Conditional immortalization and/or transformation of rat cells carrying v-abl or EJras oncogene in the presence or absence of glucocorticoid hormone

Early-passaged rat chondroblasts (RX cells) and embryonal fibroblasts (RE cells) are hardly transformed by transfection of activated human H-ras (EJras) or by Abelson murine leukemia virus v-abl oncogene. However, these cells were transformed by v-abl or EJras gene when dexamethasone (DX) was added in the culture medium as well as when co-transfected with retrovirus LTR-linked mouse c-myc gene. RX cell lines carrying v-abl (RXabl), RE cell lines carrying v-abl (REabl) and RX cell lines carrying EJras (RXEJ) were established from transformed colonies in the DX-added soft agar. In the absence and in the presence of DX, RXabl cells showed mortal and immortalized, REabl cells showed mortal and transformed, and RXEJ cells showed immortalized and transformed phenotypes, respectively. Especially, immortalization and transformation of REabl1 and REabl3 lines were switched on and off by addition and depletion of DX. v-abl or EJras mRNA levels in tested REabl, RXabl and RXEJ lines cultured without DX was not decreased compared to those cultured with the hormone. The above suggests that, like myc gene, glucocorticoid collaborates with v-abl or activated ras oncogene to transform unestablished rat cells and that the transformation phenotypes were determined not only by the introduced oncogene but by the cellular condition including their tissue origin. Transformation of senescent REabl cells in the absence of DX was tested by transfecting different oncogenes. Among nuclear oncogenes tested, only adenovirus 12 E1A gene could induce transformation of G0-arrested REabl cells in a cooperative fashion with the integrated v-abl gene.     

Expression of transformed morphology and anchorage independent growth of hamster embryo cells

Formation of morphologically transformed colonies and the abilityto grow in semi-solid agar has been compared for 3 differentcell lines from hamster embryo and for primary hamster embryocells. By manipulating the growth conditions, transformed colonymorphology and growth in agar could be induced for all celltypes studied. Conditions that induced morphologically transformedcolonies, also produced growing colonies in agar. One cell lineand the primary cells needed the presence of the tumor promoter12-O-tetra-decanoyl phorbol-13-acetate for the expression oftransformed morphology and agar growth, while the two othercell lines produced both morphologically transformed coloniesand growth in soft agar without any additions. None of the celllines would produce morphologically transformed colonies inthe presence of newborn bovine serum. Likewise, the cells weredependent on fetal bovine serum in order to grow in soft agar,except for one of the cell lines which produced a low numberof agar growing colonies in newborn bovine serum. The data indicatea close relation between morphological transformation and growthin soft agar.     

Expression of the tumor-specific and calcium-binding protein oncomodulin during chemical transformation of rat fibroblasts

An in vivo-in vitro approach to studying neoplastic development in carcinogen-exposed rat fibroblasts was evaluated. In the model described, oncomodulin (Mr 12,000; pI 3.9), a tumor-associated and Ca2+-binding protein, was used as a specific marker of malignant transformation. A rapidly proliferating granulation tissue was exposed in vivo or in vitro to potent carcinogens like N-methyl-N'-nitro-N-nitrosoguanidine and procarbazine. As an endpoint of transformation anchorage independent (AI) colony formation in the soft agar assay was chosen. Exposure to various doses of N-methyl-N'-nitro-N-nitrosoguanidine in vivo or in vitro, or to procarbazine in vivo, led to induction of AI, transformed cells. Exposure of the cells to various doses of procarbazine in vitro produced neither formation of AI cells in the agar nor expression of oncomodulin in extracts of the exposed cell population. Almost all of the chemically induced AI cell lines tested have been found to be tumorigenic in athymic mice. In contrast, a very low rate (zero to two colonies per 10(6) cells tested) of spontaneous AI populations derived from untreated cells. None of these control AI colonies yielded tumors. In our transformation assay the appearance of neoplastic phenotypes seems very rapid, probably due to the increased cell division at the time of carcinogen-exposure. Expression of oncomodulin was found in extracts of transformed cells harvested from agar colonies, derived from carcinogen-exposed granulation tissue, but not from normal, untreated fibroblasts, as shown by two-dimensional polyacrylamide gel electrophoresis and high-performance liquid chromatographic analysis, as well as 45Ca2+-transblot electrophoresis. The presence of oncomodulin in extracts of transformed cells correlates well with the chemically induced colony formation in the soft agar assay. Oncomodulin might be a suitable neoplastic marker to study chemical carcinogenesis.     

Tax protein of human T-cell leukemia virus type I is required for maintenance of the transformed phenotype.

We have isolated and characterized revertants of a clonal cell line (40MRatcl-1) of human T-cell leukemia virus type I Tax-transformed Rat1 cells. The 40MRatcl-1 cells contain a single copy of tax gene, form large colonies in soft agar, elicit tumors rapidly in nude mice and revert to the normal phenotype at low frequency. From one of its subclones (B7) bearing pSV2gpt DNA as a marker gene, four morphologically reverse-transformed cell lines were isolated. They display contact inhibition at confluency, lose the ability to form colonies in soft agar, fail to form tumors in nude mice and restore the transformed phenotype similar to that of 40MRatcl-1 cells by transfection with the tax-expression plasmid. Southern blot analysis revealed that they have lost the tax gene. Our results indicate that transformation of Rat1 cells by Tax is not the consequence of secondary mutations of cellular genes and that tax functions are directly required for establishment and maintenance of the transformed phenotype.     

Down-regulation of the insulin-like growth factor I receptor by antisense RNA can reverse the transformed phenotype of human cervical cancer cell lines

The insulin-like growth factor I receptor (IGF-IR) plays an essential role in the establishment and maintenance of transformed phenotype, and interference with the IGF-IR pathway by antisense or dominant-negative mutants causes reversal of the transformed phenotype in many rodent and human tumor cell lines. We stably transfected an IGF-IR antisense mRNA expression plasmid into human papillomavirus (HPV)-negative C33a cell line, HPV-16-positive SiHa cell line, and HPV-18-positive HeLa S3 cell line to determine whether the IGF-IR could be a target for cervical cancer cells, especially in the presence of HPV. Approximately 30-80% down-regulation of IGF-IR expression was observed by Western blot in antisense transfected clones. There was a little inhibition in monolayer growth in all cell lines. In C33a cells, wild-type and sense clones formed 92-146 colonies in soft agar after 3 weeks; antisense clones formed <12 colonies. In SiHa cells, wild-type and sense clones formed approximately 60 colonies after 5 weeks; antisense clones formed 0-3 colonies. In HeLa S3 cells, wild-type and sense clones formed 218-291 colonies in soft agar after 2 weeks; antisense clones formed 14-160 colonies. There was a good correlation between IGF-IR down-regulation level and inhibition of transformation in soft agar. Tumorigenesis in nude mice was strongly inhibited in HeLa S3 and SiHa clones transfected with the antisense. These results indicate that down-regulation of IGF-IR by antisense RNA can reverse the transformed phenotype of human cervical cancer cells, even when harboring malignant type HPVs.     

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.     

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.     

Immortalized fibroblasts from NF-kappaB RelA knockout mice show phenotypic heterogeneity and maintain increased sensitivity to tumor necrosis factor alpha after transformation by v-Ras

Activation of the NF-kappaB pathway can either promote or block apoptosis and oncogenesis in different cell types and circumstances. In this report, we show that independently derived immortalized mouse embryonic fibroblast cell lines prepared from RelA knockout mice have different phenotypes, based on their sensitivity to tumor necrosis factor alpha (TNFalpha)-induced apoptosis, morphology, ability to form colonies in soft agar, and the presence of distinct kappaB site-binding complexes. In addition, these RelA-deficient cell lines appear to have distinct alterations in the p53 pathway, which correlate with the normal vs transformed status of individual cell lines. We have also infected mouse embryonic fibroblasts lacking RelA, c-Rel or p50 with a retrovirus for the expression of v-Ha-Ras to determine whether individual NF-kappaB family members are required for Ras-mediated transformation. All three NF-kappaB-deficient cell types could be transformed by v-Ha-Ras. However, v-Ras-infected RelA-deficient cells formed colonies in soft agar at an approximately fourfold reduced efficiency compared to v-Ras-transformed control mouse 3T3 and p50-deficient cells. Ras transformation did not alter the sensitivity of RelA-deficient cells to TNFalpha-induced apoptosis, and Ras transformation did not affect the general resistance of 3T3, c-Rel-deficient, and p50-deficient cells to TNFalpha-induced apoptosis. However, TNFalpha specifically and dose-dependently decreased the ability of v-Ras-transformed RelA-deficient cells to form colonies in soft agar. These results suggest that RelA is a potential protein target for human tumors driven by oncogenic Ras mutations, but caution that inhibition of RelA may promote tumorigenesis in some circumstances.     

Growth in culture and tumorigenicity after transfection with the ras oncogene of liver epithelial cells from carcinogen-treated rats

Two epithelial cell lines designated LE/2 and LE/6 were established from cells isolated by centrifugal elutriation from the livers of carcinogen-treated rats. Both cell lines exhibit some characteristics of fetal liver cells, such as the expression of the 2.3-kilobase alpha-fetoprotein mRNA, aldolase A, and lactate dehydrogenases 4 and 5. Primary cultures contain gamma-glutamyl transferase-positive cells which do not proliferate in vitro. After the first passage, the LE/2 and LE/6 cell lines are uniformly gamma-glutamyl transferase negative. Neither cell line is transformed as assayed by morphology, anchorage-independent growth, or tumor formation in nude mice. By the 50th passage, LE/6 cells form numerous colonies in soft agar in the presence of epidermal growth factor, while no colonies grow in medium lacking this growth factor. Clonal cell populations derived from five epidermal growth factor-induced soft agar colonies were not tumorigenic in nude mice. This indicates that, although epidermal growth factor-responsive late passage cells had acquired some of the phenotypic properties commonly associated with tumor cells, these cells were not fully transformed. Transformation of LE/6 cells was accomplished by transfection of the rasH oncogene (EJ). Subcutaneous inoculation of rasH (EJ)-transfected LE/6 cells produced tumors at the site of injection with histological features of moderate to well-differentiated trabecular hepatocellular carcinomas. Tumor cell lines derived from the nude mouse tumors are gamma-glutamyl transferase positive and express alpha-fetoprotein mRNA. One clonal cell line expresses both alpha-fetoprotein and albumin mRNA. These results show that nonparenchymal liver epithelial cells transfected with an activated oncogene can give rise to differentiated hepatocellular tumors similar to those induced in livers of rats fed a carcinogenic diet.     

Oncogenic potential of Hsp72.

Hsp72 is the major heat shock-inducible protein capable of protecting cells from a variety of stresses. In non-transformed cells at normal conditions Hsp72 is expressed at very low levels. It is, however, present at elevated levels in the major fraction of tumors and in many transformed cell lines. It is commonly assumed that in tumor cells the expression of Hsp72 at elevated levels is the consequence of oncogenic transformation. In the present study we addressed an alternative possibility that Hsp72 plays an active role in the process of oncogenic transformation. We report here that when Hsp72 was expressed in the Rat-1 fibroblasts either constitutively or from an adenovirus-based construct, cells become oncogenically transformed by the following criteria: loss of contact inhibition and formation of foci characteristic for oncogenically transformed cells; acquisition of the ability to grow in an anchorage-independent manner and to form colonies in soft agar; generation of tumors upon injection into mice. Furthermore, we also report that turning off the Hsp72 expression led to the reversal of the transformed phenotype. We also show that oncogenic potential of Hsp72 is confined in its peptide binding domain since the expression of this domain alone was sufficient for oncogenic transformation of Rat-1 cells.     

Malignant transformation of human fibroblasts by a transfected N-ras oncogene

The only ras oncogene as yet identified in cells from human fibrosarcomas is N-ras, but the relationship between N-ras oncogene expression and the malignant state of these cell lines is not known. To determine if expression of an N-ras oncogene causes human cells to become malignant, we transfected the N-ras oncogene from human leukemia cell line 8402, cloned into a high expression vector pSV N-ras, into MSU-1.1 cells, a nontumorigenic, infinite life span fibroblast cell strain with a normal morphology and a stable near-diploid karyotype. The transformants formed distinct foci composed of morphologically transformed cells. Cells from such foci expressed higher than normal levels of N-ras protein, exhibited growth factor independence, and formed large colonies in soft agar at a high frequency. Injection of progeny of these focus-derived cells s.c. into athymic mice resulted in progressively growing, invasive malignant tumors (round cell, spindle cell, or giant cell sarcomas) which reached a diameter of 6 mm in 3 to 4 weeks. Injection of focus-derived or tumor-derived cells i.v. resulted in tumors in various organs of the mice. The focus-derived cell strain tested, as well as the majority of the cells derived from the tumor it produced, exhibited the same near-diploid karyotype as the parental MSU-1.1 cells. Cells transfected with an N-ras oncogene that was expressed at a normal level formed only a single, indistinct focus, and cells from that focus were not malignant.     

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.     

Constitutive overexpression of a 89 kDa heat shock protein gene in the HBL100 human mammary cell line converted to a tumorigenic phenotype by the EJ/T24 Harvey-ras oncogene

The non tumorigenic human mammary cell line HBL100 has been transformed by the EJ/T24 human bladder carcinoma Harvey(Ha)-ras oncogene. Six cell lines were established from transformed colonies. They all expressed a high level of the ras oncogene and were tumorigenic in athymic nude mice. During an in vivo passage in animals, tumour cells presenting a growth advantage were selected, and some of the tumours revealed an amplification of the transfected ras sequences. Using this model of human cell transformation, we have isolated a cDNA clone corresponding to a heat shock protein gene (hsp89 alpha). This gene, normally transcribed at a higher rate in response to serum stimulation, was found to be constitutively overexpressed in ras-transformed HBL100 cells. In contrast, a closely related hsp gene (hsp89 beta), remained sensitive to serum stimulation, in both untransformed and ras-transformed HBL100 cells. Thus, the regulation of the expression of the hsp89 genes, upon serum stimulation, involves ras-dependent and ras-independent pathways. Constitutive overexpression of the murine homolog of the hsp89 alpha was observed in NIH3T3 cells transformed by the three ras oncogenes, but not with some other oncogenes. Therefore, alteration of the hsp89 alpha gene expression is not a general characteristic of transformed cells, but seems to be linked to ras transformation.     

Cooperation of bcl-2 and myc in the neoplastic transformation of normal rat liver epithelial cells is related to the down-regulation of gap junction-mediated intercellular communication

The objectives of this study were to isolate several rat liver epithelial cell clones containing the human bcl-2 and myc/bcl-2 genes in order to study their potential cooperative effect on neoplastic transformation and gap junction-mediated intercellular communication (GJIC) and to test the hypothesis that the loss of GJIC leads to tumorigenesis. Using anchorage-independent growth as a surrogate marker for neoplastic transformation, we transfected both normal rat liver epithelial cells, WB-F344, and a WB-F344 cell line overexpressing v-myc with human bcl-2 cDNA. Those cell lines that only expressed v-myc or human bcl-2 were unable to form colonies in soft agar. However, those cell lines that overexpressed both v-myc and human bcl-2 showed varying ability to form colonies in soft agar, which did not correlate with their human bcl-2 expression level. In order to test if there was a correlation between cell line growth in soft agar and the ability to communicate through gap junctions, we performed scrape load dye transfer and fluorescence recovery after photobleaching assays. Our results show that v-myc and human bcl-2 can cooperate in the transformation of normal cells, but the degree to which the cells are transformed is dependent on the cells' ability to communicate through gap junctions.     

Effect of adeno-associated virus on transformation of NIH 3T3 cells by ras gene and on tumorigenicity of an NIH 3T3 transformed cell line

Transfection of NIH-3T3 cells with the plasmid pJ234, containing DNA from the human bladder carcinoma T24 cell line (ras gene), results in their transformation. Adeno-associated virus did not affect significantly the number of the transformed foci when different multiplicities of infection were used and when the virus was added to the cultures at different time intervals before or after transfection. A transformed cell line was derived following transfection of NIH 3T3 cells by the ras gene. Infection of these cells with adeno-associated virus resulted in a decrease in their growth rate and cloning efficiency. These infected cells showed a dose-dependent reduction in the frequency and an increase in the latent period for tumor appearance in nude mice.     

Transformation of an established mouse mammary epithelial cell line following transfection with a human transforming growth factor alpha cDNA

To determine whether the enhanced expression of transforming growth factor alpha (TGF alpha) is sufficient to induce the neoplastic transformation of an immortalized population of mammary epithelial cells, we cotransfected NOG-8 cells, a cloned mouse mammary epithelial cell line, with a simian virus 40-human TGF alpha cDNA expression vector plasmid and a pSV2neo plasmid. After cotransfection, nine G418-resistant NOG-8 colonies were cloned and expanded. All clones were subsequently analyzed for TGF alpha mRNA expression by northern blot analysis, TGF alpha secretion, anchorage-dependent growth in serum-free medium, anchorage-independent growth in soft agar, and tumorigenicity in nude mice. Three TGF alpha-transfected NOG-8 clones expressed high levels of a specific TGF alpha mRNA, secreted elevated levels of TGF alpha into the culture medium (177-595 ng/10(8) cells/48 h), exhibited an enhanced growth rate, grew aggressively as colonies in soft agar, and formed undifferentiated, invasive carcinomas in nude mice. A neutralizing mouse monoclonal antibody generated against the low molecular weight human TGF alpha peptide was able to inhibit colony formation in soft agar by TGF alpha-transfected NOG-8 clones that produced high levels by TGF alpha. This inhibition suggested that TGF alpha acted through an external autocrine loop. NOG-8 cells and NOG-8 cells transfected with a pSV2neo plasmid alone secreted very low levels of TGF alpha, failed to grow as colonies in soft agar and did not form tumors in nude mice. These results demonstrate that overexpression of a human TGF alpha cDNA in immortalized, nontransformed mouse mammary epithelial cells can induce a transformed phenotype in vitro and can facilitate tumor formation in vivo.     

Morphological transformation, focus formation, and anchorage independence induced in diploid human fibroblasts by expression of a transfected H-ras oncogene

In an attempt to determine how normal human fibroblasts respond to high expression of the T24 H-ras oncogene, we tranfected such cells with the plasmid vector pHO6T1 (D. A. Spandidos and N. M. Wilkie, Nature (Lond.), 310:469-475, 1984), containing the T24 H-ras oncogene with 5' and 3' enhancer sequences, and the aminoglycoside phosphotransferase gene which confers resistance to the drug, G418. Approximately 1.5% of the G418-resistant colonies obtained after transfection and selection consisted of cells exhibiting obvious morphological transformation; i.e., they were highly refractile and more rounded than normal fibroblasts. DNA hybridization analysis showed that the morphologically transformed cells contained the transfected T24 H-ras oncogene, and radioimmunoprecipitation analysis showed that they were expressing the T24 H-ras protein product, M, 21,000 protein. Morphologically transformed cells formed colonies in soft agar at a frequency at least 60 times higher than that of cells that had been transfected with the control plasmid containing the normal cellular H-ras gene. Cells transfected with plasmid pHO6T1 could also be identified by their ability to form distinct foci when grown to confluence in nonselective medium following transfection. This study demonstrates that normal diploid human fibroblasts in culture can be transformed by transfection with a H-ras oncogene, and that such transformation correlates with expression of the mutant Mr 21,000 protein.     

MALIGNANT TRANSFORMATION IN BALB/c-3T3 CELLS INDUCED BY CRYSTALLINE NICKEL SULFIDE

采用细胞灶法测定结晶型硫化镍对ＢＡＬＢ／ｃ－３Ｔ３诱发细胞转化的活性,结果表明当硫化镍浓度≥０．２５μｇ／ｃｍ２时转化率增高（Ｐ＞０．０５）,且有剂量反应关系。各剂量组转化灶细胞共６份经软琼脂培养试验均获阳性结果,而对照细胞则为阴性,提示硫化镍诱发的转化细胞具有锚着不依赖性,即恶性特征。