Sequential expression and cooperative interaction of c-Ha-ras and c-erbB genes in in vivo chemical carcinogenesis.

The level of expression of several cellular protooncogenes is examined at different stages of 7,12-dimethylbenzanthracene (DMBA)-induced tumor development in hamster buccal pouch epithelium (HBPE). Results presented demonstrate overexpression of c-Ha-ras gene at a very early stage of tumor development, and this elevated level of expression of the gene persists throughout the tumorigenesis process. The expression of the cellular protooncogene c-erbB, on the other hand, can be detected only after 8-10 weeks of DMBA treatment of the tissue and increases with the progression of the disease. The overexpression of c-erbB gene can be correlated with the stage of extensive proliferation and subsequent invasion of the HBPE cells into the underlying connective tissue. This sequential pattern of stage-specific expression of the two cellular protooncogenes can be observed in (i) treated tissues, (ii) stage-representative cultured cells, and (iii) NIH 3T3 transformants derived with DNA from HBPE cells. The low-level expression of c-myc and c-sis genes detected in control tissues remains unaffected, while c-fos gene activity cannot be detected at any stage of tumor development. The overexpression of c-Ha-ras gene alone in HBPE cells derived from tissues treated for 5 weeks (DM5) is not sufficient to induce tumors in athymic mice, whereas expression of c-Ha-ras and c-erbB genes at later stages of tumor development (DM10 and HCPC cells) induce histopathologically defined epithelial cell carcinoma in athymic mice within 2-3 weeks. The sequential overexpression of c-Ha-ras and c-erbB genes in a stage-specific manner and their cooperative interaction in the DMBA-induced in vivo oral carcinogenesis have been demonstrated.     

Cellular analysis of c-Ha-ras gene expression in rat liver after CCl4 administration

Expression of the c-Ha-ras proto-oncogene is specifically enhanced during liver regeneration, in parallel with increased DNA replication, which suggests that c-Ha-ras may play a role in the control of regeneration. In this study, an in situ hybridization technique was applied for analysis of expression of the c-Ha-ras gene at the cellular level during liver regeneration induced by CCl4 administration. The in situ hybridization was compared with the observation for the p21c-Ha-ras protein, the corresponding protein of the c-Ha-ras gene, by immunohistochemistry. In normal rat liver, a few hepatocytes expressed the mRNAs and the corresponding proteins without any preferential localization. Zonal heterogeneity of c-Ha-ras gene expression first became evident at 12 hr after CCl4 administration, a higher number of gene products being detected in the pericentral zone than in the periportal zone. This heterogeneity became maximal at 24 hr after CCl4 administration. Zonal heterogeneity in the level of the p21c-Ha-ras protein paralleled that in the level of gene expression. Furthermore, both hepatocytes and nonparenchymal cells participated in expression of the c-Ha-ras gene during liver regeneration.     

Expression of genes associated with dedifferentiation and cell proliferation during pancreatic regeneration following acute pancreatitis.

Pancreatic gene expression was analyzed in the rat during taurocholate-induced pancreatitis, with emphasis on the postacute phase where regeneration occurs. Increased expression of cellular oncogenes c-myc and H-ras followed a pattern typical of tissular regeneration. The c-myc protein was immunolocalized to acinar cells, in which amylase expression was concomitantly decreased. Such modifications in the program of gene expression and the presence of numerous mitotic figures confirmed participation of acinar cells in regeneration. There was, on the contrary, no evidence of duct cell proliferation and pancreatitis did not influence the expression of two mRNAs encoding ductal proteins. Expression of villin, which is a marker of the embryonic pancreas, increased by five times during pancreatic regeneration. The protein was localized to the tubular complexes, suggesting that cells forming those structures had returned to a protodifferentiated stage in which they should have recovered pluripotency. They might therefore supply the pancreas with any cell type needed to reconstitute functional parenchyma.     

Expression of cellular oncogenes in the myocardium during the developmental stage and pressure-overloaded hypertrophy of the rat heart

Proto-oncogenes have been revealed to participate in normal cell proliferation as well as in cell transformation. Since cardiac myocytes are terminally differentiated, they cannot divide except in the fetal period. To determine the role of cellular oncogenes in the growth of the heart, the expression pattern of eight cellular oncogenes during the developmental stage and pressure-overloaded hypertrophy of the rat hearts were examined in vivo. Northern blot analysis was performed with eight oncogene probes (myc, fos, Ha-ras, src, erbA, erbB, sis, myb). Pressure overload increased the levels of cellular (c-) fos, c-myc, and c-Ha-ras. An increase of c-fos and c-myc was detected at 30 minutes and 2 hours, respectively; the levels peaked at 8 hours, and they returned to baseline by 48 hours after aortic constriction. However, the level of c-Ha-ras showed a gradual increase. During the course of development, the expression of c-myc was detectable only in the embryonic stage, whereas the expression of c-fos was not detected in the fetal period, was increased after birth, and peaked in 200-day-old adults. The expression of c-Ha-ras was almost the same throughout the development. Cellular oncogenes were expressed in the heart in response to pressure overload and in a stage-specific manner. These results suggest that cellular oncogenes may participate in the normal developmental process and hypertrophy of hearts and that the cellular hypertrophy induced by pressure overload may share a similar mechanistic pathway with cell proliferation.     

Enhanced expression of epidermal growth factor receptor gene in gastric mucosal cells by the serum derived from rats treated with electroacupuncture at stomach meridian acupoints

INTRODUCTION Gastric mucosal damage is a common pathological reaction in the diseases of the digestive system. The acupuncture and moxibustion are very effective cure for this damage[1,2]. Previous experimental studies demonstrated that epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) were the most important peptides for the repair of the gastric mucosal injury[3]. Acupuncture at gastric meridian acupoint could alter gastric motility and secretion and also the co…     

Patterns of mRNA expression during early cell growth differ in kidney epithelial cells destined to undergo compensatory hypertrophy versus regenerative hyperplasia.

An increase in cell size and protein content is characteristic of cells undergoing hypertrophy and of replicating cells prior to DNA synthesis. Cell enlargement in the two situations could be regulated by similar early events with an interruption of the cell cycle occurring in hypertrophy, or the two processes could be uncoupled. In vivo models were used to compare hypertrophy induced by unilateral nephrectomy and hyperplasia induced by folic acid injection in rabbit renal cortical cells. Within 48 hr, cell volume increased in both groups but the number of cells in the cell cycle and DNA synthesis was increased only after folic acid. Patterns of mRNA expression of the following three groups of cell cycle-dependent genes were analyzed: (i) protooncogenes (c-fos, c-myc, and c-Ha-ras), (ii) structural protein genes (vimentin and beta-actin), and (iii) transport protein genes (Na+, K+-ATPase, ADP-ATP translocase, and calcyclin). mRNAs for all genes, except calcyclin and c-Ha-ras, were detected in controls. Folic acid generally induced rapid, transient increases in mRNA levels, but after unilateral nephrectomy, expression of most mRNAs showed a gradual, progressive increase. These data indicate that gene expression in the early stages of cell enlargement differs in cells destined to undergo proliferation vs. hypertrophy. The term "sustained message amplification" is proposed to describe the hypertrophied cell.     

Enhanced expression of poly(ADP-ribose) synthetase gene in malignant lymphoma

Over-expression of cellular protooncogenes has been proposed to function in the initiation and maintenance of malignancies. In order to distinguish malignant lymphoma from reactive proliferative diseases, we surveyed the expression levels of three protooncogenes(c-myc, c-fos and c-myb) in malignant lymphoma and reactive proliferative diseases. An increased level of c-myc or c-fos mRNA was observed in one case, respectively, out of three malignant lymphomata. The other cases exhibited no enhancement in protooncogenes. These oncogenes are critically regulated during differentiation, but the half-life of c-myc mRNA was very short, and the level of the mRNA decreased to the initial level very quickly. Thus, the high level of the expression of these oncogenes may not always be maintained in all malignant cells. We then examined the level of mRNA for poly(ADP-ribose) synthetase in those cases. An enhanced expression for the synthetase gene was observed in all five malignant lymphomata tested, but no increase in the level of the mRNA was observed in any reactive proliferative cases or normal lymph nodes. These results suggest that enhanced expression of poly(ADP-ribose) synthetase gene seems to be a common characteristic of protopathic malignant lymphoma. By using the characteristics of malignant lymphoma, the level of mRNA for the synthetase may be applicable for differential diagnosis of malignant lymphoma from several pathologically indistinguishable diseases.     

Cooperation of c-raf-1 and c-myc protooncogenes in the neoplastic transformation of simian virus 40 large tumor antigen-immortalized human bronchial epithelial cells.

Overexpression of c-raf-1 and the myc family of protooncogenes is primarily associated with small cell carcinoma, which accounts for approximately 25% of human lung cancer. To determine the functional significance of the c-raf-1 and/or c-myc gene expression in lung carcinogenesis and to delineate the relationship between protooncogene expression and tumor phenotype, we introduced both protooncogenes, alone or in combination, into human bronchial epithelial cells. Two retroviral recombinants, pZip-raf and pZip-myc, containing the complete coding sequences of the human c-raf-1 and murine c-myc genes, respectively, were constructed and transfected into simian virus 40 large tumor antigen-immortalized bronchial epithelial cells (BEAS-2B); this was followed by selection for G418 resistance. BEAS-2B cells expressing both the transfected c-raf-1 and c-myc sequences formed large cell carcinomas in athymic nude mice with a latency of 4-21 weeks, whereas either pZip-raf- or pZip-myc-transfected cells were nontumorigenic after 12 months. Cell lines established from tumors (designated RMT) revealed the presence of the cotransfected c-raf-1 and c-myc sequences and expressed morphological, chromosomal, and isoenzyme markers, which identified BEAS-2B cells as the progenitor line of the tumors. A significant increase in the mRNA levels of neuron-specific enolase was detected in BEAS-2B cells containing both the c-raf-1 and c-myc genes and derived tumor cell lines. The data demonstrate that the concomitant expression of the c-raf and c-myc protooncogenes causes neoplastic transformation of human bronchial epithelial cells resulting in large cell carcinomas with certain neuroendocrine markers. The presented model system should be useful in studies of molecular events involved in multistage lung carcinogenesis.     

Gene expression profiles on hypoxia and reoxygenation in rat gastric epithelial cells: a high-density DNA microarray analysis

Previous investigations have demonstrated that the cellular signaling induced by hypoxia-reoxygenation is a major pathway contributing to gastric mucosal injury induced by stress, non-steroidal anti-inflammatory drugs, and Helicobacter pylori. The aim of the present study was to perform a gene expression analysis of the gastric mucosal cellular response and to define the protective molecules in hypoxia and reoxygenation using a high-density DNA microarray analysis. Normal rat gastric mucosal (RGM-1) cells were subjected to hypoxia for 2 h, and reoxygenation was initiated by placing the cells in an environment of normoxia for 2, 4, or 8 h. Total RNA was extracted, and differences in the gene expression profiles between the normoxia and hypoxia groups or among the different durations of reoxygenation were investigated using a high-density DNA microarray. HIF-1- and apoptosis-related genes were modulated by hypoxia. Moreover, inflammation-, stress-, and wound- healing-related genes were regulated by reoxygenation following hypoxia. In particular, the expression of heat shock protein-70, amphiregulin and cyclooxygenase-2 were upregulated during reoxygenation following hypoxia, suggesting that these upregulations may play an important role in maintaining cell survival and supporting cell function.     

大鼠心肌组织中原癌基因（c—myc,Ha—ras,c—fos）表达的随龄性改变

目的　通过研究原癌基因(c-myc，Ha-ras，c-fos)在衰老心肌中的表达，探讨增龄对心肌生长相关的原癌基因的影响。方法　20月龄及6月龄健康雄性SD大鼠，以Northern杂交分析c-myc，Ha-ras，c-fos基因表达。结果　Ha-ras和c-fos在6月龄、20月龄大鼠心室肌中均表达，老龄鼠c-fos的表达水平明显高于青龄鼠(0.523±0.07，0.185±0.05)(P＜0.05)；c-Ha-ras则无显著差异(0.65±0.07，0.63±0.06)(P＞0.05)；c-myc在6月龄和20月龄大鼠心室肌中均无表达。结论　原癌基因((c-myc，Ha-ras，c-fos等)随增龄在心肌组织中的表达各不相同，探讨老年期中各基因重新激活和高表达的意义，有可能为老年心脏病的发生和心肌老化的内在机制提供一个新的解释。     

Cyclo-oxygenase-2 inhibitors suppress epithelial cell kinetics and delay gastric wound healing in rats

BACKGROUND AND AIMS: The present study examined the effects of NS-398, a specific cyclo-oxygenase-2 inhibitor, on gastric mucosal cell kinetics and gastric wound healing following acid-induced injury. METHODS: Male Sprague-Dawley rats were fasted for 24 h and then 0.6 mol/L hydrochloric acid (HCl; 1 mL) was administered into the stomach; NS-398 or indomethacin was administered to the animals 10 min after the acid. Levels of constitutive cyclo-oxygenase (COX-1) and mitogen-inducible cyclo-oxygenase (COX-2) in the gastric mucosa were analysed using western blotting and immunohistochemical staining. The grade of the lesion was assessed using planimetry and histological examination, including immunohistochemistry for proliferating cell nuclear antigen (PCNA). RESULTS: Although there was strong expression of COX-1, there was minimal expression of COX-2 in the gastric mucosa. Expression of COX-2 was enhanced mainly in surface epithelial cells and neck cells following HCl administration. Gastric mucosal ulcers and erosions healed within 48 h, during which time the proliferative zone expanded in the control animals. Indomethacin and NS-398 suppressed the expansion of the proliferative zone and delayed the healing of the gastric injury. CONCLUSION: The present study demonstrated that cyclo-oxygenase-2 inhibitors delay gastric wound healing by suppressing expansion of the mucosal proliferative zone. These results provide evidence that cyclo-oxygenase-2 has an important role in gastric mucosal regeneration.     

Papillomavirus sequences integrate near cellular oncogenes in some cervical carcinomas.

The chromosomal locations of cellular sequences flanking integrated papillomavirus DNA in four cervical carcinoma cell lines and a primary cervical carcinoma have been determined. The two human papillomavirus (HPV) 16 flanking sequences derived from the tumor were localized to chromosome regions 20pter----20q13 and 3p25----3qter, regions that also contain the protooncogenes c-src-1 and c-raf-1, respectively. The HPV 16 integration site in the SiHa cervical carcinoma-derived cell line is in chromosome region 13q14----13q32. The HPV 18 integration site in SW756 cervical carcinoma cells is in chromosome 12 but is not closely linked to the Ki-ras2 gene. Finally, in two cervical carcinoma cell lines, HeLa and C4-I, HPV 18 DNA is integrated in chromosome 8, 5' of the c-myc gene. The HeLa HPV 18 integration site is within 40 kilobases 5' of the c-myc gene, inside the HL60 amplification unit surrounding and including the c-myc gene. Additionally, steady-state levels of c-myc mRNA are elevated in HeLa and C4-I cells relative to other cervical carcinoma cell lines. Thus, in at least some genital tumors, cis-activation of cellular oncogenes by HPV may be involved in malignant transformation of cervical cells.     

Investigation of doublecortin and calcium/calmodulin‐dependent protein kinase‐like‐1‐expressing cells in the mouse stomach

Background and Aims: For lack of a definite stem cell marker, there is limited knowledge of the precise location and fate of stem cells after injury. Doublecortin and calcium/calmodulin‐dependent protein kinase‐like‐1 (DCAMKL‐1) is a putative intestinal and colon stem cell marker. Our aim was to identify DCAMKL‐1‐expressing cells in the gastric epithelium and to analyze the fate of DCAMKL‐1‐expressing cells during gastric mucosal injury and repair. Methods: Acidified ethanol was administered to wild‐type mice. DCAMKL‐1 expression were detected by immunohistochemistry and western blotting. Results: There were some DCAMKL‐1‐expressing cells in normal mouse stomachs. All the cells were located in the gastric isthmus region. All DCAMKL‐1‐expressing cells were double stained with Dolichos biflorus lectin‐expressing parietal cells and Musashi‐1‐expressing cells. The DCAMKL‐1 antigen expression decreased 12 h after injury and gradually increased to normal 4 d after injury. Conclusion: Using DCAMKL‐1 as a marker for stomach stem cells, we could describe the expression pattern of stomach stem cells during mucosal injury.     

Initiation of normal thyroid cells in primary culture associated with enhanced c-myc messenger ribonucleic acid levels

We studied expression of the c-myc and c-ras protooncogenes during the initiation of pig thyroid cells in primary cell culture. This period is associated with major changes in differentiated thyroid cell function. After 1, 2, and 3 days of culture, polyadenylated mRNA was prepared from 30-50 replicate dishes of cells. mRNA was also prepared from a portion of the intact tissue used to prepare the dispersed follicles. Expression of c-myc and c-ras mRNA was determined by Northern blot analysis using v-myc and v-ras probes labeled by nick translation. As a nononcogene control, actin mRNA was determined using a beta-actin probe. In intact thyroid tissue before follicle dispersion, c-myc (2.6 kilobases) and c-ras (1.1 kilobases) mRNA were detectable, though at relatively low levels. c-myc mRNA expression increased in cultured cells, to 430%, 670%, and 330% of tissue levels on the first, second, and third days of culture, respectively. In contrast to c-myc, c-ras oncogene mRNA expression was not significantly altered during the 3-day culture period. beta-Action mRNA expression, like c-myc, increased to 560%, 810%, and 460% of tissue levels on the first, second, and third days of culture, respectively. Southern blot analysis of thyroid tissue and cultured cell DNA indicated that gene amplification did not account for the increase in c-myc mRNA levels in cultured cells. In conclusion, c-myc, but not c-ras, oncogene expression is enhanced during the transition of thyroid follicular cells into monolayer culture. beta-Actin mRNA expression is also enhanced during the initiation of primary thyroid cell culture. These data are consistent with a role for c-myc expression in the regulation of normal thyroid cell differentiated function.     

Dissociation of cellular proliferation and c-myc expression by buttercup extract

Buttercup extract (BE), an extract of the buttercup plant (Zanthoriza simplicissima), inhibits RNA and DNA synthesis by HL-60 promyelocytic leukemia cells. Exposure of these cells to 3% BE for 48 hours results in dramatic inhibition of RNA synthesis without loss of cell viability. The effect of BE is partially reversible over 12-24 hours with the level of RNA synthesis returning nearly to control levels during this time period. DNA synthesis is also reversibly inhibited by exposure to BE. Despite the inhibition of RNA synthesis in HL-60 cells, there is no decrease in the level of c-myc mRNA, even at high BE concentrations. The level of gene-specific mRNA for the c-Ha-ras, c-fms, and c-mos genes in these cells also remained constant during exposure to BE. Ribosomal RNA is not degraded during 24 hours of BE treatment in vitro, suggesting that BE does not maintain the relative mRNA level for these genes by selective degradation of other RNA species. The inhibition of RNA and DNA synthesis by BE without a corresponding alteration in the level of expression of the c-myc gene suggests that this agent dissociates c-myc expression and cellular proliferation in these cells.