Regional chromosomal localization of N-ras, K-ras-1, K-ras-2 and myb oncogenes in human cells

The identification of transforming genes in human tumor cells has been made possible by DNA mediated gene transfer techniques. To date, it has been possible to show that most of these transforming genes are activated cellular analogues of the ras oncogene family. To better understand the relationship between these oncogenes and other human genes, we have determined their chromosomal localization by analyzing human rodent somatic cell hybrids with molecularly cloned human proto-oncogene probes. It was possible to assign N-ras to chromosome 1 and regionally localize c-K-ras-1 and c-K-ras-2 to human chromosomes 6pter-q13 and 12q, respectively. These results along with previous studies demonstrate the highly dispersed nature of ras genes in the human genome. Previous reports indicated that the c-myb gene also resides on chromosome 6. It has been possible to sublocalize c-myb to the long arm of chromosome 6 (q15-q21). The non-random aberrations in chromosomes 1, 6 and 12 that occur in certain human tumors suggest possible etiologic involvement of ras and/or myb oncogenes in such tumors.     

Absolute values of ras p21 defined by direct binding liquid competition radioimmunoassays

Several distinct and high-conserved genes comprise the ras gene family of rodents and humans, i.e., rodent Harvey and Kirsten, and human Harvey, Kirsten and neuroblastoma. Transformation, either by a point-mutation resulting in a change in one amino acid of the 21 kDa ras gene product (p21), or by increased expression of ras p21, has been demonstrated to be mediated by members of this gene family. We report here the development of direct binding liquid competition radioimmunoassays for the detection and quantitation of the ras oncogene and proto-oncogene products. Using these radioimmunoassays and ras p21 purified from Escherichia coli containing the full-length T24 mutant human Harvey ras gene protein product as a standard, we have defined the actual amount of ras p21 per micrograms of total cellular protein, or per cell, in various ras transformed and 'normal' mammalian cell lines. One of the radioimmunoassays developed is group-specific, since the antigenic determinant recognized is shared by both the point-mutated and proto-forms of Harvey, Kirsten and neuroblastoma members of the ras gene family, while the second may be termed type-selective, since it recognizes an antigenic determinant localized primarily on the Harvey ras p21. Both radioimmunoassays are interspecies, since they detect a ras p21 antigenic determinant common to cells of human and rodent origin. These studies thus describe the first means for defining absolute values of any oncogene or proto-oncogene protein product. The assays described, when used in combination with specific c-DNA probes to define specific ras proto-oncogenes or point-mutated oncogenes being expressed, will now permit truly quantitative analyses of ras p21 expression in experimental cell culture systems, animal models and human biopsy material.     

let-60, a gene that specifies cell fates during C. elegans vulval induction, encodes a ras protein

Genetic analysis previously suggested that the let-60 gene controls the switch between vulval and hypodermal cell fates during C. elegans vulval induction. We have cloned the let-60 gene, and shown that it encodes a gene product identical in 84% of its first 164 amino acids to ras gene products from other vertebrate and invertebrate species. This conservation suggests that the let-60 product contains all the biochemical functions of ras proteins. Extrachromosomal arrays of let-60 ras DNA cause cell-type misspecification (extra vulval fates) phenotypically opposite to that caused by let-60 ras loss-of-function mutations (no vulval fates), and suppress the vulvaless phenotype of mutations in two other genes necessary for vulval induction. Thus, the level and pattern of let-60 ras expression may be under strict regulation; increase in let-60 ras activity bypasses or reduces the need for upstream genes in the vulval induction pathway.     

Ras-induced hyperplasia occurs with mutation of p53, but activated ras and myc together can induce carcinoma without p53 mutation.

Using a reconstituted mouse prostate organ, the effects on endogenous p53 expression of the ras oncogene or of the ras + myc oncogenes were investigated. In this system the ras gene alone causes mild hyperplasia, but the combination of ras and myc leads to the formation of carcinomas. Surprisingly, while p53 mutations were found in cells derived from the reconstituted organs containing ras alone, no such mutations were found in the ras + myc-transformed cells. Their growth, unlike that of the cells containing ras alone, was not inhibited by transfection with plasmids encoding wild-type human p53. We suggest that expression of both activated ras and myc genes bypasses the need for p53 mutation by neutralizing the tumor suppressor activity of normal p53.     

RAS enzyme-linked immunoblot assay discriminates p21 species: a technique to dissect gene family expression

The members of the RAS gene family of protooncogenes are of implied biological significance in oncogenesis. The precise role of these genes is unclear. One difficulty has been the inability to discriminate the individual p21 protein products of various ras genes in cell lines, de novo human tumors, and related normal tissues. In this report, specific proteins of the human c-Ha-ras-1, c-Ki-ras-2, and c-N-ras genes have been detected and discriminated by the differential use of various antisera recognizing these p21s. This enzyme-linked immunoblot assay utilizes a double antibody system in which monoclonal antibodies are initially used to immunoprecipitate the p21ras proteins. Immunoprecipitates are then subjected to one-dimensional Western blot analysis utilizing other antibodies raised against p21s, coupled with nonradiolabeled enzyme-linked colorimetric detection. By direct detection, the specific products of the three human ras genes can be discriminated. In addition, we describe the generation and characterization of a new anti-p21c-N-ras-specific antibody. The simultaneous expression into protein of multiple ras genes is unequivocally demonstrated in both homogeneous cell lines and heterogeneous human tissues. This new technique is also applicable for discrimination of the protein products of other gene families.     

Activation of a human c-K-ras oncogene

The human lung carcinomas PR310 and PR371 contain activated c-K-ras oncogenes. The oncogene of PR371 was found to present a mutation at codon 12 of the first coding exon which substitutes cysteine for glycine in the encoded p21 protein. We report here that the transforming gene of PR310 tumor contains a mutation in the second coding exon. An A----T transversion at codon 61 results in the incorporation of histidine instead of glutamine in the c-K-ras gene product. By constructing c-K-ras/c-H-ras chimeric genes we show that this point mutation is sufficient to confer transforming potential to ras genes, and that a hybrid ras gene coding for a protein mutant at both codons 12 and 61 is also capable of transforming NIH3T3 cells. The relative transforming potency of p21 proteins encoded by ras genes mutant at codons 12, 61 or both has been analyzed. Our studies also show that the coding exons of ras genes, including the fourth, can be interchanged and the chimeric p21 ras proteins retain their oncogenic ability in normal rodent established cell lines.     

The ras gene family and human carcinogenesis

It has been well established that specific alterations in members of the ras gene family, H-ras, K-ras and N-ras, can convert them into active oncogenes. These alterations are either point mutations occurring in either codon 12, 13 or 61 or, alternatively, a 5- to 50-fold amplification of the wild-type gene. Activated ras oncogenes have been found in a significant proportion of all tumors but the incidence varies considerably with the tumor type: it is relatively frequent (20-40%) in colorectal cancer and acute myeloid leukemia, but absent or present only rarely in, for example, breast tumors and stomach cancer. No correlation has been found, yet, between the presence of absence of an activated ras gene and the clinical or biological features of the malignancy. The activation of ras oncogenes is only one step in the multistep process of tumor formation. The presence of mutated ras genes in benign polyps of the colon indicates that activation can be an early event, possibly even the initiating event. However, it can also occur later in the course of carcinogenesis to initiate for instance the transition of a benign polyp of the colon into a malignant carcinoma or to convert a primary melanoma into a metastatic tumor. Apparently, the activation of ras genes is not an obligatory event but when it occurs it can contribute to both early and advanced stages of human carcinogenesis.     

Integrative genomics revealed RAI3 is a cell growth-promoting gene and a novel P53 transcriptional target

In this study, differential gene expression between normal human mammary epithelial cells and their malignant counterparts (eight well established breast cancer cell lines) was studied using Incyte GeneAlbum 1-6, which contains 65,873 cDNA clones representing 33,515 individual genes. 3,152 cDNAs showed a > or =3.0-fold expression level change in at least one of the human breast cancer cell lines as compared with normal human mammary epithelial cells. Integration of breast tumor gene expression data with the genes in the tumor suppressor p53 signaling pathway yielded 128 genes whose expression is altered in breast tumor cell lines and in response to p53 expression. A hierarchical cluster analysis of the 128 genes revealed that a significant portion of genes demonstrate an opposing expression pattern, i.e. p53-activated genes are down-regulated in the breast tumor lines, whereas p53-repressed genes are up-regulated. Most of these genes are involved in cell cycle regulation and/or apoptosis, consistent with the tumor suppressor function of p53. Follow-up studies on one gene, RAI3, suggested that p53 interacts with the promoter of RAI3 and repressed its expression at the onset of apoptosis. The expression of RAI3 is elevated in most tumor cell lines expressing mutant p53, whereas RAI3 mRNA is relatively repressed in the tumor cell lines expressing wild-type p53. Furthermore, ectopic expression of RAI3 in 293 cells promotes anchorage-independent growth and small interfering RNA-mediated depletion of RAI3 in AsPc-1 pancreatic tumor cells induces cell morphological change. Taken together, these data suggest a role for RAI3 in tumor growth and demonstrate the predictive power of integrative genomics.     

Expression of Wnt5a Is Downregulated by Extracellular Matrix and Mutated c-Ha-ras in the Human Mammary Epithelial Cell Line MCF-10A

Wnt genes are involved in tumour growth and regulate cell adhesion. Some (Wnt5a and Wnt7b) are more highly expressed in human breast cancer compared to normal tissues. Wnt5a is involved in the regulation of cell movement inXenopusand is upregulated in several human cancers. Factors regulating Wnt gene expression in human breast epithelium are poorly understood, but c-erbB2 is amplified in many breast cancers and associated with rapid growth and metastasis, as is high expression of c-Ha-ras. To further understand the regulation of Wnt gene expression, this study investigated the effect of proto-oncogenes c-Ha-ras and c-erbB2, and collagen on Wnt mRNA expression, in a normal spontaneously immortalised human mammary epithelial cell line MCF-10A. Out of nine human Wnt genes investigated, Wnt5a and Wnt7b were expressed in the parental cell line, and neomycin-, c-Ha-ras- and c-erbB2-transfected cell lines. The level of Wnt5a mRNA expression was decreased 40-fold and 3-fold when parental cells were grown on collagen and in collagen, respectively. This downregulation correlated with cell branching. However, Wnt7b was not regulated by collagen. In the presence of activated c-Ha-ras, the level of Wnt5a mRNA expression was markedly decreased (> 200-fold) and cell growth rate was elevated. When treated with p21^rasinhibitor, BZA-5B, there was a moderate reversal of Wnt5a mRNA expression (2-fold) with a parallel decrease in cell growth. The data indicate that c-Ha-ras is an upstream inhibitory regulator of Wnt5a, and provide further evidence of an inverse relationship between Wnt5a mRNA expression and cell branching. This demonstrates selectivity of regulation of individual members of the Wnt gene family by the ras pathway. Overexpression of c-erbB2 had no effect on Wnt5a or Wnt7b mRNA expression. Thus, extracellular matrix and ras regulate Wnt5a, providing a mechanism for feedback of morphogenetic movements, which is relevant also to cancer biology.     

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.     

In vitro establishment is not a sufficient prerequisite for transformation by activated ras oncogenes

Activated ras genes transform REF52 cells only at low frequencies and adenovirus early region 1A collaborates with ras oncogenes to convert REF52 cells to a tumorigenic phenotype. While failure to transform did not result from an absence of ras gene expression, E1A appeared to enhance expression of transfected ras genes by approximately tenfold. However, enhanced ras expression alone does not account for collaboration by E1A since overexpression of T24 Ha-ras p21 induced morphological crisis and cell growth arrest rather than stable transformation. These results indicate that E1A contributes complementing biochemical activities that enable ras genes to transform REF52 and suggest that the role of E1A in primary cell transformation may extend beyond facilitating in vitro establishment.     

3'-End conjugates of minimally phosphorothioate-protected oligonucleotides with 1-O-hexadecylglycerol: synthesis and anti-ras activity in radiation-resistant cells

Activation of the ras oncogene has been implicated in many types of human tumors. It has been shown that downmodulation of ras expression can lead to the reversion of the transformed phenotype of these tumor cells. Antisense oligodeoxyribonucleotides (ODNs) can inhibit gene expression by hybridization to complementary mRNA sequences. To minimize toxicity associated with all-phosphorothioated ODNs and improve cellular uptake, we used partially phosphorothioate (PPS)-modified ODNs having an additional hydrophobic tail at the 3'-end (PPS-C(16)). The PPS ODNs are protected against degradation by PS internucleotide linkages at both the 3'- and 5'-ends and additionally stabilized at internal pyrimidine sites, which are the major sites of endonuclease cleavage. Here we show that anti-ras PPS-C(16) ODN retains the high sequence-specificity of PPS ODNs and provides maximal inhibition of Ras p21 synthesis with minimal toxicity even without the use of a cellular uptake enhancer. Moreover, treatment of T24, a radiation-resistant human tumor cell line that carries a mutant ras gene, with anti-ras PPS-C(16) ODN resulted in a reduction in the radiation resistance of the cells in vitro. We also demonstrate that the growth of RS504 (a human c-Ha-ras transformed NIH/3T3 cell line) mouse tumors was significantly inhibited by the combination of intratumoral injection of anti-ras PPS-C(16) ODN and radiation treatment. These findings indicate the potential of this combination of antisense and conventional radiation therapy as a highly effective cancer treatment modality.     

Multiplex polymerase chain reaction amplification and direct sequencing of homologous sequences: point mutation analysis of the ras genes.

ras proto-oncogenes are activated by point mutation in a wide variety of human and animal tumors, making ras gene analysis a major area of clinical and basic cancer research. Activating point mutations, in each of the three ras genes (Ha-, Ki-, or N-ras), usually occur in one of three specific codons (12, 13, or 61). Thus, an adequate assessment of activating ras gene mutations should include the analysis of at least nine codons. We have developed a rapid method for point mutation analysis of the ras genes, which involves simultaneous (multiplex) PCR amplification of all three homologous ras genes (in the regions surrounding codons 12-13 and codon 61) in a single reaction starting with only 1 microgram of genomic DNA. Although multiplex PCR has been previously used for unrelated sequences, we demonstrate here that multiplex PCR can also be used for highly homologous sequences. Importantly, after coamplification, each of the homologous ras genes can be individually and specifically sequenced even though the other two closely related genes are present in the same template mixture, by using high-stringency conditions permitted by Taq DNA polymerase. An automated multicycle DNA sequencing procedure is used to allow the double-stranded PCR products to be sequenced directly without the need to generate single-stranded templates, further simplifying the protocol. Our multiplex PCR amplification and direct DNA sequencing procedures should greatly facilitate more complete analyses of activating ras gene point mutations, particularly in studies involving many tumor samples.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystallization of human c-H-ras oncogene products

There is compelling evidence that cancer develops as a consequence of genetic changes (probably multiple) in some members of a selected set of cellular genes. DNA isolated from a variety of tumors, but not normal tissues, possesses the ability to malignantly transform non-tumorigenic cells. Many oncogenes responsible for such transformation have been isolated from transformed cell lines and animal and human tumors induced spontaneously, by virus, by chemical, or by radiation. The most commonly found transforming genes isolated from human tumor cells by DNA transfection assay are the ras gene family (c-H-ras, c-K-ras and N-ras). We report crystallization of several human c-H-ras oncogene proteins.     

Tumorigenesis in neurofibromatosis: new insights and potential therapies

The neurofibromatoses NF1 and NF2 are inherited cancer predisposition syndromes in which affected individuals are prone to development of mostly benign, but occasionally malignant, tumors. The NF1 and NF2 genes function as tumor suppressor genes (negative growth regulators), such that their loss of expression predisposes to tumor formation. Neurofibromin, the protein product of the NF1 gene, acts as a negative regulator of the ras proto-oncogene, to reduce cell growth. Merlin, the NF2 gene product, is involved in regulating cell proliferation and motility, and probably plays a role in integrating multiple cell-signaling pathways. By understanding the function of these tumor suppressors, we have a unique opportunity to develop targeted pharmacotherapeutic interventions for these disorders.