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.     

pLNCX-pemt2重组载体的构建及pemt2基因在大鼠肝癌CBRH-7919细胞中的表达

为进一步研究 pemt2对肝癌细胞生长抑制的作用机制提供方便的实验模型 ,构建了p LNCX- pemt2重组体 .将目的基因 pemt2连接入含有 neo抗性基因的真核细胞表达载体 p LNCX中 ,构建 p LNCX- pemt2重组子 ,并用磷酸钙沉淀法将其转入大鼠肝癌 CBRH- 791 9细胞中 ,应用PCR、Western印迹及 [3 H]SAM参入等技术对其转染、表达及活性进行鉴定 .转染 p LNCX- pemt2的大鼠肝癌细胞 ,PEMT2成功表达 (分子量为 2 2 .5k D) ;高表达克隆 PEMT2的表达量比对照组高约 5倍 ,其活性比对照组高 2 .1倍 ;细胞生长的倍增时间从 2 1 .54± 7.0 8h延长到 43.2 2± 7.1 1h.结果表明 ,p LNCX- pemt2重组体转入肝癌细胞后 ,PEMT2蛋白得到高效表达 ,明显抑制肝癌细胞生长 .     

Studies on the promoter region of the c-Ha-ras gene in FRTL5 rat thyroid cells

The functional activity of the promoter region of the rat c-Ha-ras gene was examined in FRTL5 rat thyroid cells, the cell type from which this promoter was cloned. A plasmid (p035-ras-CAT) was constructed containing the untranslated-1 exon as well as 172 base pairs (bp)5' to this exon inserted upstream of the chloramphenicol acetyl transferase (CAT) reporter gene. These 172 bp of 5'-flanking region contain two 10 bp GC box consensus sites and two CAAT boxes. Very weak promoter activity was observed in experiments involving transient transfection of FRTL5 cells with this plasmid, as well as with another plasmid (p5kb-ras-CAT) containing a much more extensive (3.5 kb) 5'-flanking region of the gene. In contrast, strong promoter activity was observed when the same plasmids were transfected into mouse 3T3 fibroblasts. When other promoters (pfos, RSV, and MMTV) were used to drive CAT activity, CAT activity in FRTL5 cells was about 10-fold less than in NIH-3T3 cells and rat embryo fibroblasts. However c-Ha-ras promoter activity was reduced out of proportion in FRTL5 thyroid cells relative to the other cell types (approximately 50-fold less). DNA gel-shift assays performed using crude extracts of FRTL5 and 3T3 nuclear proteins revealed quantitatively similar binding to the same promoter region in the c-Ha-ras 5'-flanking sequence. These data demonstrate that promoter activity of the rat c-Ha-ras gene is contained within the 172 bp 5'-flanking region of the gene. This promoter activity is expressed at a much lower level in slow-growing FRTL5 cells relative to other more rapidly growing cell types.     

Neoplastic transformation by the human gene N-myc.

Amplification and abundant expression of a gene known as N-myc are found frequently in advanced stages of human neuroblastoma and may play a role in the genesis of several malignant human tumors. Previous studies have shown that N-myc can cooperate with a mutant allele of the proto-oncogene c-Ha-ras to transform embryonic rat cells in culture. Here we show that N-myc can also act alone to elicit neoplastic growth of an established line of rat fibroblasts (Rat-1). We used recombinant DNA vectors to express either N-myc or its kindred gene c-myc in transfected cells. Both genes caused morphological transformation, anchorage-independent growth, and tumorigenicity. We noticed two variables that appeared to influence the ability to isolate cells transformed by N-myc and c-myc: the abundance in which the genes were expressed and biological selection to eliminate untransformed cells from the cultures. Our findings sustain the belief that N-myc is an authentic proto-oncogene, lend further credibility to the role of N-myc in the genesis of human tumors, and establish a convenient assay that can be used to explore further the properties of both N-myc and c-myc.     

Partial purification, properties and regulation of inosine 5''phosphate dehydrogenase in normal and malignant rat tissues.

IMP dehydrogenase (EC 1.2.1.14) was purified 180-fold from rat liver and from the transplantable rat hepatoma 3924A. The enzymes from the two sources were apparently identical; they exhibited hyperbolic saturation kinetics and an ordered, sequential mechanism, and were subject to inhibition by a number of purine nucleotides. Km values for the substrates, IMP and NAD+, were 12 and 24 micrometer respectively. IMP dehydrogenase activity in a spectrum of rat hepatomas was increased, relative to normal liver, by 2.5--13-fold; these increases correlated with tumour growth rate. Activity in two rat kidney tumours was increased 3-fold relative to that in normal renal cortex; control of activity of this enzyme is apparently altered in neoplastic cells. After partial hepatectomy, IMP dehydrogenase activity began to rise 6 h after operation, reaching a peak of 580% of normal activity by 18 h. Activity in neonatal liver, however, was only slightly higher than that in the adult. Organ-distribution studies showed highest enzyme activities in spleen and thymus. In livers of rats starved for 3 days, where all enzymes, except those involved in gluconeogenesis, showed decreased activity IMP dehydrogenase activity was increased; this change was accompanied by a rise in hepatic GTP concentrations. It is concluded that IMP dehydrogenase is a key enzyme in the regulation of GTP production, and thus involved in regulation of nucleic acid biosynthesis. The increased activity of IMP dehydrogenase in liver of starved rats may be related to the requirements for GTP for gluconeogenesis.     

Increased concentration of CTP synthetase in hepatoma 3924A: immunological evidence

CTP synthetase (UTP:L-glutamine ligase, EC 6.3.4.2) was purified 370-fold from rapidly growing rat hepatoma 3924A. A major band was demonstrated by acrylamide gel electrophoresis which corresponded to this enzymic activity. It was estimated that the enzyme was 90% pure. Antibodies were produced in rabbit using this purified hepatoma enzyme. The specificity of the anti-serum was proved by the absence of the reaction between control serum and CTP synthetase. The amount of anti-serum required to inactivate completely the cytosolic CTP synthetase of hepatoma 3924A was 11-fold of that required for normal liver which is in good agreement with the 11-fold increase in CTP synthetase activity in this hepatoma. These results demonstrate that the liver and hepatoma 3924A CTP synthetases were immunologically similar or identical and that the markedly increased enzymic activity in hepatoma 3924A reflected an increase in the enzyme protein amount. These studies provide further evidence that in the neoplastic transformation a reprogramming of gene expression takes place which is manifested in the emergence of increased concentrations of CTP synthetase which should provide selective advantages to cancer cells by increasing the capacity for this rate-limiting step in $\text{de novo}$ CTP biosynthesis.     

Tissue-specific expression is conferred by a sequence from the 5'' end of the rat albumin gene.

We have constructed a transient expression vector containing 400 bp of rat albumin gene immediate 5'-flanking sequences inserted 5' to the bacterial enzyme chloramphenicol acetyl transferase (CAT). We have transfected various clones of rat hepatoma cells representing different states of expression of the liver phenotype with this vector (pALB-cat) and also with two control vectors containing viral promoters (pSVE-cat and pRSV-cat), and measured activity of the bacterial enzyme CAT in cellular extracts 48 h later. The albumin flanking sequences are able to direct highly efficient CAT expression, compared with the control vectors, only in cells which express their own albumin gene: the albumin-negative hepatoma cells are at least 100 times less efficient in expressing CAT after transfection with the pALB-cat plasmid than are the albumin-positive ones. An unexpected result of our study is the total inability of the rat albumin flanking sequences to direct expression in albumin-producing mouse hepatoma cells.