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.     

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.     

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.     

Expression of ras and myc oncogenes in human hepatocellular carcinoma and non-neoplastic liver tissues

An immunohistochemical assay was used to assess expression of ras p21 and myc p62 oncogene products in human hepatocellular carcinoma (HCC) and non-neoplastic liver tissues. The monoclonal antibodies Y13 259 and Myc1-9E10, specific for ras p21 and myc p62 oncoproteins, were employed on paraffin-embedded sections. Most HCCs showed enhanced ras p21 and myc p62 expression, as indicated by staining intensity. Cirrhotic livers revealed increased myc p62 and occasionally increased ras p21 expression. HBsAg+ hepatocytes showed intense immunostaining for ras p21. Fibrotic, cholestatic, fetal and normal adult liver did not present enhancement of oncoprotein production. We suggest that combined over-expression of ras and myc oncoproteins may be important for the malignant phenotypic alteration in human HCC.     

Modulation of c-myc oncogene expression by phorbol ester and interferon-gamma: appraisal by flow cytometry

A flow cytometric assay was developed to examine the expression of the cellular myc oncogene in relation to cell cycle in individual cells. C-myc-oncoprotein was detected by indirect immunofluorescence using a purified sheep polyclonal antibody, anti-human-myc. Specific binding of anti-human-myc was measured by flow cytometry. C-myc oncoprotein was detected in 90% of HL-60 and 75% of Daudi cells; human hematopoietic cell lines known to express high levels of c-myc oncogene. However, c-myc protein could not be detected in the REH cell line, normal human peripheral lymphocytes or thymocytes. Nuclear DNA content was measured simultaneously using propidium iodide staining. There was an equal level of c-myc protein in G0/G1, S and G2/M phases. The extent and kinetics of c-myc oncoprotein induction have been determined following phorbol ester, 12-O tetradecanoylphorbol 13 acetate (TPA) and interferon-gamma (IFN-gamma) exposure of both HL-60 and Daudi cells. TPA produced a gradual reduction in the level of c-myc protein and arrested the cells in G0/G1 phase in HL-60 cells. However, TPA failed to reduce c-myc protein or to change cell cycle distribution in Daudi cells. Interestingly, c-myc protein levels were stimulated by exposure of both HL-60 and Daudi cells to IFN-gamma. The results indicate that flow cytometric assay of oncogene expression is feasible, fast and requires relatively few cells. It also allows for the direct correlation of modulation of oncogene expression with cell kinetics.     

P53-dependent effects of RAS oncogene on chromosome stability and cell cycle checkpoints.

Mutations activating the function of ras proto-oncogenes are often observed in human tumors. Their oncogenic potential is mainly due to permanent stimulation of cellular proliferation and dramatic changes in morphogenic reactions of the cell. To learn more on the role of ras activation in cancerogenesis we studied its effects on chromosome stability and cell cycle checkpoints. Since the ability of ras oncogenes to cause cell transformation may be dependent on activity of the p53 tumor-suppressor the cells with different p53 state were analysed. Ectopic expression of N-ras(asp12) caused in p53-deficient MDAH041 cell line an augmentation in the number of chromosome breaks in mitogenic cells, significant increase in the frequency of metaphases showing chromosome endoreduplication and accumulation of polyploid cells. Similar effects were induced by different exogenous ras genes (N-ras(asp12), H-ras(leu12), N-ras proto-oncogene) in Rat1 and Rat2 cells which have a defect in p53-upstream pathways. In contrast, in REF52 and human LIM1215 cells showing ras-induced p53 up-regulation, ras expression caused only slight increase in the number of chromosome breaks and did not enhance the frequency of endoreduplication and polyploidy. Inactivation in these cells of p53 function by transduction of dominant-negative C-terminal p53 fragment (genetic suppressor element #22, GSE22) or mutant p53s significantly increased the frequency of both spontaneous and ras-induced karyotypic changes. In concordance with these observations we have found that expression of ras oncogene caused in p53-defective cells further mitigation of ethyl-metansulphonate-induced G1 and G2 cell cycle arrest, but did not abrogate G1 and G2 cell cycle checkpoints in cells with normal p53 function. These data indicate that along with stimulation of cell proliferation and morphological transformation ras activation can contribute to cancerogenesis by increasing genetic instability.     

D-苧烯对人胰腺癌细胞法尼基蛋白转移酶、p21~(ras)膜结合的抑制作用的体外试验

目的探讨ｄ－艹宁烯在化学诱发实验性肿瘤及人实体瘤的防治作用及其机制。方法采用Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ方法观察了ｄ－艹宁烯对胰腺癌细胞中与膜结合的ｐ２１ｒａｓ蛋白表达和法尼基蛋白转移酶活性的抑制作用。同时,采用免疫组化法观察了ｄ－艹宁烯对ｐ２１ｒａｓ在细胞膜上定位的影响。并以Ｎｏｒｔｈｅｒｎｂｌｏｔｉｎｇ方法检测ｄ－艹宁烯对Ｈ－ｒａｓ癌基因在人胰腺癌细胞系中的表达。结果ｄ－艹宁烯对人胰腺癌细胞呈现的抑制作用,其机制可能与其抑制法尼基蛋白转移酶活性,从而降低ｐ２１Ｈ－ｒａｓ蛋白的异戊二烯化修饰有关。ｄ－艹宁烯可降低ｐ２１ｒａｓ蛋白的膜结合,增加胞浆ｐ２１ｒａｓ蛋白的积蓄。在经ｄ－艹宁烯处理过的胰腺癌细胞加入抗ｐ２１ｒａｓ抗体,采用免疫组化法也可观察到上述现象。结论法尼基蛋白转移酶的抑制和ｐ２１ｒａｓ蛋白膜结合与ｐ２１ｒａｓ定位的改变密切相关。ｐ２１ｒａｓ法尼基化的抑制作用改变了它在细胞内的定位,从而影响其生物学活性,但尚未发现与Ｈ－ｒａｓ癌基因表达的密切相关。     

Development of quantitative liquid competition radioimmunoassays for the ras oncogene and proto-oncogene p21 products

The ras gene family of rodents and humans is highly conserved and consists of several distinct genes, i.e., rodent Harvey and Kirsten, and human Harvey, Kirsten and neuroblastoma. This gene family mediates transformation via (1) a point-mutation resulting in the change of one amino acid in the 21 kDA ras gene product (p21) or (2) increased expression of ras p21. Group-specific, type-selective and interspecies indirect binding liquid competition radioimmunoassays (RIAs), capable of providing truly quantitative analyses of the 21 ras oncogene and proto-oncogene products, have been developed. Using purified recombinant ras p21 from Escherichia coli expressing the full-length T24 mutant human Harvey-ras gene protein product as a standard in these RIAs, we have defined the absolute numbers of pg, fM and molecules of ras p21 in: (1) E. coli expressing the point-mutated or proto-ras p21 and (2) mammalian cell lines of human and murine origin. Two of the RIAs developed can be termed group-specific in that they have the ability to detect the point-mutated and proto forms of all 3 human ras genes (Harvey, Kirsten, and neuroblastoma), while the third RIA is type-selective, since it detects an antigenic determinant located primarily on the Harvey ras p21. All 3 RIAs are interspecies-specific since they are able to detect ras p21 in rodent as well as human cells. The adaptability of the RIAs to various assay conditions and ease of methodology make these immunoassays applicable to the study of several parameters associated with ras p21 expression. These assays, used in conjunction with specific cDNA probes to identify specific ras proto-oncogenes or point-mutated oncogenes being expressed, now provide truly quantitative analysis of ras p21 in mammalian cells to further the study of the association between ras p21 expression and transformation.     

Quantitation of Harvey ras p21 enhanced expression in human breast and colon carcinomas

Molecular studies have demonstrated increased expression of the Harvey (Ha) ras oncogene in human breast and colon carcinomas. With the use of a direct-binding liquid competition radioimmunoassay (RIA), capable of providing truly quantitative analysis of the 21,000-dalton (p21) ras oncogene and protooncogene products, absolute levels of Ha-ras p21 have been determined in human breast and colon carcinomas, benign lesions, and/or their respective normal tissues. Enhanced Ha-ras expression was documented in 66% of breast and 100% of colon carcinomas as compared with their normal counterparts, with levels in breast carcinomas ranging from 10.1 to 50.4 pg ras p21/micrograms protein and those in colon carcinomas ranging from 18.4 to 51.7 pg ras p21/micrograms protein. Some dysplastic lesions of the breast and colon also contained elevated Ha-ras p21. Relative levels of Ha-ras p21 expression, detected by competition RIA, correlated with percent Ha-ras p21-positive cells as determined by immunohistochemical assays. By use of liquid competition RIA and immunohistochemical assays, it has been shown that levels of ras p21 expression did not always correlate between primary and metastatic colon lesions of the same patient. The use of the quantitative RIA and semiquantitative immunohistochemical assays, in concert with cDNA probes for identification of specific ras point-mutated oncogenes or protooncogenes, may now provide the means for definitive quantitative analyses of ras p21 in human carcinomas and benign lesions.     

Cellular proliferation and ras oncogene of p21 21,000 expression in relation to the intracellular cyclic adenosine 3':5'-monophosphate levels of a human salivary gland adenocarcinoma cell line in culture

We have found that reversible differentiation into the myoepithelial cells of a human salivary gland adenocarcinoma cell line (HSG) occurs in growth medium containing dibutyryl cAMP (dB-cAMP). In the current study, the relationship between intracellular cAMP levels and anchorage-dependent and -independent growth or ras oncogene of p21 levels was analyzed in the differentiation process toward myoepithelial cells of HSG cells cultured in the presence of dB-cAMP. Correlation between the concentrations of dB-cAMP and intracellular cAMP in the HSG cells was statistically significant. There was a significant inverse correlation between the concentrations of dB-cAMP and colony-forming ability of the cells in semisolid agar or on a plastic surface. We have found the expression of ras p21 protein in HSG cells. When HSG cells were cultured in the presence of dB-cAMP and were committed to differentiate into myoepithelial cells, it was shown by double-antibody labeling technique and/or immunoblotting that the committed cells expressed myosin with a concomitant decrease of ras p21 protein. Moreover, intracellular cAMP levels were found to be inversely associated with ras p21 content of the cells. These findings indicate that the intracellular cAMP levels regulate significantly cell proliferation and ras p21 expression in HSG cells.     

Modification of Ha-ras oncogene p21 expression and cell cycle progression in the human colonic cancer cell line HT-29

Multiparameter flow cytometric measurements of the Ha-ras oncogene product, Ha-p21, versus DNA content were used to study the effect of prednisolone, sodium butyrate, and hyperosmolality on the expression of this gene during the cell cycle of HT-29, a human colonic carcinoma cell line. In control cells the expression of Ha-p21 was cell cycle dependent; it increased during G1 and remained approximately constant as cells traversed the S- and G2 + M phases. Two compartments of G1 cells, one expressing low (G1A) and the other (G1B) high levels of Ha-p21 could be identified. Cells grown with prednisolone (1.4-2.1 microM) expressed higher Ha-p21 levels than controls. Cell cycle analysis revealed that this effect was accompanied by a change in the distribution of cells in G1 phase: whereas the proportion of cells in G1A was reduced, that of cells in G1B was increased. The steroid had no detectable effect on cells in S and G2 + M. By contrast, sodium butyrate and hyperosmolality caused a marked decrease in Ha-p21 content. This reduction was not accompanied by any modification of the proportion of cells in the cell cycle compartments. These results would suggest that Ha-p21 is not likely to be a primary regulator of cell cycle progression in HT-29 cells.     

子宫内膜癌和子宫颈癌p21与DNA倍体的研究

目的研究ｒａｓｐ２１致癌基因和ＤＮＡ倍体与子宫颈癌及子宫内膜癌的关系。方法应用细胞免疫荧光技术和流式细胞术对４０例宫颈癌、２０例慢性宫颈炎、３２例子宫内膜癌和１３例癌前病变进行了ｒａｓｐ２１致癌基因的表达量及ＤＮＡ倍体的测定。结果子宫颈癌和子宫内膜癌ｒａｓｐ２１的表达量高于慢性宫颈炎和子宫内膜癌前病变,并与二者的组织学分级有关。ＤＮＡ倍体与ｒａｓｐ２１的表达量有关。结论ｒａｓｐ２１的表达与子宫颈癌和子宫内膜癌的发生有关。检测ｐ２１蛋白和ＤＮＡ倍体有助于判断这两种肿瘤的恶性程度和预后,并应积极治疗慢性宫颈炎     

Immunocytochemical study of RAS oncoprotein in cytologic specimens of primary lung tumours

We have examined the distribution of ras p21 oncoprotein expression in cytologic specimens from 73 primary bronchial carcinomas using an immunocytochemical analysis. The cytologic preparations studied represent the two major groups of histological types of lung cancer: Small Cell Lung Carcinoma (SCLC) and Non-Small Cell Lung Carcinoma (NSCLC) (squamous cell carcinoma and adenocarcinoma). The differential expression of ras p21 oncoprotein correlated with histological classification and was found in 30% of 23 small cell lesions, 61% of 28 squamous cell lung carcinomas and 32% of 22 adenocarcinomas. The ras p21 oncoprotein was commonly expressed in NSCLC cases (48%) as compared to SCLC cases (30%).     

宫颈癌及慢性宫颈炎ras癌基因产物p21和DNA倍体的研究

应用细胞免疫荧光技术和流式细胞术对宫颈癌和慢性宫颈炎组织ras癌基因产物p21蛋白的表达量进行了定量研究，并分析了rasp21的表达量与DNA倍体和细胞增殖指数的关系。结果显示：宫颈癌rasp21的表达量（荧光指数FI）明显高于慢性宫颈炎组织，rasp21表达的荧光指数随组织学分级升高而增高。DNA倍体和rasp21的表达量有密切关系。     

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.     

Immunohistochemical analysis of the ras p21 oncoprotein in Hashimoto's thyroiditis.

We studied the ras oncogene expression using immunohistochemical detection of p21 oncoprotein in paraffin-embedded tissue sections or fine needle aspiration (FNA) material from 25 patients with Hashimoto's thyroiditis and 12 healthy individuals. We also investigated the presence of this protein in the lymphocytes of thyroid gland, as well as in peripheral blood lymphocytes. We found increased expression of p21 oncoprotein by thyroid epithelial cells in 22 patients (intensity of staining ++), whereas we observed negative or slightly positive in 9 and 3 out of 12 normal controls, respectively (intensity of staining, - or +/-). We also detected p21 oncoprotein in moderate amounts in patients' intrathyroid lymphocytes (intensity of staining +), but peripheral blood lymphocytes did not present any staining result. Our findings provided evidence that epithelial cells, as well as lymphocytes infiltrating the thyroid gland, are probably "activated" in Hashimoto's thyroiditis. The significance of this "activation" in thyroid tumorigenesis remains unknown.     

Constitutive expression of the c-H-ras oncogene inhibits doxorubicin-induced apoptosis and promotes cell survival in a rhabdomyosarcoma cell line.

Drugs used in anti-cancer chemotherapy are thought to exert their cytotoxic action by induction of apoptosis. Genes have been identified which can mediate or modulate this drug-induced apoptosis, among which are c-myc, p53 and bcl-2. Since expression of oncogenic ras genes is a frequent observation in human cancer, we investigated the effects of the c-H-ras oncogene on anti-cancer drug-induced apoptosis. Apoptosis induced by a 2 h doxorubicin exposure was measured by in situ nick translation and flow cytometry in a rat cell line (R2T24) stably transfected with the c-H-ras oncogene and in a control cell line (R2NEO) transfected only with the antibiotic resistance gene neo. Both cell lines (R2T24 and R2NEO) had nearly identical growth characteristics, including cell doubling time, distribution over the cell cycle phases and plating efficiency in soft agar. Doxorubicin exposure of the R2NEO cells led to massive induction of apoptosis. In contrast, R2T24 cells, expressing the c-H-ras oncogene, showed significantly less apoptosis after doxorubicin incubation. Doxorubicin induced approximately 3- to 5-fold less cytotoxicity in the R2T24 cells than in the R2NEO cells, as determined by clonogenic assay in soft agar. No difference was observed in intracellular doxorubicin accumulation between the two cell lines, indicating that the classical, P-glycoprotein-mediated multidrug resistance phenotype is not involved in the observed differences in drug sensitivity. In conclusion, our data show that constitutive expression of the c-H-ras oncogene suppresses doxorubicin-induced apoptosis and promotes cell survival, suggesting that human tumours with ras oncogene expression might be less susceptible to doxorubicin treatment.     

Growth-regulated surface glycoproteins of human bladder cancer

Clinical studies of human bladder cancer cells with mouse monoclonal antibodies (mAbs) have revealed two surface glycoproteins (T43 and T138) expressed by aggressive cancers and not by normal cells and a differentiation antigen (T16) expressed by normal and tumor cells of urothelial origin (Y. Fradet et al., Proc. Natl. Acad. Sci. USA, 81: 224-228, 1984; Y. Fradet et al. Cancer Res., 46: 5183-5188, 1986). To investigate further the possible association of these antigenic phenotypes with growth advantage of tumor cells, their expression, according to phases of the cell cycle and growth states (exponential and plateau phase) was studied in the human bladder carcinoma cell line T24. Expression of the p21 ras oncogene product, the Thomsen-Friedenreich antigen and the HLA class I antigen were also studied with mAbs. Multiparameter flow cytometry was used to determine antigen expression and DNA content of cells stained with mAbs and propidium iodide. Two antigens, T16 and T43, showed marked variations of their expression according to the growth status of the cells, although with an inverse profile. T16 was expressed on resting cells up to 12.5 times more than on exponentially growing cells. Conversely, T43 expression increased by a factor of 4.5 on actively proliferating cells. However, there was no preferential expression of either antigen in any one phase of the cell cycle. None of the other antigens studied, including the p21 protein, showed any density variation with either cell cycle or growth states. The results of these studies suggest that T43 may be associated with a growth advantage of tumor cells and that T16 has the characteristics of a differentiation antigen whose expression is induced on resting cells. These findings may have implications for the potential clinical use of these mAbs.     

Ras oncogene expression and DNA content in plasma cell dyscrasias: a flow cytofluorimetric study

Using bivariate flow cytofluorometry, we have determined the nuclear DNA distribution and the expression of the p21 protein (coded by the Ha-ras oncogene) in the bone marrow (BM) cells of five solid tumour patients having histologically normal BM and in those of 57 patients with plasma cell dyscrasia (28 with monoclonal gammopathies of undertermined significance, MGUS, and 29 with multiple myeloma, MM). All normal and MGUS and 21/29 (72.4%) MM BM had diploid modal DNA content and 8/29 (27.6%) MM BM had both diploid and hyperdiploid cell populations. In normal and MGUS BM, the level of the p21 oncoprotein was low and uniform in all G0/G1, S and G2 cells (median fluorescence values in arbitrary units were 6.1 and 7.5, respectively). The level of p21 was increased both in different aliquots of G0/G1 cells and in the S and G2 cells in diploid MM (median value for G0/G1 cells was 20), and especially in MM with hyperdiploid clones (median value for hyperdiploid cells was 40.5, P less than 0.005 with respect to normal and MGUS BM and less than 0.005 with respect to diploid MM BM). The p21 expression was greater in patients with advanced (stage III) than in earlier MM (stages I + II) (P less than 0.005), and it was directly related to the BMPC infiltration (r = 0.7; P less than 0.005). Since p21 expression is greater in MM than in both normal and MGUS BM, Ha-ras could be involved in the malignant plasma cell transformation that distinguishes MM from MGUS.