Localization of oncoprotein P21ras in the human liver cancer

Using the human liver cancer DNA transfected NIH/3T3 cell line, the human N-ras oncogene and the over expression of the oncoprotein P21ras was demonstrated, BALB/C mice were immunized. The spleen cells from the immunized mice were fused with SP2/0 myeloma cells. After the HAT medium selection and screening, two hybridoma cell lines, SCI-Oncogema 1 and 2, were established. In the immunoprecipitation test, the molecular weight of the protein reacting to Oncogema 1 was 21,000. This M.W 21,000 protein possessed the capability to bind with GTP, i.e. the character of P21ras. These data indicate that the Oncogema 1 is the monoclonal antibody against P21ras. Using Oncogema 1, specimens from 6 liver cancer patients were studied by immunopathology. With ABC stain, it was observed that the malignant cells in all the samples showed dark staining; the P21ras revealed over expression. Although the staining was heterogeneous, it implied that the ras oncogene was involved in the carcinogenesis of these six samples. No over expression was seen in the normal liver cells even in those around the cancerous lesion. However, dysplastic cells were moderately stained which means that the ras oncogene was activated and P21ras over expressed in these cells. The results suggest that the ras oncogene and P21ras play an important role in the early stage of liver cancer carcinogenesis.     

Partial down-regulation of protein kinase C in C3H 10T 1/2 mouse fibroblasts transfected with the human Ha-ras oncogene

Biochemical and immunological comparison of mouse C3H 10T 1/2 fibroblasts and C3H 10T 1/2 fibroblasts transfected with human activated Ha-ras oncogene indicated significantly lower levels of protein kinase C (PKC) activity and protein in the ras-transfected cells. This effect was observed in three clonal cell lines transfected with an activated ras oncogene. Cytosolic extracts of the ras-transfected cells contained calcium-activated, phospholipid-dependent protein kinase (PKC) activity at 61% of the level of activity present in C3H 10T 1/2 cells. A similarly decreased level of phorbol ester-binding activity was observed in these cells. Analysis of the subcellular distribution of PKC activity in cells failed to indicate significant differences between these cell lines. Immunoblots showed a lower abundance of the Mr 80,000 PKC in ras-transfected cell homogenates and extracts compared to C3H 10T 1/2 cells. Both C3H 10T 1/2 cells and cells transfected with ras expressed only one of the PKC isozymes as resolved by hydroxylapatite chromatography demonstrating that ras transfection of cells did not induce expression of alternative PKC isozymes. These observations indicate that PKC was partially down-regulated in ras-transfected cells, perhaps resulting from constitutively elevated levels of products of phosphatidylinositol-4,5-bisphosphate hydrolysis. Although C3H 10T 1/2 cells were previously shown to be distinct from NIH 3T3 cells in their sensitivity to transformation by the T24-ras oncogene, ras transformation appears to partially down-regulate PKC in C3H 10T 1/2 cells in a manner identical to that for ras-transformed NIH 3T3 cells. This indicates that down-regulation of PKC directly results from the expression of an activated ras oncogene independently of cellular sensitivity to transformation by expression of ras. The common action of ras transformation and phorbol esters to down-regulate PKC provides a possible mechanism for synergism during multistage carcinogenesis.     

Neoplastic Transformation of Rat Colon Epithelial Cells by Expression of Activated Human K-ras

Somatic mutations of the K- ras oncogene play an important role in colorectal carcinogenesis. We determined whether rat colon epithelial cells could be transformed by introducing retroviruses carrying the activated human K- ras oncogene alone. Primary epithelial cells from the rat distal colon were infected with retroviruses carrying wild-type and two types of activated K- ras (asp and val at codon 12) cDNAs. Cells infected with the wild-type K- ras virus showed no change in morphology and died within 3 weeks, whereas the activated K- ras virus-infected cells underwent morphological changes within 3 days and continued to proliferate. From these cells, several cell lines were subsequently established. Epithelial cells transformed by activated K- ras formed colonies in soft agar culture and tumors in athymic nude mice. Multiple copies of human K- ras genes and large amounts of K- ras mRNAs and proteins were found in the transformed cells. These data suggest that overexpression of activated K- ras transforms rat colon epithelial cells.     

Ras oncogene-induced sensitization to 1-beta-D-arabinofuranosylcytosine

Human tumor cells containing ras oncogenes display enhanced sensitivity to 1-beta-D-arabinofuranosylcytosine (Ara-C) and other deoxycytidine analogues (H-M. Koo, et al., Cancer Res., 56: 5211-5216, 1996). Human tumor cell lines with or without a ras oncogene as well as a pair of isogenic cell lines with one containing an activated ras oncogene were used to study the basis for differential sensitivity. We found that human tumor cells containing ras oncogenes upon entry into the S phase of the cell cycle underwent apoptosis in response to Ara-C treatment. By contrast, human tumor cells harboring wild-type ras alleles were only delayed in the S phase when exposed to Ara-C. Thus, the ras oncogene specifically renders human cells more sensitive to Ara-C by preventing S-phase arrest. This may occur by the ras oncogene compromising an S-phase checkpoint.     

Causal role for an activated N-ras oncogene in the induction of tumorigenicity acquired by a human cell line

ras oncogenes have been found in approximately 15% of the human tumors analyzed. However, a causal role for these genes in the tumorigenesis of human cells has yet to be shown. Tumorigenic late-passage PA-1 human teratocarcinoma cells (E-PA-1) contain an activated N-ras gene. In this report evidence is presented that nontumorigenic early passage revertant PA-1 cells (E-PA-1) contain only the germ-line protooncogene. Introduction by gene transfer of the activated L-PA-1 oncogene induces E-PA-1 cells to form tumors, suggesting that the activated N-ras oncogene has a causal role in the tumorigenesis of these cells.     

Induction of the neoplastic phenotype of EBV-established B lymphocytes by the human Ha-ras oncogene

We report on the use of human B lymphocytes immortalized by the Epstein-Barr virus (EBV) as targets for transformation by the c-Ha-ras oncogene of bladder carcinoma cells T24. Several stably transformed cell lines were obtained and their in vivo and in vitro growth properties as well as levels of expression of the ras gene were studied. The transformed phenotype in these cells was correlated to ras oncoprotein expression level; only the cell lines which overproduce p21 ras, by at least six-fold, were tumorigenic in nude mice. In this regard, our ras transformed cells behave as lymphoblastoid cells transformed by the c-myc oncogene, suggesting that c-myc and c-Ha-ras might act on the same regulatory level.     

Stimulation of exocytotic degranulation by microinjection of the ras oncogene protein into rat mast cells

To investigate the possible role of ras proteins in the secretory process, we have microinjected the proto oncogenic and oncogenic forms of the human H-ras protein into rat peritoneal mast cells. Mast cells are secretory cells which, upon appropriate stimulus, liberate histamine and other mediators of the acute inflammatory reaction by exocytotic degranulation. We report here that microinjection of the ras oncogene protein into mast cells induces exocytotic degranulation. In contrast, microinjection of similar amounts of the proto-oncogenic protein has little apparent effect on mast cells. Degranulation induced by injection of the ras oncogene protein occurs in the absence of an external stimulus and requires the presence of external calcium. The ultrastructural features of exocytotic degranulation in mast cells injected with the ras oncogene protein are similar to those seen when mast cells are activated by soluble ligands. Our results suggest that ras proteins may be involved, possibly as regulatory elements, in cellular functions that control exocytosis.     

A modified p53 overcomes mdm2-mediated oncogenic transformation: a potential cancer therapeutic agent

The antiproliferative activities of wild-type (wt) p53 are inhibited by mdm2 (murine double minute2) oncogene product. We tested growth suppression activity of p53 14/19, an engineered p53 variant, which does not bind mdm2 and is completely resistant to the inhibition by mdm2. p53 14/19, unlike wt p53, suppressed the growth of cancer cells that contain amplified mdm2 oncogene efficiently by direct DNA transfection or adenovirus-mediated gene transfer. In addition, p53 14/19 also inhibited the growth of several different cancer cell lines expressing low levels of mdm2 oncogene product as efficiently as wt p53. We further examined the antioncogenic potencies of p53 14/19 in the rat embryo fibroblast cotransformation assay. Addition of wt p53 failed to cause any significant decrease in ras plus mdm2 foci counts. In contrast, cotransfection of p53 14/19 with ras and mdm2 significantly reduced foci number. In similar experiments, cotransfection of wt p53 or 14/19 p53 resulted in significant inhibition of oncogenic transformation in rat embryo fibroblast mediated by an activated ras plus c-myc, adenovirus E1A, or human papillomavirus E7 oncogenes. Therefore, these results suggest that p53 14/19 modified tumor suppressor gene may be a promising therapeutic agent for human cancers that express abnormally high levels of mdm2 oncogene product.     

MHC class I antigen expression is inversely related with tumor malignancy and ras oncogene product (p21ras) levels in human breast tumors.

MHC class I antigens participate in the immune response by presenting peptides to CD8+ cytotoxic T cells. Decreased expression of these antigens in tumor cells may contribute to an evasion of immune system and consequently to enhanced tumor growth. However, not all tumors expressing low levels of HLA antigens show increased malignancy, probably as a result of the differential activity of the oncogenes involved in malignant transformation. The ras family of cellular oncogenes is one of the most frequently detected families of transformation-inducing genes in human solid tumors. The aim of this work is to study the expression of MHC antigens and the ras oncogene product, p21ras, in 60 primary breast tumors in order to define its clinical significance in tumor progression. HLA antigen expression and p21ras levels were measured on breast tumors using immunohistochemistry methods and enzymoimmunoassay, respectively. The results demonstrate that more invasive tumors have both a decreased expression of HLA class I antigens and higher levels of p21ras protein expression than less aggressive tumors. These findings indicate that the capacity of breast cancers to grow and metastasize is related to low levels of MHC class I antigens and enhanced p21ras expression, thus supporting the involvement of MHC and ras oncogenes in breast tumor malignancy.     

人胃癌特异性DNA结合蛋白质

作者用不同的方法都发现转化的Rat3-3细胞核中有一种能与Ha-ras癌基因上游区2.5kb特异结合的蛋白质,分子量大约为35kD。此蛋白质不能与6.6kb基因本身结合。在未转化的细胞中无此蛋白质。作者还做了20个胃癌病人手术切下的胃癌组织和周边组织,结果是胃癌组织细胞核内有与2.5kb结合的蛋白质,而周边组织没有。     

Changes in rasT24 expression do not induce changes in c-jun, jun-B, or jun-D RNA levels in rat liver epithelial cells

We used a series of rat liver epithelial (RLE) cell lines that carry a zinc-regulatable metallothionein/rasT24 fusion gene (MTrasT24) to investigate the relation of ras oncogene expression to steady-state RNA levels of the jun family of genes. In these cells, steady-state RNA levels of c-jun, jun-B, and jun-D were unrelated to rasT24 RNA levels or the phenotypic changes induced by the ras oncogene. Steady-state levels of the three jun mRNAs varied among different rasT24 transformed clones, and, although some clones exhibited concomitant induction of rasT24 and jun mRNAs, other clones exhibited no such correlation. We conclude that the effects of rasT24 in transformed RLE cells do not appear to be mediated by c-jun, jun-B, or jun-D and that studies examining only a single transformed clone may give misleading results with respect to the role of various oncogenes in the transformation process.     

Lovastatin, a cholesterol biosynthesis inhibitor, inhibits the growth of human H-ras oncogene transformed cells in nude mice

Post-translational modification of oncogenic p21ras proteins with farnesyl, a lipid intermediate in cholesterol biosynthesis, is required for p21ras membrane association and for the ability of p21ras to transform cultured cells. We have tested the ability of lovastatin, a specific inhibitor of cholesterol biosynthesis, to inhibit the growth of ras oncogene-transformed cells in vivo. Balb/c mouse 3T3 cells, transfected with H-ras oncogene from human EJ bladder carcinoma, were highly tumorigenic in nude mice. Immunoprecipitation studies with transformed EJ cells showed that lovastatin (1-100 microM) inhibited p21ras membrane association in a concentration-dependent manner and that a 10 microM concentration reduced the amount of p21ras bound to the membrane by 50%. Lovastatin also inhibited EJ cell growth in a concentration range that closely paralleled that required for inhibition of p21ras membrane association. Treatment of nude mice bearing subcutaneous (s.c.) EJ tumors with lovastatin (50 mg/kg) significantly inhibited the abilities of these tumors to grow as early as four days and, by day 12, the lovastatin treated group of animals had tumors with an average size that was 3-fold smaller than those in the saline treated group. Western blotting studies showed that lovastatin (50 mg/kg) was also able to inhibit p21ras membrane association in EJ tumors implanted s.c. in nude mice. These results demonstrate that lovastatin, an inhibitor of cholesterol biosynthesis, inhibited in vivo tumor growth of H-ras oncogene transformed cells. The results also suggest that inhibition of p21ras membrane association, an essential step in ras oncogene neoplastic transformation, is one mechanism by which lovastatin may express its antitumor activity.     

Role of the Ha-ras (RasH) oncogene in mediating progression of the tumor cell phenotype (review)

Recent experimental evidence indicates that the c-Ha-ras (rasH) oncogene may be causally involved in the etiology and evolution of specific human neoplasms. In addition, cultured cells transformed by the rasH oncogene can induce both a tumorigenic and a metastatic phenotype when expressed in appropriate cultured cells. To begin to define the molecular and biochemical mechanism(s) by which the rasH oncogene induce their effects on expression of the transformed state we have employed a cloned rat embryo fibroblast (CREF) cell line. Transformation of CREF cells with wild-type 5 adenovirus (Wt) results in transformed cells which display anchorage-independence and an increased saturation density in monolayer culture, but are non-tumorigenic in both athymic nude mice and syngeneic Fischer rats. In contrast, when CREF cells are transformed with mutant type 5 adenoviruses, such as H5hrl, or the ElA transforming gene from hrl (0-4.5), tumors are induced in both nude mice and syngeneic rats. However, hrl (0-4.5)-transformed CREF cells are not metastatic following intravenous injection into the tail vein of syngeneic rats. Insertion of an activated T24 rasH oncogene or a wild-type v-rasH oncogene into CREF, wt-transformed CREF or hrl (0-4.5)-transformed CREF cells results in acquisition of a metastatic phenotype by these cells. A mutant v-rasH oncogene (mutant 116K), which is defective in GTP binding and the induction of transformation of NIH 3T3 cells, does not induce transformation in CREF cells, but it can progress wt-transformed CREF cells to a tumorigenic-non-metastatic state. Employing this model system which displays well-defined and stable stages in the tumor cell progression lineage, we have analyzed the potential role of changes in the phosphatidylinositol (PI) cycle and phospholipase A2 (PLA2) enzyme activity during progression to a tumorigenic and metastatic phenotype. An increase in PI cycle intermediates (primarily inositol triphosphate; IP3) were observed only in the wt-transformed and hrl (0-4.5)-transformed CREF cell lines transfected with the rasH oncogene. In the case of PLA2, all rasH-transformed CREF cell lines displayed increased activity. In contrast, CREF cells transformed only by Ad5 (Wt or hrl (0-4.5)) or the 116K v-rasH oncogene did not display increased PLA2 activity similar to that observed in rasH transfected cells. Since one important metabolite generated by PLA2 is arachidonic acid, which is converted into prostaglandins and leukotrienes by cyclooxygenase or lipooxygenase, respectively, the levels of prostaglandin E2 (PGE2) in the various cell lines were monitored.(ABSTRACT TRUNCATED AT 400 WORDS)     

Early and late responses to induction of rasT24 expression in Rat-1 cells

We have used a series of Rat-1 cell lines carrying a Zn-inducible human c-Ha-ras oncogene construction (MTrasT24) to evaluate the effect of varied ras oncogene expression on the expression of genes and proteins related to morphologic transformation in vitro. In response to the expression of the ras oncogene, at least two different classes of events occur. These events, referred to as 'early and late' events, are dependent on distinctively different accumulated levels of the ras oncoprotein. Relatively low levels of activated c-Ha-ras p21 protein (1.5-2.5 times the proto-oncogene level) stimulate rapid entry of quiescent (G0) cells into the cell cycle and result in increased steady state c-myc and glucose transporter mRNA levels which are detectable as early as 3-6 h after zinc addition. In contrast, morphologic transformation develops more slowly and does not appear until 72-96 h after Zn++ stimulation in cells with very low basal levels of activated p21 (MR4 cells) and 24-48 h in cells with higher basal levels (MR5 cells). These morphologic changes depend on the accumulation of significant amounts of the ras oncoprotein (greater than 4 to 5 times the proto-oncogene level) and are accompanied by large increases in the steady state mRNA levels of transin and TGF-alpha and decreases in PDGF-receptor mRNA and fibronectin protein and mRNA levels. In addition, the level of a novel cytoplasmic protein species (referred to as p29), which is stained by a monoclonal antibody for ras, is dramatically reduced in response to these levels of activated ras protein. Thus changes in morphology and gene expression induced by rasT24 occur sequentially and are quantitatively dependent on activated ras expression.     

Immunohistochemical detection of the ras oncogene p21 product in an experimental tumour and in human colorectal neoplasms

The monoclonal antibody Y13 259 to the ras oncogene protein product p21 was used in an immunohistochemical study of ras expression in human colorectal neoplasms. The ability of the antibody to detect enhanced levels of ras expression was confirmed by its use with an experimental neoplasm known to express ras at high levels. Human colonic adenocarcinomas in general showed a similar staining intensity to that seen in normal mucosa. Adenomas however showed consistently high p21 expression as indicated by staining intensity. This suggests that elevated ras expression may be important in the development of adenomas, but that high levels need not be sustained in the conversion to invasive carcinoma.     

Syndecan-1 alterations during the tumorigenic progression of human colonic Caco-2 cells induced by human Ha-ras or polyoma middle T oncogenes.

The products of ras and src proto-oncogenes are frequently activated in a constitutive state in human colorectal cancer. In this study we attempted to establish whether the tumorigenic progression induced by oncogenic activation of p21ras and pp60c-src in human colonic Caco-2 cells is associated with specific alterations of syndecan-1, a membrane-anchored proteoglycan playing a role in cell-matrix interaction and neoplastic growth control. To this end, we used Caco-2 cells made highly tumorigenic by transfection with an activated (Val 12) human Ha-ras gene or with the polyoma middle T (Py-MT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity. Compared with control vector-transfected Caco-2 cells, both oncogene-transfected cell lines (1) contained smaller amounts of membrane-anchored PGs; (2) exhibited decreased syndecan-1 expression at the protein but not the mRNA level; (3) synthesized 35S-labelled syndecan-1 with decreased specific activity; (4) produced a syndecan-1 ectodomain with a lower molecular mass and reduced GAG chain size and sulphation; and (5) expressed heparanase degradative activity. These results show that the dramatic activation of the tumorigenic potential induced by oncogenic p21ras or Py-MT/pp60c-src in Caco-2 cells is associated with marked alterations of syndecan-1 expression at the translational and post-translational levels.     

Multistep process of neoplastic transformation of normal human fibroblasts by 60Co gamma rays and Harvey sarcoma viruses

As reported previously (Namba et al., 1985), normal human fibroblasts were transformed by 60Co gamma-ray irradiation into immortal cells with abnormal karyotypes. These transformed cells (KMST-6), however, showed a low cloning efficiency in soft agar and no transplantability. However, upon treatment with Harvey murine sarcoma virus (Ha-MSV), the cells acquired elevated clonability in soft agar and transplantability in nude mice. Ha-MSV alone, however, did not convert normal human fibroblasts into either immortal or tumorigenic cells. The Ha-MSV-transformed KMST-6 cells showed an enhanced expression of the ras oncogene, but normal and 60Co gamma-ray-transformed cells did not. Our current data suggest that gamma rays worked against normal human cells as an initiator, giving rise to chromosome aberrations and immortality, and that Ha-MSV, probably through its ras oncogene, played a role in the progression of the malignant cell population to a more malignant one showing enhanced colony formation in soft agar and tumorigenicity in nude mice.