Altered gene products are associated with activation of cellular rasK genes in human lung and colon carcinomas

Two lung and two colon carcinoma cell lines of human origin, which contained the same activated ras^K transforming gene, expressed abnormal species of p21 that were distinct from the p21 proteins expressed in normal human cells and other human carcinomas. The abnormal species of p21 expressed by three of these cell lines were indistinguishable from each other, but differed from the abnormal p21 expressed by one lung carcinoma cell line. NIH cells transformed by DNAs of these carcinomas expressed the same abnormal p21 species, indicating that these abnormal proteins were encoded by the activated ras^K genes detected by transfection. These results indicate that transforming activity of ras^K genes in human lung and colon carcinoma cell lines is activated by mutations which alter the structure of their gene products, and that activation of ras^K genes can result from different molecular alterations in different individual neoplasms.     

Activation of human raf transforming genes by deletion of normal amino-terminal coding sequences.

Three activated cellular raf genes have been detected by transfection of NIH 3T3 cells with human tumor DNAs. Blot hybridization analysis indicated that all three transforming raf genes had recombined with non-raf sequences in the vicinity of raf exon 7-intron 7, resulting in the deletion of about 40% of the normal coding sequence from the raf amino terminus. By cloning sequences upstream of the truncated raf loci we have shown that the rearrangements involve the fusion of three different 5' non-raf human sequences to the human raf gene. No rearrangements could be detected in the raf loci of the three original human tumor DNAs, suggesting that the raf genes were activated by DNA rearrangements occurring during transfection. Significant overexpression of raf mRNA was not evident in two of the three transformant lines, indicating that raf overexpression is not necessary and 5' truncation alone may be sufficient to activate the transforming potential of cellular raf genes.     

Cellular oncogenes and cancer

Human oncogenes have been identified either by the ability of normal or tumor DNAs to induce transformation of cells in culture or as the targets of chromosome translocations or DNA amplification in neoplasms. By the combination of these approaches, approximately 40 different genes have been implicated as potential contributors to the development of human neoplasms. The proteins which are encoded by these potential human oncogenes include plasma membrane proteins with tyrosine kinase activity, plasma membrane guanine nucleotide binding proteins, cytoplasmic proteins with serine/threonine kinase activity and nuclear proteins. In many tumors, more than 1 potential oncogene has been activated, suggesting that multiple genes may contribute to neoplasm pathogenesis. I will discuss the identification of these genes, their modes of activation, the diversity of their protein products and their potential roles in both neoplastic and normal cells.     

Co-transfection of normal NIH/3T3 DNA and retroval LTR sequences: a novel strategy for the detection of potential c-onc genes.

Morphologically transformed, tumorigenic cell lines were obtained after co-transfecting normal NIH/3T3 DNA and cloned 3'-long terminal repeat sequences of Moloney leukemia virus (Mo-LTR) onto NIH/3T3 recipient cells. In four such cell lines the malignant phenotype was found to be associated with single and specific Mo-LTR integration sites that were retained after serial passages through NIH/3T3 and rat 208F cells, indicating that Mo-LTR sequences are linked to the activated oncogenes. In one of these clones the activated transforming gene was identified as c-raf, the cellular homologue of a recently described retroviral oncogene. This finding not only demonstrates that the mouse c-raf gene can be activated to exhibit an oncogenic potential but also that the approach chosen in this study is suitable for the detection of potential c-onc genes. In contrast to this clone, the activated transforming genes in other cell lines appear to be different from 19 previously isolated v-onc and c-onc genes. These results demonstrate the potential of the established transformation system for the detection and isolation of previously unidentified c-onc genes.     

H-ras activation in benign and self-regressing skin tumors (keratoacanthomas) in both humans and an animal model system.

The involvement of the ras oncogenes in tumorigenesis was investigated in keratoacanthomas, which are benign and self-regressing skin tumors, both in humans and in a corresponding animal model system. Keratoacanthomas were induced on rabbit ears by repeated applications of 7,12-dimethylbenz(a)anthracene. About 60% of the tumor DNAs produced transformed foci after transfection into NIH 3T3 cells, and in all of them the transforming gene was identified as H-ras by Southern and Northern (RNA) hybridization. Immunoprecipitation experiments suggested that the transforming rabbit H-ras protein carried a mutation in codon 61. In addition, an activated H-ras gene was detected in a human keratoacanthoma by using a nude mouse tumorigenesis assay after transfection of tumor DNA into NIH 3T3 cells. This is the first report of ras activation in a benign human tumor. The transforming human H-ras gene showed a point mutation in codon 61 that would result in leucine instead of the glutamine present in the normal gene product. The finding of ras activation in tumors that are not only benign but also self-regressing indicates that activated ras genes are not sufficient to maintain a neoplastic phenotype, although they likely play a role in early stages of tumorigenesis.     

Highly frequent detection of transforming genes in acute leukemias by transfection using in vivo selection assays

DNAs from nine out of ten acute leukemia cases that were negative by in vitro focus forming assays exhibited transforming activity tested by in vivo selection assays in nude mice using transfected NIH3T3 cells. Of the nine cases, six cases contained activated N-ras genes, and one case exhibited activation of the c-K-ras gene. None of the ras gene family showed homology with the transforming genes derived from the other two cases. Our observations indicate that in vivo selection assays detect transforming genes including ras oncogenes at high frequency, and that activated N-ras genes are frequently detected in human acute leukemias.     

A transforming activity not detected by DNA transfer to NIH 3T3 cells is detected by JB6 mouse epidermal cells.

Transfection of four different mouse epidermal tumor cell DNAs into NIH 3T3 cells yielded neither morphologically altered foci nor anchorage independence. However, promotion-sensitive, but not promotion-insensitive, JB6 mouse epidermal cell lines were permissive for the expression of anchorage independence after transfection of DNA from three of these tumor cell lines. This transforming activity and the promotion-sensitive activity that confers sensitivity to promotion of transformation show differences in restriction enzyme sensitivity. In view of this difference and the differences in both recipient cells and 12-O-tetradecanoyl-phorbol-13-acetate dependence of expression, it appears that the transforming activity and the promotion-sensitive activity are specified by different genes. The JB6 promotion-sensitive cell lines may be useful for detecting and cloning transforming genes that escape detection in the NIH 3T3 cell focus assay.     

Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185

The neu oncogene, which is frequently activated in neuro- and glioblastomas of BDIX rats, was originally identified in the NIH 3T3 focus-forming assay. cDNA clones of the normal and transforming alleles of neu have been isolated. When these clones are inserted into the expression vector pSV2, they direct the synthesis of p185, the neu gene product. The transforming cDNA clone yields foci when transfected onto a NIH 3T3 monolayer, but the normal cDNA does not. The construction of in vitro recombinants between the normal and transforming cDNAs has allowed the determination of the mutation responsible for the activation of the neu proto-oncogene. A single point mutation changes a valine in the transmembrane domain of the predicted protein product insert to a glutamic acid. The DNAs from four independent cell lines containing activated neu oncogenes contain the identical mutation at this position.     

The oncogenic forms of N-ras or H-ras prevent skeletal myoblast differentiation.

Differentiation of skeletal muscle involves withdrawal of myoblasts from the cell cycle, fusion to form myotubes, and the coordinate expression of a variety of muscle-specific gene products. Fibroblast growth factor and type beta transforming growth factor specifically inhibit myogenesis; however, the transmembrane signaling pathways responsible for suppression of differentiation by these growth factors remain elusive. Because ras proteins have been implicated in the transduction of growth factor signals across the plasma membrane, we used DNA-mediated gene transfer to investigate the potential involvement of this family of regulatory proteins in the control of myogenesis. Transfection of the mouse skeletal muscle cell line C2 with the oncogenic forms of H-ras or N-ras completely suppressed both myoblast fusion and induction of the muscle-specific gene products nicotinic acetylcholine receptor and creatine kinase. Inhibition of differentiation by activated ras genes occurred at the level of muscle-specific mRNA accumulation. In contrast, proto-oncogenic forms of N-ras or H-ras had no apparent effects on the ability of C2 cells to differentiate. Myoblasts transfected with activated ras genes exhibited normal growth properties and ceased proliferating in the absence of mitogens, indicating that ras inhibited differentiation through a mechanism independent of cell proliferation. These results demonstrate that activated ras gene products mimic the inhibitory effects of fibroblast growth factor and type beta transforming growth factor on myogenic differentiation and suggest that each of these regulators of myogenesis may operate through a common intracellular pathway.     

Malignant cell transformation and oncogenes

In genome of all transforming retroviruses special genes (oncogenes) have been identified which play a key role in malignant conversion of the cells, infected with these viruses. The homologues of these genes (protooncogenes) are persist in all normal cells. During transformation protooncogenes can be activated as a result of one of following processes: insertion of promotor-like elements, mutations, translocations, amplifications or rearrangements. Using transfection technique the transforming genes were isolated from different human tumors. The activation of one of the cellular oncogenes may switch on the other genes and malignant cell transformation may be characterized as a multifactor and multistage process.     

Amplification and activation of c-Ki-ras oncogene in cell line developed from human pancreas explants.

Amplification and activation of c-Ki-ras gene was studied in normal human pancreas and a cell line (T-3) derived from normal pancreas explants exposed to methylnitrosourea (MNU) for 26 weeks. Normal genomic DNAs from pancreas and derived cell lines showed no transforming activity in NIH 3T3 cells. However, DNAs isolated from tumorigenic cell line derived from MNU treated human pancreas explants transformed NIH 3T3 cells. The hybridization profiles showed that the c-Ki-ras gene was amplified 5 fold in the tumorigenic cells (T-3). The level of mRNA specific to the c-Ki-ras gene was found to be 50-60 fold higher in the malignant cells than in normal human pancreas. These results suggest that higher expression of ras genes is due to gene amplification and/or activation, which is an important step in carcinogenesis.     

Activation of a novel human transforming gene, ret, by DNA rearrangement

A novel transforming gene was detected by transfection of NIH 3T3 cells with human lymphoma DNA. The tumor DNA induced a single focus in primary transfections, whereas DNAs of transformed NIH cells induced transformation with high efficiencies in secondary and tertiary assays. Molecular clones spanning about 37 kb of human sequence were isolated from tertiary transformant DNA. Blot hybridization indicated that the transforming gene consisted of two segments that were unlinked in both normal human and primary lymphoma DNAs. The two segments of human DNA were cotranscribed in transformed NIH cells but not in any human cells examined. The transforming gene thus appeared to be activated by recombination between two unlinked human DNA segments, possibly by cointegration during transfection.     

Identification of a precursor to phosphatidyl choline-specific B-1 cells suggesting that B-1 cells differentiate from splenic conventional B cells in vivo: cyclosporin A blocks differentiation to B-1

The origin of B-1 cells is controversial. The initial paradigm posited that B-1 and B-2 cells derive from separate lineages. More recently it has been argued that B-1 cells derive from conventional B cells as a result of T-independent Ag activation. To understand B-1 cell differentiation, we have generated Ig transgenic (Tg) mice using the H and L chain genes (VH12 and Vkappa4) of anti-phosphatidyl choline (anti-PtC) B cells. In normal mice anti-PtC B cells segregate to B-1. Segregation is intact in VH12 (6-1) and VH12/Vkappa4 (double) Tg mice that develop large numbers of PtC-specific B cells. However, if B-1 cell differentiation is blocked, anti-PtC B cells in these Tg mice are B-2-like in phenotype, suggesting the existence of an Ag-driven differentiative pathway from B-2 to B-1. In this study, we show that double Tg mice have a population of anti-PtC B cells that have the phenotypic characteristics of both B-2 and B-1 cells and that have the potential to differentiate to B-1 (B-1a and B-1b). Cyclosporin A blocks this differentiation and induces a more B-2-like phenotype in these cells. These findings indicate that these cells are intermediate between B-2 and B-1, further evidence of a B-2 to B-1 differentiative pathway.     

Methylation of satellite sequences in mouse spermatogenic and somatic DNAs.

The distribution of 5-methyl cytosine (5-MeC) residues in a highly repetitive sequence, mouse major satellite, was examined in germinal versus somatic DNAs by digestion with the methylation sensitive isoschizomers Msp I and Hpa II and Southern blot analysis, using a cloned satellite probe. DNA from liver, brain, and a mouse fibroblast cell line, C3H 10T1/2, yielded a multimeric hybridization pattern after digestion with Msp I (and control Eco RI) but were resistant to digestion with Hpa II, reflecting a high level of methylation of the satellite sequences. In contrast, DNA from mature sperm was undermethylated at these same sequences as indicated by the ability of Hpa II to generate a multimeric pattern. DNAs from purified populations of testis cells in different stages of spermatogenesis were examined to determine when during germ cell differentiation the undermethylation was established. As early as in primitive type A, type A, and type B spermatogonia, an undermethylation of satellite sequences was observed. This suggest that this highly specific undermethylation of germ cell satellite DNA occurs very early in the germ cell lineage, prior to entry into meiosis.     

Activity of some proteinases and glycosidases in human leukemic lymphoid cells at various stages of differentiation.

Activities of some glycosidases and proteinases in human leukemic lymphoid cells at various stages of differentiation have been compared. It was found that cells with different immunological phenotypes gave different enzymic spectra. Glycosidases and proteinases in lymphoid cell precursors had higher activity level than the enzymes in mature T- and B- cells. In cells of B- lineage, all activities were lower than in common precursor of lymphoid cells. In T-cells at the earlier stages of thymic differentiation, activities of all proteinases and most of glycosidases were higher than in common precursor cells whereas in mature T-helpers and T-suppressors the activities were markedly lower. Most of hydrolases in mature T-cells were twice more active than the enzymes in mature B-cells. The opposite-directional changes in activities of some hydrolases at the earlier stages of differentiation of lymphoid cells along B- or T- cells pathways are suggested.