as 24 h after inoculation with a further increase at later times. Protooncogenes c-fos and c-myc, which have been implicated in G1/S phase transition, have also been investigated in VSMC by Northern blot analysis. At low cell density, calf serum stimulated c-fos and c-myc mRNA expression was comparable in WKY and SHR cells whereas at high cell density, c-fos induction was higher in VSMC from SHR. SHR VSMC respond more to mitogenic stimulation and to environmental (e.g., heat) stress, particularly when growing near saturation density. hsp70 constitutes a gene family responsive to environmental stimuli (heat) and to mitogenic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous activation of heat shock protein (hsp 70) and nucleolin genes during in vivo and in vitro prereplicative stages of rat hepatocytes.

Rapidly growing cells usually have high levels of ribosome biogenesis. The sequential expression of protooncogenes during the transition of quiescent hepatocytes to the replicative stage was assumed to be followed by activation of cellular genes related to cell growth such as ribosome biosynthesis. First, the expression of major nucleolar protein (nucleolin or C23) and major heat-shock protein (hsp 70) genes was examined during rat liver regeneration. hsp 70 may function in cell growth and has a characteristic nucleolar location after heat shock. Both nucleolin and hsp 70 mRNA began to increase simultaneously after peaks of c-fos and c-myc, showed a peak 6 h after partial hepatectomy, and declined to the control levels around 20 h. That is, the peaks of nucleolin and hsp 70 mRNA precede the peak of ribosome formation (12-20 h) and DNA replication (24 h). Second, the behavior of nucleolin and hsp 70 mRNA was examined in primary cultured hepatocytes during their G0-G1 transition. Although the amounts of c-myc mRNA reached a plateau around 20 h after the initiation of culture and remained at these levels, DNA synthesis has never been found to start without the addition of EGF and insulin to this system. Both nucleolin and hsp 70 mRNA began to increase at around 20 h (prereplicative stage) and simultaneously decreased in inverse proportion to DNA synthesis induced by these growth factors. Thus, it is possible that the simultaneous enhancement of nucleolin and hsp 70 genes as described above is not merely coincidental, but is important biologically during the transition of quiescent hepatocytes to proliferative cells.     

IGF-I is a mitogen involved in differentiation-related gene expression in fetal rat brown adipocytes

Fetal rat brown adipocytes at time zero of culture constitute a population of cells of broad spectrum, as estimated by cell size, endogenous fluorescence and lipid content, and show an intrinsic mitogenic competence. They express constitutively early growth-related genes such as c-myc, c-fos, and beta-actin, tissue specific-genes such as the uncoupling protein (UCP) and the lipogenic marker malic enzyme (ME). Fetal brown adipocytes bear a high expression of insulin-like growth factor receptor (IGF-IR), and show a high affinity IGF-I specific-binding to its receptor, and a high number of binding sites per cell. After cell quiescence, insulin-like growth factor I (IGF-I) was as potent as 10% FCS in inducing DNA synthesis, cell number increase, and the entry of cells into the cell-cycle. In addition, IGF- I or 10% FCS for 48 h increased the percentage of [3H]thymidine-labeled nuclei as compared to quiescent cells. Single cell autoradiographic microphotographs show typical multilocular fat droplets brown adipocytes, resulting positive to [3H]thymidine-labeled nuclei in response to IGF-I. IGF-I increased mRNA expression of the early- response genes c-fos (30 min), c-myc (2 and 24 h), and H-ras (4 and 24 h). 10% FCS also increased c-fos and c-myc, but failed to increase H- ras as an early event. IGF-I or 10% FCS, however, similarly increased the mRNA late expression of c-myc, H-ras, c-raf, beta-actin, and glucose 6-phosphate dehydrogenase (G6PD) at 72 h, as compared to quiescent cells. IGF-I or FCS maintained at 24 h or increased at 48 and 72 h UCP mRNA expression. The results demonstrate that IGF-I is a mitogen for fetal rat brown adipocytes, capable of inducing the expression of early and late growth-regulated genes, and of increasing the lipogenic marker ME and the tissue-specific gene UCP, suggesting the involvement of IGF-I in the differentiation as well as in the proliferation processes.     

Effects of epidermal growth factor and platelet-derived growth factor on c-fos and c-myc mRNA levels in normal human fibroblasts

The mRNA levels of two proto-oncogenes, c-fos and c-myc, were determined in human foreskin fibroblasts exposed to epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) in a serum-free, defined medium (MCDB 104). Untreated, quiescent cells were found to have low or undetectable levels of c-fos and c-myc mRNA. Within 10 min after the addition of EGF or PDGF the c-fos mRNA level increased, reached a peak at 30 min, and then declined to the control level after 60 min. The level of c-myc mRNA increased somewhat later and peaked after 8 h in cultures treated with either of the growth factors. The c-myc mRNA level remained elevated throughout the 24 h of investigation. The concentrations of EGF and PDGF required for a maximal effect on c-fos or c-myc expression were found to be similar to those that give maximal effect on cell proliferation. Both c-fos and c-myc mRNA expression were super-induced by the addition of cycloheximide. The addition of neutralizing PDGF antibodies to cultures that had received PDGF 4 h earlier inhibited the subsequent increase in the c-myc mRNA level, indicating that the effect of PDGF on c-myc expression is not caused by a "hit and run," mechanism. Density-inhibited cells responded to EGF and PDGF by an increase in c-fos and c-myc mRNA levels in the absence of any mitogenic response. The present results conform to the view that the c-fos and c-myc proto-oncogenes may be important (or necessary) but not sufficient for the initiation of DNA synthesis. Moreover, the finding that both EGF and PDGF increase c-fos and c-myc expression supports our previous suggestion that these two growth factors may in part act via a common intracellular pathway in the prereplicative phase of human fibroblasts.     

Expressions of the c-Ha-ras and c-myc genes in rat liver tumors

Expressions of the c-Ha-ras and c-myc genes were studied by Northern blotting of total RNA from primary tumors and non-tumorous parts of the liver of rats given diet containing 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) and from established rat hepatoma cell lines. The expression of the c-Ha-ras gene was found to be high in the primary tumors, non-tumorous parts of 3'-Me-DAB-treated livers and hepatoma cell lines. In contrast, the c-myc gene was expressed at a high level only in primary tumors and hepatoma cell lines. During 3'-Me-DAB treatment, the c-Ha-ras mRNA level in the liver increased by day 5 and then remained high. Increase in expression of the c-Ha-ras gene in regenerating liver was confirmed. These findings suggest that increase in expression of the c-Ha-ras gene is related to proliferation of hepatocytes, whereas expression of the c-myc gene is associated with hepatocarcinogenesis.     

H-ras gene is expressed at the G1 phase in primary cultures of hepatocytes

The expression of c-H-ras and proliferating cell nuclear antigen (PCNA) in primary cultures of rat hepatocytes was determined in order to elucidate the relationship between the c-H-ras gene and the S phase of the cell cycle. In cells treated with EGF, elevation of c-H-ras expression was detected at the 22nd, 34th, 44th, and 54th h after plating, PCNA expression and DNA synthesis were detected at the 44th and 54th h. In cells without EGF treatment, only c-H-ras expression was detected at the 44th and 54th h. In our previous report, we showed that c-myc expression increased within several hours after plating, suggesting that isolated hepatocytes traverse from G0 to G1 under culture conditions, regardless of EGF treatment. These results clearly showed that the c-H-ras gene of adult rat hepatocytes was expressed in the mid-to-late G1 phase of the cell cycle as well as in the early S phase in primary culture.     

An altered repertoire of fos/jun (AP-1) at the onset of replicative senescence.

With multiple divisions in culture, normal diploid cells suffer a loss of growth potential that leads to replicative senescence and a finite replicative capacity. Using quantitative RT-PCR, we have monitored mRNA expression levels of c-fos, c-jun, JunB, c-myc, p53, H-ras, and histone H4 during the replicative senescence of human fibroblasts. The earliest and the largest changes in gene expression occurred in c-fos and junB at mid-senescence prior to the first slowing in cell growth rates. The basal level of c-fos mRNA decreased to one-ninth that of the early-passage levels, while junB declined to one-third and c-jun expression remained constant. The decline in the basal c-fos mRNA level in mid-senescence should lead to an increase in Jun/Jun AP-1 homodimers at the expense of Fos/Jun heterodimers and may trigger a cascade of further changes in c-myc, p53, and H-ras expression in late-passage senescent fibroblasts.     

Developmental and growth-related regulation of expression of serine dehydratase mRNA in rat liver

In rat liver, serine dehydratase mRNA is undetectable in the late prenatal period, but its level increases rapidly after birth to a transient peak, and then after decrease gradually increases again to a maximum 2 weeks after birth that is slightly higher than that of adult liver. To determine whether mature quiescent hepatocytes proliferate without loss of differentiated functions, we measured the serine dehydratase mRNA contents in regenerating liver and primary cultured hepatocytes from adult rats. Partial hepatectomy resulted in a dramatic decrease in the mRNA content within 24 h and then its recovery within a week. In subconfluent cultures of adult rat hepatocytes that did not grow even in the presence of mitogens, serine dehydratase mRNA was maintained at a high level. However, when the hepatocytes were cultured at low cell density without added mitogens, their serine dehydratase mRNA content decreases to a quarter of that of subconfluent cultures. The possibility that the expression of serine dehydratase mRNA is regulated in G0/G1 transition before entry into the S phase and the relationship of the mRNA with growth are discussed.     

Expression of c-myc oncogene in rat liver by a dietary manipulation

After rats deprived of protein for several days are fed a meal containing protein, hepatic DNA replication is induced. When nuclear DNA synthesis is stimulated in the normally quiescent rat liver by a dietary manipulation, we examined the changes of the steady-state levels of messenger RNA for c-myc. Levels of c-myc mRNA are gradually elevated approximately 4 to 5-fold above normal in the livers of rats that are fed for several days a diet that lacks protein. After a nutritional shift from a protein-free diet to a diet containing 50% casein, the levels of c-myc mRNA decrease rapidly by 2 h and returned to approximately basal levels after 8 h. Our results suggest that c-myc expression during the prereplicative stage of liver is likely to reflect events associated with entry and progression of hepatocytes into the cell cycle.