Activated Ha-ras Gene Research Articles

Disruption of Stat3 reveals a critical role in both the initiation and the promotion stages of epithelial carcinogenesis.

Constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been found in a wide spectrum of human malignancies. Here, we have assessed the effect of Stat3 deficiency on skin tumor development using the 2-stage chemical carcinogenesis model. The epidermis of Stat3-deficient mice showed a significantly reduced proliferative response following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) because of a defect in G1-to-S-phase cell cycle progression. Treatment with the tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA) resulted in a significant increase in the number of keratinocyte stem cells undergoing apoptosis in the bulge region of hair follicles of Stat3-deficient mice compared with nontransgenic littermates. Notably, Stat3-deficient mice were completely resistant to skin tumor development when DMBA was used as the initiator and TPA as the promoter. Abrogation of Stat3 function using a decoy oligonucleotide inhibited the growth of initiated keratinocytes possessing an activated Ha-ras gene, both in vitro and in vivo. In addition, injection of Stat3 decoy into skin tumors inhibited their growth. To our knowledge, these data provide the first evidence that Stat3 is required for de novo epithelial carcinogenesis, through maintaining the survival of DNA-damaged stem cells and through mediating and maintaining the proliferation necessary for clonal expansion of initiated cells during tumor promotion. Collectively, these data suggest that, in addition to its emerging role as a target for cancer therapy, Stat3 may also be a target for cancer prevention strategies.

Analysis of Ha-ras Gene in the B6C3F1 Mouse Liver by Non-RI PCR-SSCP.

The codon 61 of the activated Ha-ras gene is detected most frequently among mutations of proto-oncogenes in spontaneously-occurring hepatocellular tumors in B6C3F1 mice. In the present study, the ras mutations and their patterns in normal liver tissue, foci of cellular alteration (FCA), and hepatocellular tumor from untreated mice were analyzed using the non-RI PCR-SSCP method. The Ha-ras gene with mutated codon 61 was detected in 1.6% (2/122) from normal liver tissues, 16.7% (6/36) from spontaneous FCA tissues, 30.4% (48/158) from spontaneous hepatocellular tumor tissues, and 7.7% (3/39) from normal portion in spontaneous hepatocellular tumor tissues. Furthermore, mutation in the Ha-ras gene was compared between spontaneous and induced hepatocellular proliferative lesions including hepatocellular tumor from archival materials at our Center. Although no clear difference was found between genotoxic carcinogen and non-genotoxic carcinogens in the incidence of Ha-ras gene mutation in the induced tumors, the incidence of Ha-ras gene mutation in FCA, which was induced by genotoxic carcinogen, was higher than that in FCA induced by non-genotoxic carcinogens. These data suggested that to analyze the incidence of Ha-ras gene mutation in proliferative lesions in the livers of B6C3F1 mice, especially in the FCA is useful in evaluation of hepatocarcinogenicity of the chemicals.

Activated Ha-Ras but Not TPA Induces Transcription through Binding Sites for Activating Transcription Factor 3/Jun and a Novel Nuclear Factor

We report the identification of a 20-base pair sequence mediating induced transcription in response to an activated Ha-ras gene and epidermal growth factor (EGF) but not 12-O-tetradecanoylphorbol-13-acetate stimulation. This signal-specific nuclear target is present in the long terminal repeat of a mouse VL30 retrotransposon expressed in epidermis. Functional studies and in vitro binding analyses using cultured keratinocytes (Balb/MK) reveal that the response element is composed of two cooperating sequence motifs in juxtaposed position, both of which are targets for induced binding activity 1-2 h after EGF stimulation. Of many different activating transcription factor/cAMP-responsive element binding protein/activating protein 1 factors tested, one part of the sequence selectively binds endogenous proteins immunologically related to activating transcription factor 3 (ATF3) and Jun isotypes. The other sequence is a target for a nuclear factor showing binding specificity unrelated to factors known to mediate EGF- or ras-induced transcription as determined by its sequence specificity and by antibody experiments. This component has been characterized and partially purified by gel filtration chromatography and velocity centrifugation revealing a Stokes radius of 43.6 A and a sedimentation coefficient of 9.7 S in solution. Based on these parameters, a molecular mass of 178,000 Da was calculated. The results indicate that the specific binding of ATF3/Jun and a previously uncharacterized factor account for signal-specific transcription in response to EGF or an activated Ha-ras gene in a cell type in which the cooperative action of an activated Ha-ras gene and 12-O-tetradecanoylphorbol-13-acetate cause tumor growth.

Transcriptional regulatory and response mapping of the rat Ha-ras upstream sequence using primary mammary epithelial cells.

The rat Ha-ras upstream sequence (-2876 to +986 bp relative to the most 5' transcriptional start site) was transcriptionally mapped at a gross level. Ha-ras upstream sequence and 5' unidirectional deletion reporter constructs were transfected via particle bombardment into primary cultures of rat mammary epithelial cells. Analyses of Ha-ras reporter expression show that a fragment extending from -2876 to -2110 bp contains a positive regulatory sequence. The majority of Ha-ras expression is attributed to this sequence, since its deletion results in a 4-fold decrease in expression. The Ha-ras gene was also assessed for autoregulation using similar co-transfection experiments. Wild-type and activated (codon 12 G-->A transition) Ha-ras expression vectors, transcriptionally driven by Ha-ras upstream sequence, were co-transfected with Ha-ras upstream sequence deletion reporter constructs. The activated Ha-ras gene product induced its own transcription 2-fold, targeting the regulatory region between -2119 and -313 bp.

Augmented expression of cytokines in mouse epidermal tumor cells and its possible involvement in the induction of hematopoietic alterations

Mice with skin tumors induced either by 7,12-dimethylbenz[a]anthracene complete carcinogenesis or subcutaneous injection of a carcinogenic keratinocyte cell line showed moderate to severe splenomegaly as a result of an increase in splenic granulocyte-macrophage and erythroid (erythroid burst-forming unit) progenitors. To test whether the observed alterations involve the release of soluble factors by the epidermal component of skin tumors, we used an in vitro approach. A series of mouse keratinocyte cell lines resembling progressive stages of skin carcinogenesis and carrying either normal or activated Ha-ras genes were assayed for their ability to produce the factors required for colony growth of hematopoietic-committed progenitors. Only the conditioned media of keratinocytes harboring activated Ha-ras genes were able to support the growth of granulocyte-macrophage colony-forming units. In addition, preincubation of normal bone-marrow cells with conditioned media from the transformed epidermal cell lines stimulated in vitro amplification of the hematopoietic granulocyte-macrophage progenitor compartment. To identify the possible factors responsible for the activities detected in the keratinocyte-conditioned media, we performed northern blot analysis using the cytokine probes granulocyte colony-stimulating factor, macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, stem cell factor, interleukin-1 alpha, interleukin-3, and tumor necrosis factor-alpha. The cell lines expressed different cytokine mRNA combinations that positively correlated with the colony-stimulating activity detected in the corresponding conditioned medium. These results suggest that transformed epidermal tumor cells in vivo may alter normal hematopoiesis as a consequence of the production of cytokines that act in autocrine or paracrine loops probably related to tumor growth.

The human Ha-ras oncogene induces genomic instability in murine fibroblasts within one cell cycle.

Many human tumors contain an activating mutation in one of the ras protooncogenes. Additionally, these tumor cells are often heteroploid and characterized by chromosome breaks and rearrangements that are consequences of the genomic instability that is thought to contribute to tumor progression. The concurrence of ras mutations and genomic instability in tumors prompted us to ask whether selective induction of an activated Ha-ras gene could render a genome unstable. The NIH 3T3 cells used in this study contained mutant p53 genes and carried a selectively inducible activated (EJ) Ha-ras transgene under the control of bacterial lactose regulatory elements. When stably transfected cells were induced to express activated Ha-ras by isopropyl beta-D-thiogalactoside administration, there was a marked increase in the number of gross chromosomal aberrations including acentric fragments, multicentric chromosomes, and double minutes, which occurred within the time frame of a single cell cycle from the time of induction. To confirm that these aberrations occurred within the first cell cycle after mutant Ha-ras induction, the cells were arrested in G1 phase by serum depletion and, subsequently, released by administration of isopropyl beta-D-thiogalactoside or serum. The mitoses from cells released with isopropyl beta-D-thiogalactoside contained a 3-fold elevation in the fraction of chromosomes containing aberrations compared to mitoses from parallel cell cultures that were released with serum. Thus, the induction of activated Ha-ras gene expression in these cells results in genomic instability that can be detected as aberrant chromosomes at the next mitosis.

Sequencing analysis of Ha‐, Ki‐, and N‐ras genes in rat urinary bladder tumors induced by N‐[4‐(5‐nitro‐2‐furyl)‐2‐thiazolyl]formamide (FANFT) and sodium saccharin

Male F344 rats were fed N[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) for up to 4 wk, then given the basal diet with or without 5% sodium saccharin for up to 100 wk. In a previous study, we demonstrated point mutations in codons 12 and 61 of Ha-ras gene among eleven transitional cell carcinomas (TCC), one undifferentiated carcinoma, and two sarcomas of the urinary bladder (Mol Carcinogen 3:210-215, 1990). In this study, Ha-ras, Ki-ras, and N-ras sequences were examined by polymerase chain reaction (PCR) and direct DNA sequencing. The results confirm the point mutation in codon 61 (CAA to CGA in 5 TCCs and to CTA in one TCC) of the Ha-ras gene. Mutation at codon 12 was not confirmed. No mutation was found in the Ki-ras gene. Sequences of the N-ras gene exons 1 and 2 were determined, and no mutations was detected. These results suggest the involvement of activated Ha-ras gene, but not Ki-N or N-ras gene, in rat urinary bladder carcinogenesis induced by FANFT. Subsequent sodium saccharin administration did not affect the changes in Ha-ras gene.

Implication of RNA structure on antisense oligonucleotide hybridization kinetics.

A 47-nucleotide transcript of the activated Ha-ras gene was prepared and determined, by enzymatic structure mapping, to form a stable hairpin structure. Six antisense decaribonucleotides were designed, and association constants (Ka) for the hairpin- and length-matched complements were measured. Two of the antisense oligonucleotides targeted to the loop had nearly equal affinity for the transcript compared to the complement. The others, including one oligonucleotide complementary to the 3' side of the single-stranded loop, bound 10(5)-10(6)-fold less tightly to the transcript than to the short complement. We propose the difference in affinity is due to the target structure, both the secondary structure of the stem and the structure in the loop. Measurement of the bimolecular association rate constant, k1, and the dissociation rate constant, k-1, for these oligonucleotides indicates the observed relationship between affinity and structure is primarily due to k1.

Detection of mutant Ha-ras genes in chemically initiated mouse skin epidermis before the development of benign tumors.

An activated Ha-ras oncogene has been consistently found in chemically initiated benign and malignant mouse skin tumors, and an activated ras oncogene has been shown to initiate the process of mouse skin carcinogenesis. However, the exact timing of mutational activation of the Ha-ras gene relative to application of the chemical carcinogen is not known. A sensitive mutation-specific PCR technique was used to experimentally address the timing of Ha-ras gene mutational activation. This technique can detect mutant Ha-ras alleles in the presence of a very large excess of normal ras alleles. Activated Ha-ras genes with 61st codon A----T mutations were found in the epidermis of mice 1 week after topical initiation with 7,12-dimethylbenz[a]anthracene or urethane by using this assay. These results were confirmed by Xba I restriction fragment length polymorphism analysis and direct DNA sequencing. One week after initiation is 1-2 months before the appearance of benign papillomas that harbor activated Ha-ras oncogenes when the initiated mice are promoted with the tumor promoter phorbol 12-myristate 13-acetate. Our data support the hypothesis that initiated epidermal cells containing an activated Ha-ras gene can remain dormant in the skin until a tumor promoter induces regenerative hyperplasia that allows for outgrowth of these cells with an activated ras oncogene to give rise to a benign papilloma.

Ras oncogene activation in mammary carcinomas induced by N-methyl-N-nitrosourea in Copenhagen rats.

The occurrence of Ha-ras and Ki-ras oncogenes was investigated in mammary tumors produced by treating genetically resistant Copenhagen (Cop) rats with N-methyl-N-nitrosourea. G35-->A codon 12 mutations in both Ha-ras and Ki-ras genes were analyzed by a polymerase chain reaction/liquid hybridization and gel retardation assay. More than half of the adenocarcinomas analyzed contained an activated Ha-ras gene. This was also the predominant mutation in similar tumors from susceptible Buf/N rats, suggesting a common mechanism of initiation. In contrast, only two of 15 mammary adenosquamous carcinomas from the Cop rats contained an activated Ha-ras gene, suggesting a different initiation mechanism for most of these tumors. Ki-ras activation was found in none of five and one of five adenocarcinomas from Buf/N and Cop rats, respectively, and in none of 13 adenosquamous carcinomas from Cop rats. These results suggest that Ki-ras activation does not play a major role in the initiation of the mammary tumors.

Alterations in protein kinase C isozymes alpha and beta 2 in activated Ha-ras containing papillomas in the absence of an increase in diacylglycerol.

The levels of protein kinase C (PKC) activity, PKC isozymes, as well as the level of endogenous diacylglycerols (DAG) were examined in early emergence mouse skin papillomas and compared to the levels in the epidermis. The papillomas were derived from a two-stage carcinogenesis protocol in which mice were initiated with 7,12-dimethylbenz[a]anthracene (DMBA) and promoted twice weekly for only 12 weeks with 12-O-tetradecanoylphorbol-13-acetate (TPA). As expected, greater than 90% of these early emergence papillomas contained an activated Ha-ras gene with an A----T transversion in the 61st codon. There was a TPA-independent, irreversible decrease in total PKC activity (70%) in the early emergence papillomas compared to that in the epidermis. Immunoblot analysis of epidermis and papillomas taken 4 weeks following the cessation of TPA treatment, a time when PKC catalytic activity has completely recovered to control level in epidermis but not in papillomas, revealed that the levels of PKC-alpha and PKC-beta 2 were dramatically decreased in the cytosol of the papillomas, while the levels of these two isozymes in the particulate fraction were approximately equal to the epidermis. PKC-delta, -epsilon and -zeta immunoreactive proteins were present in both epidermis and papillomas and only minor changes were observed in the papillomas. PKC-delta and PKC-epsilon displayed a particulate fraction localization in both the epidermis and papillomas, while PKC-zeta was found in both subcellular fractions. We were unable to detect PKC-gamma in mouse epidermis or papillomas. Since the level of DAG has been shown to be elevated in some ras-transformed cells, we examined DAG levels in the papillomas, as an increased DAG level could explain the constitutive decreases in the levels of PKC. Measurements of cellular DAG indicated that there was no elevation in the total pool of DAG in the early emergence papillomas. These data demonstrate an irreversible decrease in and alteration of the subcellular distribution of PKC-alpha and beta 2 in DMBA-initiated/TPA-promoted papillomas. These changes are TPA-independent, and occur in the absence of an elevation in the total pool of endogenous DAG. These alterations of PKC isozymes may be important early events in multistage tumorigenesis.

Carcinogen-induced mutations in the mouse c-Ha-ras gene provide evidence of multiple pathways for tumor progression.

A number of mouse skin tumors initiated by the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), 3-methylcholanthrene (MCA), and 7,12-dimethylbenz[a]anthracene (DMBA) have been shown to contain activated Ha-ras genes. In each case, the point mutations responsible for activation have been characterized. Results presented demonstrate the carcinogen-specific nature of these ras mutations. For each initiating agent, a distinct spectrum of mutations is observed. Most importantly, the distribution of ras gene mutations is found to differ between benign papillomas and carcinomas, suggesting that molecular events occurring at the time of initiation influence the probability with which papillomas progress to malignancy. This study provides molecular evidence in support of the existence of subsets of papillomas with differing progression frequencies. Thus, the alkylating agents MNNG and MNU induced exclusively G ---- A transitions at codon 12, with this mutation being found predominantly in papillomas. MCA initiation produced both codon 13 G ---- T and codon 61 A ---- T transversions in papillomas; only the G ---- T mutation, however, was found in carcinomas. These findings provide strong evidence that the mutational activation of Ha-ras occurs as a result of the initiation process and that the nature of the initiating event can affect the probability of progression to malignancy.

Point mutation in codons 12 and 61 of the ha‐ras gene in rat urinary bladder carcinomas induced by N‐[4‐(5‐nitro‐2‐furyl)‐2‐thiazolyl]formamide

Male F344 rats were fed N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) for up to 4 wk, then were given the basal diets (Prolab 3200 or AIN-76A) with or without 5% sodium saccharin for up to 100 wk. Eleven transitional cell carcinomas (TCCs), one undifferentiated carcinoma, and two sarcomas of the urinary bladder were examined for the expression of ras gene product, p21, by immunohistochemical staining and western blot analysis. Point mutation in codons 12 or 61 of the Ha-ras genes amplified by polymerase chain reaction was examined by a slot-blot screening procedure using allele-specific oligonucleotide probes. Immunohistochemical staining showed enhanced immunoreactivity with the antibody to ras p21 in seven TCCs and one undifferentiated carcinoma. Western blot analysis showed faster migration of the p21 band in 6 of 11 TCCs. Oligonucleotide hybridization revealed the point mutation in codon 12 of Ha-ras gene (GGA----GTA in 1 TCC) and in codon 61 (CAA----CGA in 5 TCCs and CAA----CTA in 1 TCC). Two mutations in codons 12 and 61 coexisted in one tumor, which were found to be present in different Ha-ras alleles. The incidence of Ha-ras gene mutations were similar in groups treated with (3 of 6) or without (3 of 8) sodium saccharin. These results suggest the involvement of activated Ha-ras gene in rat urinary bladder carcinogenesis induced by FANFT.

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.

Isolation and characterization of the 5' flanking region of the mouse c-Harvey-ras gene.

The complete 5' flanking region of the murine c-Ha-ras gene was cloned and sequenced. An untranslated exon (-1) was identified and the promoter region of the gene located. Like the rat and human homologues, the murine promoter is GC rich and contains several GC boxes together with a CAAT element, but lacks a TATA box, an arrangement similar to that found in many housekeeping genes. From primer extension studies, the gene was shown to have three transcriptional start sites, whose positions differ from those previously found for the human gene. No alterations in these start sites were detected between the normal gene and activated Ha-ras genes from mouse skin tumors. A region of strong homology between mouse, rat, and human Ha-ras genes exists within the large intron separating exon (-1) from the first coding exon. In addition, from chloramphenicol acetyltransferase assays, the upstream region has promoter activity which appears to be enhanced by the inclusion of sequences within this intron.

Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines.

A rabbit antiserum was prepared against the C-terminal peptide of 21 amino acids from the human heat shock protein hsp70. These antibodies were shown to be specific for this highly inducible heat shock protein (72 kilodaltons [kDa] in rat cells), and for a moderately inducible, constitutively expressed heat shock protein, hsc70 (74 kDa). In six independently derived rat cell lines transformed by a murine cDNA-genomic hybrid clone of p53 plus an activated Ha-ras gene, elevated levels of p53 were detected by immunoprecipitation by using murine-specific anti-p53 monoclonal antibodies. In all cases, the hsc70, but not the hsp70, protein was coimmunoprecipitated with the murine p53 protein. Similarly, antiserum to heat shock protein coimmunoprecipitated p53. Western blot (immunoblot) analysis demonstrated that the hsc70 and p53 proteins did not share detectable antigenic epitopes. The results provide clear immunological evidence for the specific association of a single heat shock protein, hsc70, with p53 in p53-plus-ras-transformed cell lines. A p53 cDNA clone, p11-4, failed to produce clonable cell lines from foci of primary rat cells transfected with p11-4 plus Ha-ras. A mutant p53 cDNA clone derived from p11-4, SVKH215, yielded a 2- to 35-fold increase in the number of foci produced after transfection of rat cells with SVKH215 plus Ha-ras. When cloned, 87.5% of these foci produced transformed cell lines. SVKH215 encodes a mutant p53 protein that binds preferentially to the heat shock proteins of 70 kDa compared with binding by the parental p11-4 p53 gene product. These data suggest that the p53-hsc70 protein complex could have functional significance in these transformed cells.

Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines.

A rabbit antiserum was prepared against the C-terminal peptide of 21 amino acids from the human heat shock protein hsp70. These antibodies were shown to be specific for this highly inducible heat shock protein (72 kilodaltons [kDa] in rat cells), and for a moderately inducible, constitutively expressed heat shock protein, hsc70 (74 kDa). In six independently derived rat cell lines transformed by a murine cDNA-genomic hybrid clone of p53 plus an activated Ha-ras gene, elevated levels of p53 were detected by immunoprecipitation by using murine-specific anti-p53 monoclonal antibodies. In all cases, the hsc70, but not the hsp70, protein was coimmunoprecipitated with the murine p53 protein. Similarly, antiserum to heat shock protein coimmunoprecipitated p53. Western blot (immunoblot) analysis demonstrated that the hsc70 and p53 proteins did not share detectable antigenic epitopes. The results provide clear immunological evidence for the specific association of a single heat shock protein, hsc70, with p53 in p53-plus-ras-transformed cell lines. A p53 cDNA clone, p11-4, failed to produce clonable cell lines from foci of primary rat cells transfected with p11-4 plus Ha-ras. A mutant p53 cDNA clone derived from p11-4, SVKH215, yielded a 2- to 35-fold increase in the number of foci produced after transfection of rat cells with SVKH215 plus Ha-ras. When cloned, 87.5% of these foci produced transformed cell lines. SVKH215 encodes a mutant p53 protein that binds preferentially to the heat shock proteins of 70 kDa compared with binding by the parental p11-4 p53 gene product. These data suggest that the p53-hsc70 protein complex could have functional significance in these transformed cells.

Alteration of the tumorigenic and metastatic properties of neoplastic cells is associated with the process of calcium phosphate-mediated DNA transfection.

During the course of studies designed to assess the effect of human Ha-ras gene expression on the malignant behavior of transfected mouse tumor cells we noted that the process of Ca3(PO4)2-mediated DNA transfection was itself associated with profound alterations in tumorigenic or metastatic behavior. The cell line used as a recipient for these studies was a tumorigenic nonmetastatic CBA/J mouse mammary adenocarcinoma line called SP1. When cotransfected with plasmids containing the neo gene (pSV2neo) and the activated Ha-ras gene (pT24-c3), cells from the pooled (5-10 colonies) G418-resistant colonies gave rise to spontaneous lung metastases in 85% of mice after subcutaneous inoculation. However, we noted that 17% of control mice inoculated with G418-resistant pSV2neo-transfected SP1 cells also had lung metastases and that this number approached 100% as the inoculum comprised a greater pool size (50-100 colonies). When cell lines established from isolated pSV2neo-transfected colonies were examined, 3/16 were found to be metastatic. We also found that 3/16 clones grew slowly, or not at all, in CBA/J mice, whereas they grew readily in athymic (nude) mice. The increase in immunogenicity of two out of three of these latter clones was accompanied by expression of the class I H-2Dk major histocompatibility complex antigen that was not detectable in the parental SP1 cells. At least some of these results would appear to be due to exposure to Ca3(PO4)2 alone, as we found that it resulted in 5/20 (25%) clones manifesting metastatic properties. Our results suggest that heritable changes in malignant behavior of transfected tumor cells can be observed at high frequency subsequent to the process of Ca3(PO4)2-mediated DNA transfection, and these changes may be brought about in part by inherited disturbances in expression of recipient cell genes.

Suppression and re-expression of transformed phenotype in hybrids of HA-ras-1-transformed rat-1 cells and early-passage rat embryonic fibroblasts.

Rat-1 cells which had been transformed with the activated Ha-ras-1 gene from human EJ bladder carcinoma cells were fused with diploid embryonic rat fibroblasts. Four selected cell hybrids expressed the human transforming gene product p21 at levels of 10 to 30% compared to 100% in the transformed parental cells. The hybrid cells, however, exhibited normal morphology, anchorage requirement for proliferation, and largely extended latency periods of tumorigenicity in newborn rats. Tumorigenic hybrid derivatives contained lower numbers of chromosomes than the tetraploid parental hybrids. DNA of the non-tumorigenic cell hybrids transformed Rat-1 cells to anchorage-independent proliferation as expected for the transforming human Ha-ras gene present in the donor DNA. We conclude that the transforming properties of the activated Ha-ras gene in Rat-1 cells can be suppressed at the post-translational level by the presence of the genome from diploid embryonic rat fibroblasts but additional controls of expression of the transforming gene are likely to exist. Normal cells contain suppressor gene(s) which safeguard these cells against transformation by the product of the transforming Ha-ras-1 oncogene.

Detection and identification of activated oncogenes in spontaneously occurring benign and malignant hepatocellular tumors of the B6C3F1 mouse.

Species- and strain-specific spontaneously occurring tumors have been observed in rodents maintained under normal laboratory conditions. Elucidation of the molecular mechanisms associated with the development of these spontaneous tumors may provide a better understanding of tumor development associated with exposure to chemical carcinogens. In view of the high frequencies of oncogene activation shown in rodent tumors induced by known chemical carcinogens, we have investigated oncogene activation in spontaneous tumors of the B6C3F1 mouse and Fischer 344/N rat by DNA transfection techniques. A marked difference in the presence of activated oncogenes in spontaneous rat tumors versus spontaneous mouse liver tumors was observed in this study. All rat tumors tested failed to yield activated oncogenes (0/29), whereas 30% (3/10) of mouse hepatocellular adenomas and 77% (10/13) of hepatocellular carcinomas scored positive by DNA transfection. These transforming genes were identified as an activated Ha-ras gene in all the adenoma transfectants and in 8 of the 10 carcinoma transfectants. The two remaining hepatocellular carcinomas contained transforming genes that appear not to be members of the known ras gene family. The B6C3F1 mouse liver system might provide a very sensitive assay not only for assessing the potential of a chemical to activate a cellular proto-oncogene, but also for detecting various classes of proto-oncogenes that are susceptible to mutational activation.