Regulation of c-fos and c-jun protooncogene expression by the Ca(2+)-ATPase inhibitor thapsigargin.

Thapsigargin, a non-phorbol-ester-type tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca(2+)-ATPase. We used this drug to analyze the involvement of Ca2+ and Ca(2+)-ATPases in the control of growth- and transformation-related genes. Here we show that treatment of mouse NIH 3T3 fibroblasts with thapsigargin induced rapid expression of the c-fos and c-jun protooncogenes. Inhibition or depletion of protein kinase C partially diminished the c-fos but not the c-jun response. Furthermore, thapsigargin could synergize with the tumor promoter phorbol 12-myristate 13-acetate to induce c-fos but not c-jun. However, thapsigargin had no effect on basal or phorbol ester-induced protein kinase C activity. Our results indicate that Ca2+ is a potent second messenger that controls expression of growth- and transformation-related genes. Since inhibition of the endoplasmic reticulum Ca(2+)-ATPase results in a strong induction of these genes, our data suggest that this Ca2+ pump may act as a negative regulator of cell growth.     

Cardiac myocyte hypertrophy is associated with c-myc protooncogene expression.

The mechanism of hormonally induced cell hypertrophy is unknown. Stimulation of cardiac myocytes by alpha 1-adrenergic agents, phorbol esters, and serum induces an increase in the cell size of nondividing cardiac myocytes in primary culture. Expression of the c-myc gene, known to be increased in growth factor-induced cell division, was studied in this model of cell hypertrophy. The alpha-adrenergic agonist norepinephrine (0.002-20 microM) increased levels of c-myc-encoded mRNA to 10-fold over control levels. This increase was detectable at 30 min, peaked at 2 hr, and returned to baseline by 6 hr after stimulation. The norepinephrine response was abolished by the alpha 1-antagonist terazosin (2 microM) but was not affected by the beta-adrenergic antagonist propranolol (2 microM) and was only slightly (25%) attenuated by the alpha 2-adrenergic antagonist yohimbine (2 microM). Serum and the phorbol ester tumor promoter phorbol 12-myristate 13-acetate also enhanced c-myc expression in cardiac myocyte cultures. These findings show that the induction of cardiac myocyte hypertrophy is associated with enhanced expression of the c-myc gene and suggest that hormonally induced cell hypertrophy and cell division share common mechanistic pathways.     

Localization and regulation of c-fos and c-jun protooncogene induction by systolic wall stress in normal and hypertrophied rat hearts.

The effect of changes in left ventricular (LV) systolic force generation on cardiac c-fos and c-jun protooncogene expression was studied by using isolated beating hearts from male Wistar rats. An isovolumic buffer-perfused heart preparation was utilized in which coronary flow and heart rate were held constant and increments in LV balloon volume were used to generate defined levels of LV systolic wall stress. Using Northern and slot-blot analyses, we found that LV tissue from control hearts that generated high levels of LV systolic wall stress expressed 3- to 4.4-fold higher c-fos and c-jun mRNA levels in comparison with tissue from the respective flaccid right ventricles, and in comparison with LV tissue from hearts that generated minimal LV systolic wall stress. To distinguish the role of passive LV diastolic wall stretch from active LV force generation, we found that distension of the LV balloon per se did not have a significant effect on protooncogene induction in hearts perfused with 2,3-butanedione monoxime, which prevents systolic cross-bridge cycling and force generation. In additional hearts studied at a constant LV balloon volume to generate an LV end-diastolic pressure of 10 mm Hg, c-fos mRNA levels were proportional to the magnitude of peak LV systolic wall stress (r = 0.823, P less than 0.05). In these protocols, Fos protein was localized by immunohistochemistry in myocyte nuclei with minimal staining in fibroblasts and vascular smooth muscle. When c-fos and c-jun mRNA expression was compared in hearts with chronic LV hypertrophy due to ascending aortic banding and age-matched control hearts that generated similar incremental levels of LV systolic wall stress, significantly lower levels of c-fos and c-jun mRNA were measured in the hypertrophied hearts. However, there was no difference in protooncogene mRNA expression in response to stimulation by the Ca2+ ionophore A23187. These data suggest that, in this isolated isovolumic beating heart preparation, the active generation of an acute increment in LV systolic force independent of passive diastolic myocardial stretch causes a rapid induction of both c-fos and c-jun, which is down-regulated in the presence of established LV hypertrophy.     

Elevated c-myc protooncogene expression in autosomal recessive polycystic kidney disease.

The polycystic kidney diseases (PKDs) are a group of disorders characterized by the growth of epithelial cysts from the nephrons and collecting ducts of kidney tubules. The diseases can be inherited or can be provoked by environmental factors. To investigate the molecular basis of the abnormal cell growth associated with PKD, c-myc protooncogene expression was studied in a mouse model for autosomal recessive PKD. Homozygous recessive C57BL/6J (cpk/cpk) mice develop massively enlarged cystic kidneys and die from renal failure shortly after 3 weeks of age. Quantitative dot blot and RNA blot hybridization experiments in which whole kidney poly(A)+ RNA was hybridized with a c-myc RNA probe showed a 2- to 6-fold increase in c-myc mRNA at 2 weeks, and a 25- to 30-fold increase in c-myc mRNA at 3 weeks of age in polycystic mice, as compared to normal littermates. c-myc expression was also examined under two conditions in which kidney cell growth was experimentally induced in normal adult mice: compensatory renal hypertrophy and tubule regeneration following folic acid-induced renal cell injury. While compensatory hypertrophy resulted in only a small (less than 3-fold) increase in c-myc, folic acid treatment gave rise after 24 hr to a 12-fold increase in c-myc mRNA. The induction of c-myc by folic acid is consistent with increased cellular proliferation in regenerating tubules. In contrast, polycystic kidneys show only a minimal increase in cellular proliferation over that seen in normal kidneys, while c-myc levels were found to be markedly elevated. Thus, the level of c-myc expression in cystic kidneys appears to be out of proportion to the rate of cell division, suggesting that elevated and potentially abnormal c-myc expression may be involved in the pathogenesis of PKD.     

Effects of glucocorticoids on expression of the fos protooncogene in AtT-20 cells.

Glucocorticoid regulation of expression of the protooncogene fos has been examined in AtT-20 cells at both the RNA and protein levels. When cells were incubated continuously in the presence of dexamethasone, an early (30 min) rise in the expression of fos mRNA was observed, which declined by 1 h, but rose again after 2 h of hormone treatment. Six hours after hormone treatment, fos mRNA levels had returned to control levels in spite of the continued presence of dexamethasone. Serum treatment resulted in a sustained increase in fos mRNA levels; however, the glucocorticoid and serum effects were additive. Dexamethasone and/or serum both increased the steady state levels of fos protein. Glucocorticoid treatment of AtT-20 cells results in complex changes in fos expression, but does not affect their viability or growth rate; these results suggest that fos may play a role in mediation or modulation of glucocorticoid effects other than growth.     

Sustained expression of the human protooncogene MYCN rescues rat embryo cells from senescence.

Amplification of the human gene MYCN may play a role in the malignant progression of human neuroblastomas. In pursuit of this possibility, previous studies have shown that the abundant expression of MYCN in cultured cells can elicit several aspects of the transformed phenotype. We now extend those findings by demonstrating that rat embryo cells transfected with MYCN can proliferate for at least 200 generations. Isolation of established cells was dependent on high expression of MYCN and on biological selection to eliminate untransfected cells. The established cells were not tumorigenic in syngeneic rats or athymic mice, failed to grow in soft agar, and required relatively high concentrations of serum for proliferation in culture. Our results show that enhanced expression of MYCN can rescue normal cells from senescence, add to the credentials of MYCN as an authentic protooncogene, and identify an additional biological activity that can be used in the characterization of MYCN.     

Ionizing radiation induces heritable disruption of epithelial cell interactions

Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. As a consequence, we have proposed that an additional factor contributing to IR carcinogenesis is the potential disruption of critical constraints that are imposed by normal cell interactions. To test this hypothesis, we asked whether IR affected the ability of nonmalignant human mammary epithelial cells (HMEC) to undergo tissue-specific morphogenesis in culture by using confocal microscopy and imaging bioinformatics. We found that irradiated single HMEC gave rise to colonies exhibiting decreased localization of E-cadherin, beta-catenin, and connexin-43, proteins necessary for the establishment of polarity and communication. Severely compromised acinar organization was manifested by the majority of irradiated HMEC progeny as quantified by image analysis. Disrupted cell-cell communication, aberrant cell-extracellular matrix interactions, and loss of tissue-specific architecture observed in the daughters of irradiated HMEC are characteristic of neoplastic progression. These data point to a heritable, nonmutational mechanism whereby IR compromises cell polarity and multicellular organization.     

Ionizing radiation accelerates aortic lesion formation in fat-fed mice via SOD-inhibitable processes.

Ionizing radiation promotes formation of reactive oxygen species, including the superoxide anion (O2-). To evaluate whether O2- or O2--mediated perturbations may contribute to the known atherogenic effects of radiation, we examined aortic lesion formation in irradiated C57BL/6 mice and evaluated the effects of CuZn-superoxide dismutase (CuZn-SOD) overexpression. Ten-week-old mice were exposed to a 2-, 4-, or 8-Gy dose of 250-keV x-rays to the upper thorax and then placed on a high-fat diet for 18 weeks. Based on quantitative lipid staining of serial sections of the proximal aorta, mean lesion area was increased with increasing radiation dose and was 3-fold greater in 8-Gy-irradiated than sham-irradiated mice (7800+/-2140 versus 2635+/-709 micrometer(2), P<0.05). These effects were absolutely dependent on a high-fat diet, which had to be introduced within 1 to 2 weeks of the radiation exposure, suggesting the early involvement of atherogenic lipoproteins that were elevated in response to the diet. The importance of radiation-induced oxidative stress was supported by the observation of a 2-fold lower mean lesion area in irradiated CuZn-SOD transgenic mice than in their irradiated, nontransgenic littermates (3026+/-1590 versus 6102+/-1834 micrometer(2), P<0.05). Lucigenin-enhanced chemiluminescence, used as an index of aortic O2- concentrations, was significantly elevated in the postradiation period, and this response was reduced in CuZn-SOD transgenics. On the basis of these results, we propose that radiation may be a useful tool for initiating oxidative or redox-regulated events that promote atherogenesis and for testing the antiatherogenic properties of antioxidants.     

Sequential protooncogene expression during regeneration in rat stomach

Cellular protooncogenes such as c-myc and c-Ha-ras may play important roles in the control of regeneration of the stomach. In this study, in situ hybridization histochemistry and immunohistochemistry were used to determine how these protooncogenes and the corresponding oncoproteins are expressed at the cellular level during gastric regeneration after mucosal injuries caused by indomethacin. In addition, cells in the S-phase were immunohistochemically detected by means of 5-bromo-2'-deoxyuridine incorporation. Expression of the c-myc gene was localized to nuclei and reached a maximum at 3 h, and that of the c-Ha-ras gene was localized to cytoplasm with a peak at 6-12 h after treatment on the mucous neck, parietal, chief, and enterochromaffinlike cells around the lesions. The distribution of cells in the S-phase roughly coincided with that of cells in which expression of the protooncogenes was detected. In conclusion, various types of gastric mucosal cells participated in the sequential regulated expression of cellular protooncogenes during regeneration of the rat stomach.