Role of activated c-H-ras oncogene in the induction and progression of immortal liver epithelial cell lines derived from normal C3H mice

The nature of 15 immortal mouse liver epithelial cell (MLEC) lines established from normal C3H mice was investigated specifically in terms of ras oncogene activation. Neither transforming activity nor point mutation within codon 61 of c-H-ras could be demonstrated in any of the cell lines by DNA transfection in a NIH/3T3 cell system or by the direct sequencing method after polymerase chain reaction, respectively. Acrylamide gel migration analysis of ras p21 products did not show any shift from the normal. Transplantation experiments demonstrated only 2 out of the 15 lines to be tumorigenic in nude mice. When 4 of the non-tumorigenic MLEC lines were transfected with cloned activated c-H-ras containing a point mutation within codon 61, they all became tumorigenic, the resultant neoplasms being hepatocellular carcinomas often associated with albumin mRNA expression. Our results thus indicate that ras activation is not necessary for immortalization or even for transformation of mouse liver cells in culture, and that ras activation may be an event during the progression process in mouse hepatocarcinogenesis in vivo.     

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.     

Enhanced ras oncogene mediated cell transformation and tumorigenesis by adenovirus 2 mutants lacking the C-terminal region of E1a protein

Mutants of adenovirus 2 E1a defective in coding for the C-terminal 61 or 67 amino acids of a 243 amino acid (243R) protein are defective in immortalization of primary baby rat kidney (BRK) cells. However, they cooperate with T24 ras in oncogenic transformation more efficiently than wt. BRK cells transformed by the E1a C-terminal mutants and T24 ras induce rapidly growing tumors in syngeneic rats and athymic mice whereas cells transformed by the wt 243R and ras oncogene are not tumorigenic in syngeneic rats and can only induce slowly growing tumors in athymic mice. Cells transformed by the E1a mutants and ras oncogene also induce rapid metastatic tumors whereas cells transformed by the wt 243R and T24 ras can not do so. The increased tumorigenic ability exhibited by the 243R mutants does not appear to be due to differential levels of expression of p21 ras. Our results suggest that the C-terminal region of the 243R protein may have a novel function in suppression of cell transformation, tumorigenesis and tumor progression.     

BRCA1 partially reverses the transforming activity of the ras oncogene

The BRCA1 gene is associated with hereditary breast and ovarian cancers. BRCA1 fits the model of a classic tumor suppressor gene, a hypothesis supported by recent work demonstrating that expression of BRCA1 inhibits growth of breast and ovarian cancer cell lines. The present study was designed to test the potential of BRCA1 to reverse the transforming activity of the ras oncogene. The v-Ha ras oncogene was cloned downstream of the retrovirus LTR and stably expressed in Rat-1 cells (Rat-1/ras). Rat-1/ras (R/R) cells were fully transformed as indicated by change in morphology, colony formation in soft-agarose and tumor induction in nude mice. BRCA1 was stably expressed in R/R cells under the CMV promoter (R/R-BRCA1). The expression of ras and BRCA1 was confirmed by Western blot using monoclonal antibodies (mAbs) specific to ras and BRCA1, respectively. R/R-BRCA1 cells grew slower than the negative control, which was R/R cells transfected with vector alone (R/R-pCEP4). R/R-BRCA1 cells generated approximately 5 to 10 times less colonies in a soft-agarose assay compared to the negative control. When injected into nude mice, R/R-BRCA1 cells exhibited a delayed onset of tumorigenesis and generated smaller tumors compared to R/R or R/R-pCEP4 cells. These data strongly suggest that BRCA1 partially reverses the transforming activity of the v-Ha ras oncogene indicating that BRCA1 can bypass the effects of the v-Ha ras oncogene on cell growth. BRCA1, therefore, may be used in therapy of tumors arising due to activation of v-Ha ras oncogene.     

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.     

PTEN基因转染的子宫内膜癌细胞对阿霉素诱导凋亡的敏感性

目的 探讨PTEN基因是否增强子宫内膜癌细胞系Ishikawa细胞对阿霉素的敏感性。方法 将PTEN基因转染前、后的Ishikawa细胞分别暴露于系列浓度的阿霉素,以MTT法测定这些细胞对阿霉素的敏感性,并通过Hoechst33258染色荧光显微镜观察细胞凋亡情况。同时,以免疫沉淀Western blot法观察阿霉素对Bad和Akt／PKB蛋白磷酸化的影响。结果 阿霉素以剂量依赖方式诱导全部细胞系的细胞凋亡,但对表达PTEN的克隆细胞所诱导的凋亡比对亲本株Ishikawa细胞更明显。低浓度阿霉素（0．1μmol／L）不引起PTEN蛋白缺失的Ishikawa细胞凋亡,但却能诱导表达PTEN蛋白克隆细胞凋亡;高浓度阿霉素（1μmol／L）诱导全部细胞系的细胞凋亡,但凋亡的发生率在表达PTEN的克隆细胞中明显高于亲本株Ishikawa细胞。在表达PTEN的克隆细胞中,磷酸化Akt／PKB和磷酸化Bad（Ser-136）的水平均下降。阿霉素降低了全部细胞系的磷酸化Akt／PKB和磷酸化Bad（Set-136）水平,但在表达PTEN的克隆细胞中降低最明显。结论 PTEN基因转染明显地增强了Ishikawa细胞对阿霉素的化学敏感性,阿霉素可能是通过下调P13k／Akt／PKB信号途径而与PTEN蛋白共同发挥凋亡诱导作用。     

Osteopontin is required for full expression of the transformed phenotype by the ras oncogene

The secreted phosphoprotein osteopontin (OPN) is strongly associated with the process of neoplastic transformation, based both on its pattern of expression in vivo and in vitro and on functional analyses. We have used 3T3 cells derived from wildtype and OPN-deficient mice and transformed by transfection with oncogenic ras to assess the role of OPN in transformation in vitro and in tumorigenesis in vivo. There was no effect of an absence of OPN on the ability of the cells to undergo immortalization or to form morphologically transformed foci following ras transfection. Wildtype and OPN-deficient cell lines were established from such foci, and lines with similar ras mRNA levels selected for further analysis. Ras-transformed cell lines from both wildtype and OPN-deficient mice could form colonies in soft agar indicating that this process can occur in the absence of OPN. However, the ability of the OPN-deficient cell lines to form colonies was reduced as compared to wildtype cell lines. Tumorigenesis in syngeneic and nude mice was assessed for a subset of cell lines that formed colonies efficiently in soft agar. Cell lines unable to make OPN formed tumors in these mice much more slowly than wildtype cells, despite similar growth of the cells on plastic and in soft agar. Taken together, these results indicate that maximal transformation by ras requires OPN expression, and implicate increased OPN expression as an important effector of the transforming activity of the ras oncogene.     

Reversible suppression by nalidixic acid of anchorage-independent growth of mouse cells transformed by 3-methylcholanthrene or an activated c-Ha-ras gene

Effects of nalidixic acid and its derivatives were investigated on mouse cells transformed by methylcholanthrene or an activated c-Ha-ras oncogene. Our findings were as follows. Nalidixic acid preferentially suppressed growth in soft agar of transformed Balb/3T3 mouse cells induced by methylcholanthrene. The suppressive effect of nalidixic acid on growth in soft agar was reversible. Nalidixic acid reversibly reduced saturation density of these transformed cells. Oxolinic acid and pipemidic acid, which are derivatives of nalidixic acid, were less effective than nalidixic acid in suppressing growth in soft agar. Nalidixic acid suppressed growth in soft agar of NIH/3T3 mouse cells transformed by an activated c-Ha-ras, without affecting the amount of ras p21 proteins as detected by an immunoblotting analysis using a monoclonal antibody. These results show that nalidixic acid reversibly suppressed the expression of transformed phenotypes that were already being expressed.     

Malignant transformation of a mouse liver epithelial cell line by transfection of an activated c-H-ras gene with a point mutation at codon 12

In order to scrutinize the reason why in mouse liver systemonly activated H-ras gene with a point mutation at codon 61but not codon 12 is frequently seen although the latter mutationis highly frequent in methylnitrosourea-induced rat mammarytumors, transforming activity of these two types of mutatedH-ras gene was investigated utilizing an immortalized but notfully transformed mouse liver epithelial cell line MLE-10, establishedin our laboratory. MLE-10 cells were transfected with activatedhuman c-H-ras gene having a point mutation at either codon 12(PT24) or 61 (PSK2), together with PSV2neo, or with PSV2neoonly. G418 resistant colonies, propagated separately, gave riseto 6, 3 and 6 lines respectively. All the PT24 and PSK2-transfectedcell lines were growth capable in both soft agar and nude mousesubcutis, with similar growth rate and morphological featureswhereas none of the cell lines transfected with the PSV2neoonly revealed such growth capability. The results thus revealedthat c-H-ras with a mutation at codon 12 has oncogenic activityto the mouse hepatocyte, although after immortalization, atthe degree similar to the same gene with a mutation at codon61.     

Tumorigenicity of T24 urinary bladder carcinoma cell sublines

Two sublines of the T24 human urinary bladder carcinoma cell line which differ in tumorigenicity in nude mice have been studied (T24A and T24P). T24A obtained directly from the American Type Culture Collection is non-tumorigenic while T24P obtained after multiple passages in several NCI laboratories produces tumors in 100% of inoculated mice. T24P cells differ morphologically from T24A, have a higher saturation density, are less serum-dependent for growth, and are more sensitive to ouabain toxicity. Cytogenetic studies show that the 2 sublines differ significantly in chromosome number, with a modal chromosome range of 76-89 in T24A and a modal chromosome number of 48-51 in T24P. Southern blot analysis of MspI cleaved T24A and T24P DNAs with the H-ras SmaI probe indicates that both contain only the activated mutant allele originally described in T24. Northern blot analysis shows equal amounts of the 1.2kB ras polyadenylated message, and immunoblotting with rasHa antibody demonstrates no significant difference in the amounts of ras proteins. These results indicate that 2 sublines of a ras oncogene-containing tumor cell line can differ greatly in tumorigenicity and other in vitro characteristics of transformation, and yet have similar expression of the ras oncogene. The fact that the tumorigenic cell line contains fewer chromosomes suggests that tumorigenicity may be related to the loss of some regulatory gene.     

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.     

Decreased sensitivity of drug-resistant cells towards T cell cytotoxicity.

Killing of target cells by cytotoxic T cells is mediated by induction of apoptosis requiring functional death pathways. Kill is mediated either by the CD95 or the perforin/granzyme pathway. We found that SH-EP neuroblastoma cells are preferentially killed via CD95, while in the T leukemia cell line CEM CD95 and perforin/granzyme are involved. In both types of cell lines, cells resistant to CD95- and drug-induced apoptosis are crossresistant to cytotoxic T cell kill. Resistant cells show decreased apoptosis and deficient activation of caspases indicated by decreased cleavage of the prototype caspase substrate PARP. Preincubation with the caspase inhibitor zVAD-fmk strongly decreased LAK cell kill in sensitive cells. Although parental CEM cells could be sensitized for LAK kill by preincubation with doxorubicin, resistance could not be reverted in doxorubicin or CD95 resistant CEM cells. These data demonstrate the crossresistance in induction of apoptosis by different cytotoxic regimens in tumor cells and may have implications for the immunotherapy of tumors in which apoptosis resistance was induced by previous chemotherapy.     

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.     

Expression of the normal H-ras1 gene can suppress the transformed and tumorigenic phenotypes induced by mutant ras genes

The transformed phenotype of rat 208F cells transfected with the T24 H-ras1 oncogene is suppressed by simultaneous or subsequent transfection with the normal H-ras1 gene. The suppressed cells express both the normal and mutant forms of ras p21 but the normal form predominates. Rare transformed cells obtained after simultaneous transfection express mainly the T24 p21. Some suppressed cells induce tumours in nude mice after a long lag period and these tumour cell lines have much reduced expression of normal p21. The normal H-ras1 gene also suppresses the transformed phenotype induced by mutant N-ras, albeit less effectively. The tumorigenicity of the EJ bladder carcinoma cell line, which contains only the T24 mutant allele of H-ras1, is also suppressed following transfection with the normal H-ras1 gene. The results suggest that transforming alleles of ras genes do not behave in a fully dominant manner and that expression of the normal allele at elevated levels can lead to suppression of the transformed and tumorigenic phenotypes.     

LS14 cells: a model for chemoresistance in liposarcoma

Liposarcoma, a malignancy of adipose tissue, is the most common soft tissue sarcoma. Patients whose primary tumor cannot be resected or those who have developed metastasis, have poor prognosis since liposarcomas are highly resistant to chemotherapy. We recently generated a spontaneously immortalized cell line, named LS14, from a patient with metastatic liposarcoma. Our goal was to compare the responsiveness of LS14 and SW872 liposarcoma cells to anti-cancer drugs and explore mechanisms of chemoresistance. Using complementary assays for cell viability and number we found that SW872 cells responded robustly to relatively low concentrations of doxorubicin, cisplatin and vinblastine. This reduction in cell viability was due to apoptosis, as evident by phosphatidylserine exposure and caspase 3 cleavage. In contrast, only a high dose of doxorubicin or combination therapy effectively reduced LS14 cell viability and induced apoptosis. LS14 cells showed a higher expression of Bcl-2 and Bcl-xL, but a lower expression of survivin and Bax, than SW872 cells, suggesting that anti-apoptotic proteins contribute to chemoresistance in LS14 cells. Although LS14 cells did not form colonies in soft agar, they generated large tumors and metastases in SCID mice, establishing their tumorigenicity in vivo. In conclusion, LS14 cells are much more resistant to chemotherapy than SW872 cells, making them an excellent model for exploring the efficacy and mechanism of action of anti-cancer drugs in liposarcomas.