Antipain-induced suppression of oncogene expression in H-ras-transformed NIH3T3 cells

Antipain (AP; 50 micrograms/ml) inhibits transformation of NIH3T3 cells after transfection with an activated H-ras oncogene. To determine whether AP effects on transformation are associated with alterations in oncogene expression, NIH3T3 cells were cotransfected with an activated H-ras oncogene and the selectable marker gene aph, and gene expression was quantified. Fifty percent of geneticin-resistant colonies which were exposed to AP failed to express the transformed phenotype as determined by their inability to grow in soft agar. Northern blot analysis of the transformed and nontransformed colonies revealed that suppression of H-ras transformation by AP was associated with a decrease in expression of the exogenously transfected H-ras gene by approximately 4-fold. Expression of the endogenous oncogene c-myc was decreased by approximately 2.5-fold, to levels seen in untransfected cells. AP-treated colonies that retained the transformed phenotype had levels of oncogene expression that were similar to untreated ras-transformed colonies. Southern blot analysis revealed no effects of AP on incorporation or copy number of the H-ras gene.     

Oncogene-mediated multistep transformation of C3H10T1/2 cells

We have examined the response of the mouse embryonic cell line C3H10T1/2 to transfection with the activated human c-H-ras oncogene and the gag-myc oncogene from avian myelocytomatosis virus 29. C3H10T1/2 cells are not morphologically transformed following transfection with the gag-myc oncogene. A low level of focus formation is observed following transfection of the c-H-ras oncogene. When C3H10T1/2 cells are cotransfected with the ras and myc oncogenes, focus formation is increased by an average of 13 fold. In addition, C3H10T1/2 ras/myc foci have a distinct, transformed morphology which correlates with an increased potential for anchorage-independent growth. Although morphological transformation in this system is largely a function of ras oncogene expression, our studies demonstrate that it is potentiated by the presence of a functional gag-myc protein. Oncogene-mediated multistep transformation, which was first described in primary embryo cultures, is not a general property of established cell lines. The C3H10T1/2 cell line is an exception and provides a model system in which partially transformed phenotypes, in a progression toward malignant transformation, can be isolated and studied.     

Different malignant phenotypes induced in a stable, subdiploid, benign epithelial clone by DNA transfection.

Progression to malignancy in carcinomas has been studied in a stable, benign, subdiploid, cloned epithelial cell line (A5P/B10) sensitive to Geneticin at 100 micrograms/ml. A total of 28 cell lines were selected for Geneticin - resistance and inoculated into the footpads of syngeneic animals following co-transfection with pSV2neo and genomic DNA, or transfection with plasmid constructs containing neo and the activated Ha-ras oncogene. The behavior of 12 cell lines cotransfected with normal genomic DNA and inoculated into 146 footpads was the same as the A5P/B10 cells. Low grade primary tumors were produced in 122 footpads by 13 cell lines transfected with Ha-ras, and a proportion (61/122) produced well-differentiated lymph node metastases. One of 3 cell lines cotransfected with genomic DNA from a malignant cell line (BC1) produced 8 anaplastic primary tumors with anaplastic metastases. Cell lines from lymph nodes involved by these anaplastic tumors were sensitive to Geneticin, and genomic DNA from 2 clones of these cells failed to produce a malignant phenotype when co-transfected into the A5P/B10 cells. These results indicated that the progression to a malignant phenotype induced in benign cells from a spontaneous epithelial tumor by co-transfection with genomic DNA from malignant cells was different from that induced by the ras oncogene.     

No acquisition of metastatic capacity of R1H rhabdomyosarcoma upon transfection with c-Ha-ras oncogene

The effect of the activated c-Ha-ras oncogene on invasiveness and formation of spontaneous metastases was studied using the rhabdomyosarcoma R1H of the rat. R1H tumor cells which are able to grow in vitro and produce tumors upon subcutaneous injection in syngeneic WAG/Rij rats were transfected with the c-Ha-ras (EJ) oncogene and the neomycin gene for selection. Two R1H cell lines harboring and expressing the human c-Ha-ras oncogene, one cell line containing the neomycin gene only, and the parent R1H cell line were compared. The expression of the transfected c-Ha-ras oncogene was assessed by Northern blot analysis and by flow cytometry using antibodies against ras p21. No difference in tumor growth rate and morphology was observed for the transfected and untransfected cell lines. Tumor volume doubling time was about 2 days in R1H-ras as well as in R1H parent tumors. Formation of spontaneous metastases was tested by excising the tumors when they had reached a volume of 2 cm3; after that the animals were observed up to 12 months. The excised tumors still contained and expressed the transfected ras oncogene as proved by Southern blot analysis and antibody staining using anti-ras p21. In contrast to most previous work on ras-transfected tumorigenic cells the R1H-ras tumors did not acquire invasive growth potential or increased metastatic capacity.     

Anti-receptor antibodies reverse the phenotype of cells transformed by two interacting proto-oncogene encoded receptor proteins

The neu oncogene product, p185neu, is a tyrosine kinase receptor with structural similarity to the epidermal growth factor (EGF) receptor. We have recently described that coexpression of EGF receptors and high levels of normal p185c-neu lead to transformation of rodent fibroblasts. Anti-EGF receptor and anti-p185neu monoclonal antibodies inhibited tumorigenic growth of these transformants implanted into nude mice. These monoclonal antibodies also suppressed focus formation of the cells transformed by the synergistic action of these receptor proteins in vitro. However, EGF enhanced focus formation and stimulated cell growth when added to cells transfected just with the EGF receptor encoding cDNA. These data suggest that receptor specific effectors may have potentially useful applications in cancer therapy for neoplasms which demonstrate increased receptor densities. In addition the data suggest novel differences in the actions of tyrosine kinases when acting alone or in concert with other receptors.     

Human oncogenes detected by a defined medium culture assay

Oncogenes in DNAs from human tumor cell lines have been detected by a new transformation assay. Cellular DNAs are transfected into NIH3T3 murine fibroblasts, and transformed cells are selected by maintaining cell cultures in a defined medium lacking platelet-derived or fibroblast growth factors. DNAs from eight of 17 human tumor cell lines have yielded transformants by this method. Activated cellular ras genes account for three of the transforming activities. The SAOS2 osteosarcoma cell line contains an activated oncogene distinct from 18 known oncogenes. Another cellular oncogene was detected as the consequence of a fortuitous transfection-mediated DNA rearrangement.     

Effects of triiodothyronine and tamoxifen on cell transformation induced by an activated c-Ha-ras oncogene

We have found that the thyroid hormone 3,5',3'-triiodo-L-thyronine stimulates the transformation of Rat 6 fibroblasts when these cells are transfected with an activated human c-Ha-ras oncogene (T24). 3,5',3'-Triiodo-L-thyronine did not further augment the stimulation of oncogene-induced transformation obtained with a phorbol ester tumor promoter (12-O-tetradecanoylphorbol-13-acetate) or a factor from fetal calf serum. On the other hand, tamoxifen, an antiestrogen that also inhibits protein kinase C, markedly inhibited Ha-ras-induced cell transformation in the presence of 12-O-tetradecanoylphorbol-13-acetate or fetal calf serum. Time course studies and Southern blot analyses of DNAs isolated from transformed foci provided evidence that 3,5',3'-triiodo-L-thyronine and tamoxifen do not exert their effects simply by enhancing or inhibiting integration of the transfected oncogene into cellular DNA. These findings indicate that hormonal factors can modulate the ability of an activated Ha-ras oncogene to transform cells. They may be relevant to the process of multistage carcinogenesis in vivo.     

Overexpression of ha-ras oncogene transforms rodent fibroblasts with low-frequency but not human-diploid fibroblasts

Activated Ha-ras oncogenes were introduced into early passage normal human adult dermal fibroblasts, 149BR and established mouse Swiss 3T3 fibroblasts by retroviral-mediated gene transfer or by DNA transfection, respectively. The presence and expression of Ha-ras oncogenes was followed by Southern and Northern blotting and immunoprecipitation. Overexpression of the human mutant c-Ha-ras1 T24 oncogene in mouse cells induced morphological transformation, focus formation and growth in soft agar with low frequency. Tumourigenicity studies showed that the malignant transformation of these cells by p21Ha-ras T24 oncoprotein was concentration-dependent and it required additional events suggesting that in vitro establishment is not a sufficient prerequisite for tumourigenic conversion by activated Ha-ras oncogenes. In contrast, constitutive expression of the viral Ha-ras oncogene in human diploid fibroblasts failed to immortalise or transform them. All ras-expressing human cells remained flat, anchorage-dependent and non-tumourigenic suggesting that these cells are resistant to transformation by activated oncogenes. The role of Ha-ras oncogenes in the transformation of mammalian cells in discussed.     

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.     

Loss of transforming growth factor beta 1 (TGF-beta 1)-induced growth arrest and p34cdc2 regulation in ras-transfected epithelial cells.

Transforming growth factor beta 1 (TGF-beta 1) is a potent inhibitor of mink lung epithelial (CCL64) cell growth in culture. The fact that many transformed epithelial cells have escaped from negative growth control by TGF-beta 1 suggests that transfected epithelial cells may be an appropriate model for investigating the growth-inhibitory mechanism of TGF-beta 1. We transfected CCL64 cells with a mouse c-myc oncogene (pSVc-myc1), a mutated Harvey-ras (Ha-ras) oncogene, or a combination of both. The results indicate that cells transfected with c-myc alone exhibit normal morphology and maintain sensitivity to TGF-beta 1 growth arrest, but are unable to form colonies in soft agar in the presence or absence of TGF-beta 1. Cells transfected with Ha-ras, or co-transfected with c-myc, display a transformed morphology, grow spontaneously under anchorage-independent conditions and acquire a complete resistance to growth inhibition by TGF-beta 1. Affinity cross-linking of [125I]TGF-beta 1 to cell-surface receptors from these transfectants revealed that all three TGF-beta receptor types were present and no significant differences in [125I]TGF-beta 1 labeling of these receptors was observed. Since we have previously demonstrated that modulation of p34cdc2 kinase is a marker for TGF-beta 1 growth inhibition, we investigated p34cdc2 activity in the CCL64 transfected clones. The results show that in the control CCL64 cells and in the myc-transfected clones TGF-beta 1 regulation of p34cdc2 activity is maintained. In the ras- and ras + myc-transfected cells p34cdc2 phosphorylation and histone H1 kinase activity is significantly increased and regulation by TGF-beta 1 is lost.     

Hormone receptors and cathepsin D levels in human breast epithelial cells transformed by chemical carcinogens and c-Ha-ras transfection

Summary The objective of this work was to determine whether transformation of the human breast epithelial cell line MCF-10F by the chemical carcinogens 7, 12-dimethylbenz(a)anthracene (DMBA) or benzo(a)pyrene (BP), or c-Ha-ras oncogene transfection, influence the expression of epidermal growth factor receptor (EGFR), estrogen (ER) or progesterone (PR) receptors, and the content of cathepsin-D (Cath.D). MCF-10F control cells did not express any of the phenotypes of neoplastic transformation, whereas carcinogen-treated cells and clones derived from the latter formed colonies in agar-methocel, and exhibited increased chemotaxis and chemoinvasion. Clone BP-1E was also tumorigenic in SCID mice. The BP1 cell line transfected with mutated c-Ha-ras oncogene, named BP1-Tras, became more aggressive after transfection and decreased the latency time to tumorigenesis. Radioligand binding and immunocytochemical reactions were utilized for determining the receptors and Cath.D content of control and carcinogen-treated cells and their derived clones. MCF-10F cells contained 37 fmol/mg of protein of EGFR, ER and PR were undetectable, and Cath.D content was 70 fmol/mg protein. EGFR content was significantly higher in D3-1 and BP1-E cell lines vs the control MCF-10F and the other DMBA and BP clones, correlating positively with the emergence of the transformation phenotype. Whereas EGFR levels were not significantly different in BP1-Tras cells when compared with BP1-E, the former were more tumorigenic in SCID mice, an observation suggesting an alternative pathway in these cells in the formation of tumors. PR, ER, and Cath.D content was not modified by either carcinogen treatment or ras transfection in most of the clones and subclones, except clone BP2 that has a significant increase in ER content, and was not associated with any of the neoplastic phenotypes. These data allowed us to conclude that the level of EGFR is associated with the expression of carcinogen-induced transformation phenotypes whereas ER, PR, and Cath.D did not seem to be modified during the process of chemically induced or c-Ha-ras enhanced transformation of MCF-10F cells.     

Ha-ras oncogene induction of invasion and metastasis is associated with the activation and redistribution of protease(s) in rat-kidney cells

To investigate the role of oncogenes in the development of metastatic ability, the Ha-ras oncogene was transfected into murine kidney cell line NRK and an alternative murine cell line C127 cells. The resulting transfectants, NRK-Ha and HC127, were assayed for their invasive capability in an in vitro invasion assay and compared with their parental cell lines. All transfected cells were demonstrated to express a 0.6 kb mRNA for the ras oncogene by Northern blot analysis. Using a modified Boyden chamber, we showed that the motile ability of the cell lines, transfected versus non-transfected, were not altered by the introduction of the Ha-ras oncogene. However, ras transfected NRK-Ha cells were able to penetrate and invade through an artificial basement membrane coated filter when compared with the non-transfected parental cell line. Neither the C127 cells nor its ras transfected HC127 cells were able to migrate or penetrate through an artificial basement membrane coated filter. Using immunoblot analyses, we determined that invasive NRK-Ha cells expressed a mature form of protease cathepsin B with a molecular weight between 25-28 kD. Furthermore, using subcellular fractionation and immunoblot, we localized this mature form of cathepsin B to the plasma membrane fraction of the invasive NRK-Ha cells. The plasma membrane fraction of invasive NRK-Ha cells was able to degrade basement membrane components such as fibronectin and the patterns seen were similar to those of purified cathepsin B degradation. Non-invasive NRK, C127 and HC127 cells had only the precursor form of cathepsin B with a molecular weight between 38-45 kD and were unable to degrade basement membrane components. We suggest that the presence and expression of the Ha-ras oncogene alone does not confer metastatic capability, but rather that the ras oncogene must signal specific intracellular redistribution of proteases such as cathepsin B in order to promote tumor cell invasiveness.     

Increased sensitivity of nontumorigenic fibroblasts expressing ras or myc oncogenes to malignant transformation induced by 5-aza-2'-deoxycytidine.

The hypomethylating chemotherapeutic drug 5-aza-2'-deoxycytidine (5AzadC) has been shown to induce cell differentiation in some systems, while promoting neoplastic transformation in others. Using both in vitro and in vivo models, we have explored the relationship between oncogene expression and the susceptibility of cells to malignant transformation by 5AzadC. The study involved several nontumorigenic subclones of NIH3T3 fibroblasts, including cells transfected with deregulated c-myc, as well as phenotypic revertants expressing v-Ki-ras or long terminal repeat-activated c-Ha-ras. Transient 5AzadC treatment of the oncogene-bearing cell lines was associated with a rapid and efficient neoplastic transformation. In some cases, over 50% of the cell population exhibited loss of contact inhibition of growth within 1 week of treatment. The transformants were capable of forming s.c. tumors and experimental lung metastases in recipient nude mice. In contrast, 5AzadC failed to induce malignant properties in control 3T3 cultures transfected with the bacterial neor gene; rather, treatment of these cells was associated with differentiation into adipocytes and myotubes. The differential response to 5AzadC was also observed in vivo, in mice first inoculated s.c. with the premalignant cells and then treated with 5AzadC 24 h later. In agreement with the in vitro model, tumor development in mice correlated with the presence of cells with activated ras or myc oncogenes. Cytidine analogs that do not inhibit DNA methylation (i.e., 6-azacytidine and 1-beta-D-arabinofuranosyl cytosine) had no effect on cell phenotype. The results indicate that exposure of cells to 5AzadC can lead to tumor progression both in vitro and in vivo and suggest that preexisting alterations in oncogene expression may facilitate the evolution of cancerous growth induced by hypomethylating agents.     

胃癌相关基因GCRG213小干扰RNA表达载体的构建及其对胃癌细胞的影响

目的探讨胃癌相关基因GCRG213小干扰RNA(siRNA)转染对胃癌细胞MKN45的影响。方法设计两对针对GCRG213可转录为siRNA的DNA片段,退火后插入siRNA表达载体IMG-800。测序正确的重组子IMG-800-1(含干扰片段1)、IMG-800-2(含干扰片段2)和空载体经脂质体转染人胃癌细胞系MKN45细胞,G418筛选获得稳定转染的细胞株。采用半定量RT-PCR及Western免疫印迹法,比较转染不同质粒的MKN45细胞中GCRG213在mRNA和蛋白质水平上的表达差异。选取稳定转染不同质粒的MKN45细胞,采用细胞计数法绘制细胞生长曲线,流式细胞仪分析细胞的增殖状态,Annexin V FITC/PI双标记法检测凋亡细胞,平板克隆形成实验、裸鼠移植瘤实验分析转染细胞成瘤性。结果经测序证实,干扰片段退火后正确插入小干扰RNA表达载体IMG- 800,组成重组子IMG-800-1和IMG-800-2。重组子IMG-800-1、IMG-800-2和空载体经脂质体转染人胃癌细胞系MKN45细胞,G418筛选获得稳定转染的细胞株。与对应的空载体比较,RT-PCR结果显示,转染siRNA的MKN45细胞中,mRNA的表达分别下调44.9%和49.5%;Western印迹法结果显示,转染siRNA的MKN45细胞中,蛋白的表达分别下调55.3%和64.2%。与转染空载体的细胞相比,转染siRNA的MKN45细胞生长增殖速度明显减慢,细胞周期表现为处于G0～G1期的细胞有所增加,G2/M期和(或)S期的细胞比例减少,细胞凋亡率增加,细胞克隆形成数量减少,裸鼠体内成瘤性降低。结论胃癌相关基因GCRG213 siRNA转染,可抑制肿瘤细胞的生长和增殖,促进肿瘤细胞的凋亡,抑制肿瘤细胞的成瘤性。     

Transformation of human breast epithelial cells by c-Ha-ras oncogene

To determine whether the c-Ha-ras oncogene plays a role in the initiation of mammary carcinogenesis, an immortalized human breast epithelial cell line, MCF-10A, was transfected with the plasmid vector pHo6T1 containing the T24 Ha-ras oncogene and the aminoglycoside phosphotransferase gene, which confers resistance to geneticin. Transfected cells exhibited an altered pattern of growth and tridimensional morphology in collagen gel. They also exhibited anchorage-independent growth and loss of requirement for hormones and epidermal growth factor; in addition, they expressed invasiveness and increased collagenolytic activity in an in vitro system and became tumorigenic in irradiated nude mice, all properties indicative of malignant transformation. Transformed cells contained the mutated c-Ha-ras oncogene and expressed the p21 mutated protein. These data indicate that the c-Ha-ras oncogene is capable of inducing malignant phenotypes in immortalized human breast epithelial cells.