Neoplastic progression by EJ/ras at different steps of transformation in vitro of human uroepithelial cells.

The biological effects of expression of mutant ras at different stages of human uroepithelial cell (HUC) tumorigenesis were tested after transfection of EJ/ras into nonestablished HUC and three isogeneic cell lines representing different steps in HUC transformation in vitro. Transfection with EJ/ras failed to immortalize diploid HUC and also failed to cause tumorigenic conversion of a near-diploid SV40-immortalized HUC line (SV-HUC) except at one of six nude mouse inoculation sites. In contrast, EJ/ras-transfected aneuploid low-grade squamous cell carcinoma cells formed undifferentiated, invasive carcinomas at four of six inoculation sites. Furthermore, EJ/ras accelerated tumor growth in MC-ppT11-HA2, an aneuploid high-grade transitional cell carcinoma line, as determined by decreased tumor latent periods and doubling times. These results suggest that EJ/ras contributes to progression, possibly by accelerating tumor growth, but does not in itself cause tumorigenic transformation of uroepithelial cells. To test whether chromosome losses accompanied EJ/ras transformation of SV-HUC, the karyotype of the one SV-HUC tumorigenic transformant obtained (above) was examined. This tumor cell line showed losses of chromosome arms 3p, 10p, 11p, and 18, all of which have been hypothesized to contain genes that suppress cancer development. Therefore, these results also provide new evidence suggesting that genetic losses may be required for mutant ras to contribute to HUC tumorigenic progression.     

Timing of induction of tgf-alpha in ras induced human bladder neoplastic progression

Introduction of a H-ras oncogene into an SV-40 immortalized human urothelial cell lines (SV-HUC) results in morphologically altered cell clones which acquire tumorigenic potential following serial passaging in culture. Early and late passage cells, from individual ras transfected clones exhibiting different tumorigenic potential, display increased growth factor synthesis in mitogenic assays. Northern blot analysis revealed induction of TGF-alpha mRNA concomitant with the introduction of a H-ras oncogene with no modulation in EGF receptor expression observed throughout neoplastic progression. Consistent with completion of an autocrine loop, down modulation and activation of EGF receptors was observed in early passage cells coincident with TGF-alpha expression. In this human urothelial progression model TGF-alpha secretion follows the introduction of a H-ras oncogene prior to the acquisition of tumorigenic potential.     

Tumorigenicity of the cyc- variant of the S49 murine lymphoma deficient in the Gs-alpha subunit of adenylate cyclase

S49 cyc- lymphoma cells contain a mutation resulting in loss of a functional guanine nucleotide regulatory protein rendering their adenylate cyclase refractory to most stimuli. S49 wild-type and cyc- clones were used in the present study to investigate the possible association of altered cAMP metabolism with tumorigenicity and metastatic potential. The S49 clones were implanted i.v., i.p., and intracerebrally in both athymic nude mice and syngeneic, immunocompetent BALB/c mice. Both S49 clones gave rise to tumors when inoculated into athymic mice, and no differences were observed in the tumorigenicity or metastatic potential of S49 wild-type and cyc- cells. Implantation of S49 clones in syngeneic BALB/c mice gave rise to few tumors except when administered intracerebrally, where wild-type cells were more tumorigenic than cyc- cells. This raises the possibility of differences in immunogenicity between the S49 clones. Analysis of cell lines derived from tumors grown in athymic mice showed that they retained the phenotype of the S49 clones used for inoculations. The results indicate that, despite differences in adenylate cyclase responsiveness, S49 wild-type and cyc- cells are both highly tumorigenic and metastatic.     

The transformation of primary and established mouse mammary epithelial cells by p21-ras is concentration dependent

We constructed a retroviral vector (pZSR) which is capable of simultaneously expressing the neomycin resistance gene and the viral ras oncogene. Primary mammary gland epithelial cells were prepared from mid-pregnant mice and infected with this virus. Cell lines with epithelial cell characteristics could be established with a low frequency. High expression of p21 v-ras was observed in these cells. They are tumorigenic and form soft agar colonies dependent on the presence of EGF and insulin in the growth medium but progress to hormone independent growth at higher passage numbers. A cloned cell line of non-tumorigenic, established mammary epithelial cells (NOG8) was also infected with the v-ras expressing virus. Individual cell clones expressing increasing amounts of p21 v-ras were selected. The level of p21 v-ras expression directly influences the morphology of the epithelial cells in culture, determines their cloning efficiency in soft agar and their tumorigenicity.     

Expression of normal and mutant ras proteins in human acute leukemia

The expression of normal and mutant ras genes in human acute leukemias was assessed by the direct analysis of p21ras polypeptides, using immunoprecipitation with monoclonal antibodies. High-resolution two-dimensional gel electrophoresis permits the identification of a wide array of activated ras alleles encoding proteins with single amino acid substitutions at any of several positions. The products of three ras genes, H-ras, N-ras, and K-ras, were detected in each of 33 specimens of fresh leukemic cells. The normal K-ras and N-ras polypeptides were substantially more abundant than H-ras p21 in all samples. In over three-fourths of the cases the total amount of p21ras exceeded that seen in control hematopoietic cell lines. The level of ras expression did not correlate simply with clinical parameters, although the two samples with the most abundant p21ras were obtained from patients with relapsed T-cell acute lymphocytic leukemia (ALL). Abnormal p21ras, consistent with oncogenic activation, was found in eight patients. Six of 11 samples from acute myelocytic leukemia (AML) patients displayed a mutant N-ras p21, while only one of 20 ALL specimens had abnormal N-ras, and one had a mutant H-ras. In every case the mutant protein comprised a minority of total p21ras. In two T-cell ALL cell lines both normal and activated N-ras gene products were expressed at equal levels. By contrast, in five fresh AML samples the abnormal N-ras protein was several-fold less abundant than the normal N-ras p21. This finding implies that only a proportion of leukemic cells in an individual patient may carry the mutant ras oncogene.     

Characterization of human uroepithelial cells immortalized in vitro by simian virus 40

Normal human uroepithelial cells (HUC) were transformed with simian virus 40 (SV40) in vitro. SV40-transformed HUC (SV-HUC) were selected by their ability to survive senescence which normally occurs in HUC between passages 4 and 6. At passage 6, 100% of SV-HUC stained positive for SV40 T-antigen. The epithelial nature of SV-HUC was confirmed by positive staining for human cytoplasmic keratins in all cells. SV-HUC have altered growth characteristics compared to HUC including the capacity to grow on plastic, independent of a collagen-gel substrate; loss of the dependence on medium supplements for optimal growth, loss of the dependence on feeder cells for growth at clonal density, and an apparently unlimited lifespan in culture (greater than 2 years). Although SV-HUC have an increased percentage of viable cells and increased saturation density compared to HUC, the generation time of SV-HUC during log phase is similar to that of HUC. Cultures of SV-HUC are epithelial in appearance and show some morphological heterogeneity in cell size and shape. At the ultrastructural level, SV-HUC have numerous alterations such as, irregularly shaped nuclei and nucleoli, pleomorphic microvilli, and the lack of a glycocalyx on the cell surface. In addition, SV-HUC does not stratify in culture, suggesting an inability to differentiate. Unlike HUC, SV-HUC are capable of growth in soft agarose, a property which increased with serial passage. Yet, through at least P50, SV-HUC remained nontumorigenic as determined by the inability to form tumors in athymic nude mice. This cell line of human epithelial origin may be suitable for studying the conversion of cells to tumorigenicity by subsequent treatment with another oncogenic agent.     

Multiple phenotypes induced by mutant ha-ras in thyroid epithelial-cells - correlation with level of expression

To investigate the role of ras mutations in thyroid epithelial tumorigenesis, we introduced wild-type or mutant v- or c-Ha-ras genes into a sub-cloned rat thyroid follicular epithelial cell line using retroviral vectors and a neutral selection method (G418 resistance). Mutant, but not wild-type, ras induced a spectrum of clonal phenotypes. Interestingly, many clones showed an unchanged phenotype despite ras expression at levels greater than that of the endogenous gene. Further increase in ras expression was associated with altered morphology, loss of thyroid-specific differentiation (thyroglobulin synthesis) and growth factor independence, but not. anchorage-independence or tumorigenicity. The mutation site (codon 12 or 61) did not have a significant influence. The results clearly emphasize the critical importance of expression level in determining the phenotypic effect of mutant ras on epithelial cells.     

Direct tumorigenic conversion of human gallbladder carcinoma cells by v-src but not by activated c-H-ras oncogene

The roles of activated ras and src oncogene products in the acquisition of fully neoplastic phenotype by human gallbladder adenocarcinoma cells were investigated by co-transfecting non-tumorigenic HAG-1 human gallbladder carcinoma cells with the pSV2neo plasmid and a plasmid carrying either activated c-H-ras or v-src oncogene. G-418-resistant clones were isolated and assessed for the acquisition of anchorage-independent growth potential. Neither the 10 established clones transfected with pSV2neo alone nor the 17 clones transfected with activated c-H-ras, including 4 clones expressing the mutated p21^H-ras protein, could form colonies in soft agar. By contrast, out of 10 clones transfected with v-src, 2 formed colonies in soft agar and produced tumors in athymic nude mice, the resulting progressive neoplasms being poorly differentiated adenocarcinomas. These tumorigenic clones were shown to have v-src DNA and mRNA levels with p60^v-src protein, but there were no significant chromosomal alterations following tumorigenic conversion. Moreover, herbimycin A, a selective src-kinase inhibitor, markedly reduced clonogenic growth of these cells in soft agar rather than monolayer growth, suggesting that anchorage-independent growth of the v-src-transformed HAG-1 cells might be driven directly by p60^v-src kinase activity. Taken together, our data suggest that the fully neoplastic conversion of HAG-1 cells depends on src-related tyrosine-kinase activity, but not solely on the function mediated by activated ras, thus providing evidence of an src-related signaling pathway for the acquisition of tumorigenic potential by human gallbladder adenocarcinoma cells. © 1995 Wiley-Liss, Inc.     

c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo but lacks correlation with malignancy

Spontaneously immortalized human skin keratinocytes (HaCaT) were transfected with the c-Ha-ras (EJ) oncogene via a plasmid construct which also contained the selectable neomycin gene. Clones were selected on the basis of G418 resistance. Those clones that had stable integrants of Ha-ras fell into 3 classes with respect to tumorigenicity. Class I clones were nontumorigenic, i.e., formed nodules which rapidly regressed. This phenotype is identical to that seen with parental HaCaT cells. Class II clones formed slowly growing, highly differentiated cystic or papillomatous-type benign tumors, and class III clones formed highly differentiated, locally invasive squamous cell carcinomas. The clones of all three classes exhibited similar morphology and growth potential in culture and retained the ability to reconstitute an epidermis-like stratified epithelium in transplantation experiments. Only the malignant clones showed locally invasive growth. Both the benign and the malignant clones exhibited higher levels of ras integration and variable levels of mutated p21 protein product. Thus, expression of the cellular Ha-ras oncogene in these human epithelial cells significantly altered growth regulation, resulting in varying degrees of growth potential in vivo, ranging from benign to malignant tumors. However, no direct correlation was seen between high levels of p21 expression and malignant growth.     

Differential tumorigenicity of 3T3 cells transformed in vitro with polyoma virus and in vivo selection for high tumorigenicity

BALB/c 3T3 cells transformed in vitro with a temperature-sensitive mutant of polyoma virus were cloned. Forty-eight clones examined demonstrated heterogeneity with respect to doubling-time in vitro and tumorigenicity in syngeneic mice in vivo. Observation periods that lasted in certain cases as long as 2 years showed that some clones exhibited a relatively high tumorigenicity, i.e., they yielded a relatively high incidence of tumors following a small inoculum of cells and a relatively short latency period. Other clones were relatively low tumorigenic: even high tumor cell inocula yielded a relatively low tumor incidence following a relatively long latency period. These results indicate that at least in this system variation in tumorigenicity is generated independently of host factors. An intraclonal heterogeneity with respect to the length of the precancer latency period was seen. Some tumors appeared relatively early following inoculation of cloned cells, whereas others appeared considerably later following an identical inoculum of the same clone. Cloned in vitro transformed cells were passaged once in syngeneic mice and recultured. The single in vivo passage cycle augmented considerably the tumorigenicity of these cells as compared to their in vitro maintained clonal ancestors. The increased tumorigenicity of the in vivo passaged cells is due, most probably, to the in vivo induction and/or selection of high tumorigenic intraclonal variants. The survival time of mice bearing high tumorigenicity variants was very similar to that of mice bearing low tumorigenicity variants.     

Elevated expression of an exogenous c-myc gene is insufficient for transformation and tumorigenic conversion of established fibroblasts

Two established rat fibroblast lines, differing only by their number of generations in culture, show dramatically different responses to the elevated c-myc expression delivered by an efficient murine c-myc retrovirus vector. Thus, a late passage (60 generation) FR3T3 line acquires a transformed and tumorigenic phenotype upon introduction of this activated c-myc gene as indicated by its altered morphology, high efficiency of focus formation, soft agar clonability, saturation density in monolayer culture, and short latency of tumorigenicity in syngeneic hosts. Remarkably, none of these characteristics, except for an increased refractility in monolayers and an epidermal growth factor (EGF)-dependent agar clonability, were observed in a variety of early passage (10 generation) FR3T3 c-myc clones. BALB/c A31 fibroblasts transfected with this c-myc retroviral vector behaved essentially the same as the FR3T3 early line except for their inability to grow in suspension in response to EGF. However, transformation and tumorigenic conversion of each of these three fibroblast lines was achieved by an activated ras oncogene. Hence, elevated c-myc expression is insufficient for transformation of established fibroblasts but depends upon other acquired cooperating functions which are not necessary for ras induced transformation. We also demonstrate that endogenous c-myc expression remains unaffected even in clones expressing a 100-fold excess of exogenous c-myc RNAs demonstrating that c-myc autoregulation is not operative in these cells.     

Human bladder and colon carcinomas contain activated ras p21. Specific detection of twelfth codon mutants

Members of the ras family of proto-oncogenes code for 21,000-dalton molecular weight protein products (p21s). Transformation of cells from the normal to the malignant phenotype in experimental studies has been associated with point mutations within the coding region for these ras proteins. Recent reports demonstrate that 40% of human colon cancers and 20% of acute leukemias contain ras mutations in the twelfth or thirteenth codon that can result in amino acid substitutions at these positions in the p21 products. Similarly, studies of ras mRNA detected 40% of human colon tumors with twelfth codon c-Ki-ras mutant mRNA. The authors previously developed a nonradioactive double-antibody enzyme-linked immunoblot assay (ELIBA) for detection of normal and mutant ras p21. They have adapted that technology to specifically detect twelfth codon activated ras p21 utilizing mutation-specific antisera. In this report the authors show that one of seven de novo human bladder cancers and four of seven colon cancers express a twelfth codon activated ras p21. These results document that mutations at both the DNA and mRNA levels are ultimately translated into an abnormal protein product present in human tumors.     

Modified responsiveness of v-Ha-ras-transfected rat fibroblasts to growth factors and a tumor promoter

The correlation of the phenotypic changes of v-Ha-ras transfected cells with the expression of p21ras and the modified responses to growth factors and a tumor promoter were examined. Transfection of the v-Ha-ras gene together with the neomycin-resistance gene into 208F rat fibroblasts yielded transformed clones characterized by morphological changes, anchorage-independent growth, and tumorigenicity in nude mice. The degrees of these biological alterations were parallel with the expression of mRNA and protein of the ras gene. In ras-transformed cells, anchorage-independent growth was stimulated by epidermal growth factor (EGF), insulin, bombesin, and fibroblast growth factor, whereas in the parental 208F cells, anchorage-independent growth was observed only in the presence of EGF, and there were many fewer EGF-induced colonies than those in the ras-transformed clones. A tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) also augmented anchorage-independent growth of ras-transformed cells and induced morphological changes in monolayer cultures without altering the expression of the ras gene or phosphorylation of the p21ras protein. Retinoic acid inhibited the TPA-induced anchorage-independent growth. These results showed a good correlation of the expression of p21ras with the phenotypic changes and the increased sensitivity of the p21ras-expressing cells to the stimulation of growth factors and tumor promoter.     

Analysis of oncogene, tumor suppressor gene, and chromosomal alterations in HeLa x osteosarcoma somatic cell hybrids.

Using a series of tumorigenic and non-tumorigenic somatic cell hybrids that resulted from the fusion of the human osteosarcoma cell line OHS50-P16T (P16T) with the HeLa cell line D98OR, we investigated the role that genetic mutations, including alterations of oncogenes, tumor suppressor genes, and chromosomes, play in P16T tumorigenicity. Analysis of a previously identified oncogene mutation, c-myc amplification, in the P16T cell line demonstrated that both the tumorigenic and non-tumorigenic hybrids contained the amplified c-myc gene. Analysis of previously identified P16T tumor suppressor gene alterations, p53 mutation, and loss of RB1 expression demonstrated that the mutated p53 gene was selectively maintained in both the non-tumorigenic and tumorigenic hybrids, whereas loss of RB1 expression was not maintained in either the non-tumorigenic or tumorigenic hybrids. Chromosomes 11, 13, 17, and 22 were analyzed for loss of heterozygosity (LOH) to characterize the status of these previously described chromosomal alterations in the tumorigenic and non-tumorigenic hybrids. Loss of HeLa D98OR chromosome 22, with maintenance of P16T chromosome 22, was observed in the tumorigenic hybrids, a result confirmed by LOH analysis, which demonstrated the specific loss of HeLa chromosome 22 genetic material in the tumorigenic segregants. Together, these results demonstrated that amplified c-myc, mutant p53, and RB1 genes seem to be important in osteosarcoma tumorigenicity and that an additional altered gene or genes on chromosome 22 may play a key role in osteosarcoma tumorigenicity.     

Differences in GTPase-activating protein activity between liver tumors and normal liver tissue in mice.

The intrinsic GTPase activity of the cellular protein p21ras is strongly increased by two cytosolic proteins, the GTPase-activating protein (GAP) produced by the neurofibromatosis type 1 gene (NF1-GAP) and a GAP of 120 kDa molecular mass (p120-GAP). The GAP-mediated stimulation of p21ras GTPase activity was measured in cytosol obtained from carcinogen-induced liver tumors and normal liver tissues of mice of two strains, namely C3H/He and C57BL/6J. For this purpose, cytosolic extracts were incubated with recombinant human p21ras complexed to [gamma-32P]GTP and the time-dependent decrease in p21ras bound radioactivity was measured. Liver cytosolic extracts mediated an increase in the GTPase activity of wild-type p21ras. There were great differences between tumor and normal tissues in the maximal velocity (Vmax) and in the apparent Michaelis constant (KM) of the p21ras GTPase reaction. Both Vmax and apparent KM were decreased in the liver tumors. Cytosolic extracts isolated from liver tumors that harbored point mutations in codon 61 of the c-H-ras gene did not differ in their activity from extracts obtained from non-mutated liver tumors. Since both GAP proteins are important cellular regulators of the ras signaling pathway and probably also effectors of p21ras, the observed differences in GAP activity may be of relevance for the tumorigenic process in mouse liver.     

Clonal selection within metastatic SP1 mouse mammary tumors is independent of metastatic potential

Our previous studies using randomly integrated plasmid DNA as unique clonotypic markers of SPI mouse mammary tumor cells transplanted into syngeneic CBA/J or nude mice demonstrated reproducible selection and eventual overgrowth of the primary transplant tumors by genotypically distinct metastatic subclones. Two independent metastatic SPI clones, neo5 and ras1, were shown to exhibit "clonal dominance" relative to the non-metastatic SPI tumor-cell population. These results suggested that the capacity for preferential growth within the tumors may be related to cellular properties associated with metastatic ability. To investigate the clonal interactions of metastatic SPI clones present within the same tumor mass, we have analyzed tumors composed of paired mixtures of neo5 and ras1. The tumors were monitored for the relative proportion of each clone by Southern blot analysis. The ras1 clone was found to dominate over the neo5 clone in the majority of tumors examined, even when present as 1% of the mixed inoculum. This represents a 20- to 50-fold enrichment of ras1, while the proportion of neo5 within the tumors was reduced at least 5-fold. No evidence for selection of either clone was seen during co-culture in vitro. Neo5 and ras1 are indistinguishable with respect to tumorigenic and metastatic potential when inoculated separately into different mice, suggesting that clonal dominance is independent of metastatic ability. Analysis of the metastases resulting from mixed inocula indicates that it is possible for a subpopulation representing less than 1% of the primary tumor mass to give rise to metastases. This also suggests that the process of metastasis within metastatic tumors is independent of clonal dominance.     

Enhanced spontaneous metastasis of mouse carcinoma cells transfected with an activated c-Ha-ras-1 gene

To investigate whether the presence of an activated ras oncogene influences the ability of tumour cells to metastasize, the c-Ha-ras-1 oncogene cloned from EJ/T24 cells was introduced into MT1 Cl.5/7 mouse mammary carcinoma cells. Since the MT1 Cl.5/7 cells are already tumorigenic but have a low metastatic capacity, this experimental design allows a distinction to be made between the effects of the ras gene on metastasis and tumorigenicity. MT1 Cl.5/7 containing the EJ c-Ha-ras-1 metastasized more readily and to more tissue sites than control cells (2.8 sites/mouse vs 0.9 sites/mouse). The metastases expressed the EJ c-Ha-ras-1 p21 ras proteins; however, one metastasis was discovered that had lost the expression of the c-Ha-ras-1 gene. When these cells were re-tested for metastasis, the rate of metastasis was indistinguishable from that of controls. This observation, coupled with a demonstration that lung colonization potential following intravenous inoculation is unaffected by the presence of the activated ras gene, argues that the effect of mutant ras genes is exerted on the ability of cells to escape from the primary tumour, rather than on a survival in the circulatory systems and ability to seed a second site.     

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.