Transformation of normal homologous cells by a spontaneously activated Ha-ras oncogene

Several tumor-derived oncogenes have been shown to independently act as complete carcinogens following transfection into target cells from established tissue culture lines. However, the number and types of oncogenes required to transform primary cultures of normal mammalian cells is unclear. To clarify this issue in a simplified model system, we transfected genomic DNA from a naturally occurring rat tumor into NIH/3T3 cells as well as into early passage rat embryo fibroblasts. The 3T3 cells were transformed with high efficiency to malignant phenotypes; the rat embryo cells were transformed at lower frequencies following cotransfection with a selectable neomycin resistance marker and treatment with Geneticin (G418). The transformed rat cells had cancerous phenotypes as determined by in vitro, cytogenetic, and in vivo criteria. Moreover, the transformed mouse and rat cells contained new tumor DNA-derived nucleotide sequences homologous to the activated human Ha-ras oncogene. Elevated levels of Ha-ras-specific mRNA, as well as enhanced expression of the Mr 21,000 oncogene product, were detected in the transformed cells. Therefore, under well-defined experimental conditions, a spontaneously activated Ha-ras oncogene from a naturally occurring tumor was able to independently transform normal, homologous cells to a malignant phenotype.     

Detection of activated ras oncogenes in human thyroid carcinomas

Focus formation following DNA transfection of mouse 3T3-Vill cells was used to search for the presence of activated oncogenes in human thyroid tumors. Oncogenes belonging to the ras family were detected in four out of six thyroid carcinomas (Ki-ras in one anaplastic tumor and one follicular moderately differentiated tumor and Ha-ras and N-ras in two papillary tumors). Normal thyroid tissue samples obtained from two patients, one with an anaplastic tumor and one with a benign adenoma, and samples from 4 benign adenomas and from one toxic goiter of a patient with Graves' disease gave negative results. In one case, restriction enzyme analysis demonstrated the presence of a mutation in codon 12 of the activated Ha-ras oncogene. Our data show that all three ras proto-oncogenes can become activated in malignant thyroid tumors.     

Cellular transformation by a unique isolate of human papillomavirus type 11.

Infection with human papillomavirus type 11 (HPV 11) is associated with benign epithelial proliferations and rarely with malignant and metastasizing tumors. Because of the biological diversity displayed in tissues infected with HPV 11, we have examined the capacity of various isolates of HPV 11 to transform cultured cells and compared their molecular differences by DNA sequence analysis. Five isolates of HPV 11 were examined for their ability to transform primary neonatal rat kidney epithelial cells and NIH 3T3 mouse fibroblasts in DNA transfection experiments using calcium phosphate precipitation. Included in these studies are the prototype isolate from a laryngeal papilloma (HPV 11P); HPV 11VC from a verrucous carcinoma of the penis; HPV 11Epi from the viral episomes of a primary squamous cell carcinoma; and two integrated genomes (HPV 11Int 1 and HPV 11Int 2) of the metastases. Only HPV 11VC cotransfected with the oncogene Ha-ras transformed neonatal rat kidney epithelial cells with an efficiency comparable to that of HPV 16 DNA. HPV 11VC DNA alone transformed NIH 3T3 cells. Analysis of the DNA sequence of HPV 11P and 11VC revealed 16 single nucleotide changes in the upstream regulatory region and open reading frames E1, E2, E4, and E5, five resulting in amino acid substitutions. This is the first demonstration of cellular transformation by a natural isolate HPV 11 DNA in vitro and illustrates that minimal changes in the DNA sequence of certain viruses confer oncogenicity to what are normally nontransforming viruses.     

Activation of the cellular Harvey ras gene in mouse skin tumors initiated with urethane

Mouse skin tumors, benign papillomas, and squamous cell carcinomas (SCCs) were initiated by a single topical application of urethane followed by repeated promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Using the NIH 3T3 focus forming assay, dominant transforming activity was detected in DNA isolated from SCC samples. Rearranged and amplified copies of the c-Ha-ras gene were detected in NIH 3T3 transformant cell lines, indicating that an activated Ha-ras gene had been transferred to the NIH 3T3 recipient cells. Analysis of p21ras from the transformant cell lines suggested that the activating ras mutation was present in codon 61. Ultimately, the Ha-ras gene was shown to be activated by a specific A----T transversion at the second position of codon 61. This mutation was detected in both benign papillomas and SCCs, suggesting the activation occurred early in tumor development. The results demonstrate a highly consistent activation of the Ha-ras oncogene by a specific point mutation, suggesting a functional role for an activated ras gene in the initiation of mouse skin tumors by urethane.     

Malignant conversion of murine squamous papilloma cell lines by transfection with the fos oncogene

Murine papilloma cell lines 308 and SP-1 have been used as recipients for transfected oncogenes to investigate malignant conversion. These cell lines express an activated c-rasHa gene with a codon 61 mutation and produce squamous papillomas when transplanted as skin grafts onto nude mice. They are not tumorigenic by subcutaneous injection. Both papilloma cell lines were stably transfected with plasmid DNA containing either a rearranged murine plasmacytoma-derived c-myc (minus exon 1), adenovirus 5 E1A, FBJ v-fos or a human c-fos/FBJ v-fos chimera, using cotransfection with the neomycin resistance gene contained in pSV2neo to select for transformants. Southern and northern blotting analysis confirmed the uptake and expression of exogenous DNA in both G418-selected cell lines and in the derived tumors. Unlike the E1A- and myc-containing plasmids, both fos constructs caused malignant conversion in either cell line, as defined by the squamous cell carcinoma histology of tumors from grafted cells and the development of carcinomas after subcutaneous injection into athymic nude mice. Immunofluorescence analysis for specific keratin gene expression indicated that tumors derived by introduction of either of the fos oncogenes were devoid of staining for K1, a 67 kDa epidermal keratin that is expressed in papillomas but not in squamous carcinomas. Tumors from E1A, myc, or pSV2neo transfectants expressed K1, although in a focal distribution. The malignant phenotype induced by the fos oncogene constructs was not associated with the ability to form agar colonies in vitro or to express gamma-glutamyl transpeptidase in the tumors. Since both 308 and SP-1 were sensitive to the fos oncogene for malignant conversion and insensitive to E1A or myc, it is possible that fos may cooperate with the endogenous-activated c-rasHa gene to convert these cells to malignancy. However, since gamma-glutamyl transpeptidase activity is found in the majority of chemically induced mouse skin carcinomas that possess an activated c-rasHa gene, fos activation may not be a common pathway for spontaneous malignant conversion.     

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.     

Transfection of a non-metastatic diploid rat mammary epithelial cell line with the oncogenes for EJ-ras-1 and polyoma large T antigen

Transfection of rat mammary (Rama) 37 epithelial cells which yield non-metastasizing adenomas in syngeneic Wistar-Furth rats with a drug resistance plasmid containing both the neo gene and EJ-ras-1 (pSV2neo.ras) or with pSV2neo and a plasmid encoding the large T Antigen (pLT214) of polyoma virus yields drug-resistant transformants with a frequency of 10(-5). Representative transformants have been propagated in neo-selecting medium to yield various cell lines. The 7 lines transfected with pSV2neo.ras (EJ1 set) and the 10 lines co-transfected with pSV2neo and pLT214 (LT1 set) all produce tumours at subcutaneous (s.c.) sites with a shorter median latent period than tumours produced by the parental Rama 37 cells. In addition, the LT1 set of transformants yields a higher incidence of tumours than the Rama 37 cells. No metastases are produced when any of the oncogene transformants are inoculated s.c. into rats. However, when an EJ1 representative is inoculated intravenously (i.v.), tumour deposits are found in the lungs of the host animals. In contrast, other Rama 37 variants that metastasize from s.c. sites fail to produce any metastases when inoculated i.v. The oncogene transfectants contain integrated DNA that hybridizes to neo and to the requisite oncogenic DNAs; the pattern of hybridizing bands to the transfected genes and their expression as mRNA is complex, and is presented in detail.     

Infection of rat liver epithelial cells with v-Ha-ras: correlation between oncogene expression, gap junctional communication, and tumorigenicity

The role of v-Ha-ras oncogene in tumorigenesis in an in vitro/in vivo model system was studied by investigating the expression of the Ha-ras gene, gap junctional intercellular communication, and tumorigenicity as endpoints. Infection of a Fischer 344 rat liver epithelial cell line (WB 344) with a retrovirus containing the v-Ha-ras oncogene resulted in altered cell morphology and decreased contact sensitivity. Gap junctional intercellular communication in v-Ha-ras infected WB cells (WBHa-ras), assessed by fluorescence redistribution after photobleaching (FRAP), microinjection/dye transfer, and scrape-loading/dye transfer techniques, was markedly decreased compared with the level in control WB cells. Injection of 10(7) WBHa-ras cells into the portal vein of male F344 rats caused multiple focal hepatic lesions within 1 and 2 wk, merging to large invading tumors after 3 and 4 wk. Examination of the methylation pattern of the Ha-ras gene in WBHa-ras and control WB cells showed that the infected Ha-ras gene was relatively hypomethylated in comparison to the normal cellular Ha-ras gene, indicating a greater potential for expression. There was an increased level of Ha-ras mRNA in hepatomas as compared with both adjacent nontumor liver tissue and liver tissue obtained from normal animals. Three cell lines derived from three different primary hepatic tumors induced by an injection of WBHa-ras cells in a F344 rat displayed similar growth characteristics, levels of gap junctional communication, and methylation patterns as the original WBHa-ras cells. The results of these studies have established a strong positive correlation between expression of the Ha-ras oncogene, reduced gap junctional intercellular communication, decreased contact sensitivity, and tumorigenicity of the v-Ha-ras-infected rat liver epithelial cells.     

Immune response to progressor variants derived from transfection of an ultraviolet radiation-induced C3H mouse regressor tumor cell line with activated Harvey-ras oncogene

Skin cancers induced in mice by UV radiation often exhibit a regressor phenotype. In order to determine how tumors escape the immune defenses of the normal immunocompetent host, we sought to isolate progressor variants from a UV radiation-induced C3H mouse regressor fibrosarcoma cell line, UV-2240, by transfection with an activated Ha-ras oncogene. A cotransfection protocol using pSV2-neo DNA, which confers resistance to the antibiotic G418, was used to select transfected cells. Injection of Ha-ras-transfected UV-2240 cells s.c. into immunocompetent C3H mice produced tumors in four of 36 animals. In contrast, UV-2240 cells transfected with pSV2-neo DNA alone or mock transfected with CaPO4 did not produce tumors in normal C3H mice. DNAs from cell lines established from Ha-ras-induced tumors contained unique Ha-ras sequences in addition to those sequences endogenous to UV-2240 cells. However, the Ha-ras-induced progressor variants did not overexpress the Mr 21,000 protein. The Ha-ra-induced progressor variants produced experimental lung metastasis in both normal C3H and nude mice, although they induced more lung nodules in nude mice than in normal C3H mice. In addition, all four Ha-ras-induced progressor variants produced significantly more experimental lung metastases in nude mice than did the parent UV-2240 cell line. However, both the parental UV-2240 cell line and the Ha-ras-induced progressor variants expressed similar levels of H-2Kk and H-2Dk antigens and were immunologically cross-reactive, as determined by in vitro cytotoxic T-lymphocyte and in vivo immunization-challenge assays. These results indicate that the progressor phenotype of the Ha-ras-induced tumor variants is not due to loss of tumor-specific transplantation or Class I major histocompatibility complex antigens. This implies that some tumor cells can escape the immune defenses of the normal immunocompetent host by mechanisms other than loss of tumor-specific transplantation and Class I major histocompatibility antigens.     

Immunity to murine leukemia induced in susceptible mice by transfected mouse fibroblasts

A cultured cell line of mouse fibroblasts was transfected with DNA from murine leukemia cells expressing a previously characterized tumor-associated antigen. Antigen-positive cells were used as immunogens in an immunotherapy protocol to determine if they stimulated resistance to the malignant proliferation of the leukemia in susceptible mice. For the experiments, LM(TK-) mouse fibroblasts, a thymidine kinase-deficient mouse cell line, were cotransfected with DNA from ASL-1 murine leukemia cells and the plasmid pSV2neo conferring resistance to Geneticin. Integration of the plasmid into cellular DNA was confirmed by restriction digest blot analysis. A/J mice, highly susceptible to the malignant proliferation of passively transferred ASL-1 leukemia cells, were immunized with the transfected cells. Animals receiving two prior injections of antigen-positive transfected cells and then challenged with an injection of viable ASL-1 cells survived longer than animals in the unprotected control group or in the group receiving immunizations with LM(TK-) cells transfected with plasmid only (p less than 0.01). Some of the mice appeared to have rejected the tumor and lived more than 80 days. One group of protected animals rechallenged with a second injection of ASL-1 cells, 40 days after the first, survived for more than 50 additional days, without evidence of recurrent disease.     

Detection and identification of activated oncogenes in human skin cancers occurring on sun-exposed body sites

High-molecular-weight DNA isolated from eight fresh human skin cancers occurring on sun-exposed body sites were assayed for their ability to transform NIH 3T3 cells. A cotransfection protocol using pSV2-neo DNA, which confers resistance to the antibiotic G418, was used to select cells that had taken up the transfected DNA. About 2 weeks after transfection, G418-resistant colonies were pooled and injected s.c. into athymic nude mice. The NIH 3T3 cells transfected with DNA from six of the human skin cancers induced tumors in nude mice. DNAs from all six tumor cell lines contained human alu sequences. Southern blot hybridization with ras-specific probes revealed that DNAs from the four alu-rich tumors contained the human Ha-ras oncogene, in addition to that of the NIH 3T3 controls. In contrast, DNAs from the other two tumors did not contain any of the known oncogenes tested, except those endogenous to NIH 3T3 cells. DNAs from three of four first cycle tumorigenic transformants gave rise to morphologically transformed foci when assayed in a second cycle of transfection. DNAs from all three secondary transformants contained discrete human alu sequences, and in addition, contained Ha-ras sequences similar to those present in their respective primary transformants. Interestingly, DNA from both primary and secondary transformants of one particular human squamous cell carcinoma contained highly amplified copies of the Ha-ras oncogene. These results suggest that activation of the Ha-ras oncogene may be common in human skin cancers originating on sun-exposed body sites. Further characterization of the Ha-ras oncogenes present in these human skin cancers may provide information on the molecular mechanisms by which UV radiation of the sun induces human neoplasms on exposed body sites.     

Correlation of growth capacity of cells in hard agarose with successful transfection by the activated c-Ha-ras oncogene and in vivo proliferative capacity at metastatic sites

The purpose of this study was to determine whether the degree of anchorage-independent growth of rodent or human cells in increasing concentrations of agarose correlated with successful transfection of the cells with an activated c-Ha-ras oncogene and tumorigenicity in nude mice. NIH 3T3 cells, C3H 10T1/2 fibroblasts, four clones of the murine K-1735 melanoma with different metastatic capacities and the TE85 human osteogenic sarcoma line were transfected with plasmids containing the 6.6-kilobase BamHI fragment of the mutant human c-Ha-ras gene and the neo gene, which confers resistance to neomycin (pSV2-neoEJ). Cells transfected with pSV2-neo, a plasmid containing the neo gene, served as controls. Cells from parental or transfected lines (selected by Geneticin) were plated into medium containing 0.3%, 0.6% 0.9%, or 1.2% agarose. These cells were also injected subcutaneously and intravenously into nude mice. The production of tumor cell colonies in dense agarose (greater than or equal to 0.6%) correlated with successful transfection with pSV2-neoEJ and production of experimental metastases in the lung of nude mice. We conclude that the degree of anchorage-independent growth of cells predicts successful transfection with activated c-Ha-ras oncogene and tumorigenic behavior in vivo. Thus this technique may be useful for the detection of cells transfected with transforming oncogenes.     

Differentiation-dependent expression of provirus-activated int-1 oncogene in clonal cell lines derived from a mouse mammary tumor

The int-1 mammary oncogene is frequently activated by proviral insertion in mouse mammary tumors. To characterize the target cell for the oncogenic action of int-1, we have isolated permanent cell lines with distinct morphologies and differentiation characteristics, starting from a tumor with a rearranged int-1 gene. Polygonal cells had retained many differentiation markers of epithelial cells and produced adenocarcinomas upon transplantation in syngenic mice. Sphere-forming-cuboidal cells are poorly differentiated and produced anaplastic tumors. Cuboidal and elongated cells were negative for epithelial markers. Cuboidal cells were poorly tumorigenic, but elongated cells produced highly malignant sarcoma-like tumors. In all lines, the int-1 gene was identically rearranged due to insertion of proviral DNA of the Mouse Mammary Tumor Virus, but the expression of int-1 varied with the state of differentiation of the cells. Polygonal cells contained relatively high levels of int-1 RNA, which were not influenced by steroid hormones. In the sphere-forming-cuboidal cells, expression of int-1 was low but inducible by dexamethasone. In the cuboidal and elongated cells no expression of int-1 was detectable, showing that the continued expression of int-1 was not required for progression to more malignant cells. By immunoprecipitation, two int-1 protein species, of 42 and 40 kD were identified in polygonal and in sphere-forming-cells but not in the culture media.     

Peripheral benzodiazepine receptors in androgen sensitive dunning rat prostatic adenocarcinoma

Several lines of evidence implicate the ras oncogene in tumorigenesis. However, changes in ras oncogene is uncommon in prostate cancer. We evaluated tumors from 55 patients with metastatic prostate cancer (50 lymph nodes, 5 bone metastases), 10 patients with localized cancers and 35 diethylstilbestrol treated primary tumors. Also, 15 patients with benign prostatic hyperplasia and 23 with prostatic intraepithelial neoplasia (PIN) were investigated for ras p21 expression. Avidin biotin immunoperoxidase was used on formalin-fixed, paraffin-embedded tissues with the Pan-ras (Ab-1) monoclonal antibody. Antibody titration demonstrated expression of ras p21 in none of the benign, PIN or DES-treated primary tumor specimens. However, 30% of untreated primary tumors and 94.5% of metastatic tumors (94% of lymph node metastases, 100% of bone metastases) showed expression (p=0.00002). Semi-quantitative evaluation of ras protein expression revealed a significant correlation with Gleason score in lymph node metastases (p=0.001). This study suggests a possible role of ras oncogene in prostate cancer progression, metastasis and androgen independency.     

Selection for spontaneous metastasis after calcium phosphate-mediated transfer of DNA

The ras oncogene has been shown to induce metastatic potential in a wide variety of cells. However, one cell line, C127, when transformed by ras (HC127), proved to be an exception in that the transformed cells gained the ability to form tumors in nude mice, but these tumors did not form spontaneous metastases. Because C127 cells do not metastasize after ras transfection, these cells can be used to identify other factors which contribute to the development of metastatic potential. Specifically, these cells can be used as recipients in DNA transfer experiments utilizing DNA from other cells in which H-ras has been used to induce the metastatic phenotype, thus allowing the search for genes in addition to ras itself which may be necessary for metastasis. A system utilizing genomic DNA transfer into C127 cells transformed by ras has been developed. These cells were used as the recipient for genomic DNA in cotransfection with pSV2neo followed by selection both in the experimental i.v. assay and in the spontaneous metastasis assay in nude mice. DNA from two lines with metastatic potential (both transformed by ras genes) gave rise to metastatic clones, whereas DNA from the recipient cells, HC127, NIH 3T3 cells, and two human tumor cell lines, CHP126 and A2058, failed to give rise to transfectants which could metastasize. The metastases after the first cycle were put into culture, and DNA was extracted from these cells and used in a second cycle of transfection. The capacity to metastasize was also transferred in the second cycle. Of seven metastatic clones examined, four showed no detectable rearrangements of the transfected v-H-ras gene, but in three of these clones, there were rearrangements of this gene, which was originally present in the recipient cell, HC127. This indicates that in a subset of the selected clones there may have been selection for rearrangements of the host genome rather than introduction of foreign DNA sequences, and that such effects must be considered in gene transfer experiments. It is also possible that the transfer of particular genomic DNAs are leading to genetic instability in these experiments. mRNA levels were compared in the metastatic variants and the parental cells; H-ras-specific RNA was raised from 4- to 22-fold in the metastatic cell lines, regardless of rearrangement of the v-H-ras gene.     

Low incidence of Ha-ras oncogene mutations in human epidermal tumors

Activation of the Ha-ras oncogene by point mutations has been suggested to play a role in animal skin carcinogenesis models. In this study, we investigated the significance of the Ha-ras mutations in human epidermal tumors. DNAs from paraffin-embedded tissues of benign and malignant human epidermal tumors (27 samples from 25 patients) were prepared and examined for point mutations of codons 12, 13 and 61 of Ha-ras gene by polymerase chain reaction and oligonucleotide hybridization. Only one sample of basal cell carcinoma and one sample of keratoacanthoma were found to carry an A to T transversion at the second position of codon 61. This low incidence of Ha-ras mutations suggests that the mutational activation of the gene may not be primarily involved in human epidermal tumorigenesis.     

Isolation and characterization of clonal cell lines from a transplantable metastasizing rat mammary tumor, TR2CL

The metastasizing rat mammary cell strain from the Ludwig Institute for Cancer Research (London Branch) which was originally developed from a benign rat mammary tumor induced by N-methyl-N-nitrosourea (CAS: 684-93-5), yielded single-cell-cloned lines of isometric epithelial cells [rat mammary (Rama) 600-Rama 621] and one line of elongated cells (Rama 622); the former had a higher estrogen receptor content than the latter. All the representative epithelial cell lines tested (Rama 600, 603, and 617) failed to convert to elongated, myoepithelial-like cells or droplet cell/doming, alveolar-like cells in vitro. All representative cell lines tested induced tumors in syngeneic F344/N rats and CBA nu/nu mice, but only the epithelial lines metastasized to lungs and local lymph nodes in rats and to lungs in nude mice. The involved lungs and lymph nodes contained mainly intravascular thrombi and deposits in the subcapsular sinus, respectively. Tumors and metastases from the representative epithelial cell lines contained acinar and glandular structures together with an elongated cellular component. The Rama 622 tumors contained mainly spindle cells. Antisera to rat milk fat globule membranes and human keratins stained some of the epithelial and elongated cells in the Rama 600 tumors; less staining was observed in the Rama 622 tumors. None of the tumor cells stained with antiserum to myosin. Anti-laminin serum delineated a fragmented basement membrane in glandular elements and stained weakly the cytoplasm of the more elongated tumor cells. Ultrastructural analysis confirmed the identity of epithelial cells in the Rama 600 tumors, but no well-differentiated myoepithelial cells were seen in either type of tumor. Since nonmetastasizing epithelial cells isolated directly from carcinogen-induced benign rat mammary tumors can differentiate to myoepithelial-like cells in vitro or when growing as tumors in animals, it is suggested that the development of the malignant phenotype is associated with a loss of this differentiating ability.