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PubMed1993

Denaturing gradient gel electrophoresis (DGGE) assay for K-ras and N-ras genes: detection of K-ras point mutations in human lung tumour DNA.

Point mutations in the ras oncogenes are very common in lung cancers as well as in many of the other solid tumours. To effectively examine the occurrence of these mutations in a large number of tumour samples, we have applied denaturing gradient gel electrophoresis (DGGE) for the analysis of point mutations of the K-ras and N-ras genes, using GC-clamped, PCR-amplified DNA fragments. Among the 68 tumour DNA samples, we detected 14 mutations in the K-ras gene. This was 78% of the mutations identified by oligonucleotide hybridization. Altogether, eight of the nine different kinds of base substitutions found in the tumour samples were detected by the DGGE assay, representing substitutions at codons 12, 13, and 61 of the K-ras gene. Six of the detected mutations were guanine to thymine transversions at codon 12; this was the most common type of alteration. On the basis of our experience, the present non-radioactive DGGE analysis seems to be readily applicable for detection of the mutations in the K-ras and N-ras genes. Types of ras gene mutations frequent in adenocarcinomas of the lung are also discussed.

Base Sequence↗
PubMed1986

A transforming ras gene can provide an essential function ordinarily supplied by an endogenous ras gene.

Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot. However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not. These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.

Animals↗
PubMed1997

PPRAS1PPRAS2 AND PPRAP1 GENES, MEMBERS OF A RAS GENE FAMILY FROM THE TRUE SLIME MOLD PHYSARUM POLYCEPHALUM ARE DEVELOPMENTALLY REGULATED

The expression patterns of two true ras genes, Ppras1 and Ppras2and one rap gene, Pprap1were examined in four Physarum polycephalum developmental stages: uninucleate amoebae, plasmodia (multinucleate syncytia), spherules (a vegetative, dormant stage) and fruiting bodies. Ppras1 and Pprap1 are expressed in all stages examined with the maximum levels of their transcripts in amoebae and fruiting bodies, respectively, and the minimum levels in plasmodia, whereas the Ppras2 transcript is only detectable in amoebae and fruiting bodies. The results obtained indicate that P. polycephalum is an organism possessing a developmentally regulated ras gene family and presents a convenient system to study the role of ras/rap genes in control of growth and differentiation of lower eukaryotic organisms.Copyright 1997 Academic Press Limited Copyright 1997Academic Press Limited

Journal Article↗
PubMedDate not supplied

Second primary cancers after resection of lung adenocarcinoma with ras gene mutation.

BACKGROUND: Ras gene mutations are associated with a poor prognosis in patients with adenocarcinoma of the lung. However, the association between cases with ras gene mutation and an occurrence of second primary cancer is unknown. MATERIALS AND METHODS: We examined 89 adenocarcinoma of the lung obtained from patients treated by curative resection, and four second primary cancers, for mutation at codons 12, 13, and 61 of three ras oncogenes using polymerase chain reaction and oligonucleotide hybridization techniques. RESULTS: Ras gene mutations were detected in 13 cases. Six patients died from recurrent disease within 3 years. Of the seven patients who survived over 5 years, three patients demonstrated a metachronous second primary cancer after the operation for lung cancer, and one patient had synchronous laryngeal cancer. Two of the cases with second primary cancer demonstrated the ras gene mutation. One had the same mutation as that of the primary lung cancer, and the other had a different mutation from the first lung cancer. CONCLUSION: Ras gene mutations play an important role in tumor progression of lung adenocarcinoma and also might have an role in the carcinogenesis in respiratory tract cancer.

Adenocarcinoma↗
PubMed2002

ras Gene mutations and expression of Ras signal transduction mediators in gastric adenocarcinomas.

OBJECTIVE: To investigate ras gene alteration in human gastric adenocarcinomas and its potential relationship to ras signal transduction mediators. DESIGN: Genomic DNA from 104 gastric tumors were analyzed by sequencing of polymerase chain reaction-amplified products for the presence of ras mutations. All the samples were further investigated with the use of immunohistochemical analysis for ERK1 and ERK2. SETTING: Tertiary care teaching hospital. PATIENTS: Seventy patients from a Korean population and 34 from a Midwestern US population composed of white Americans and African Americans. RESULTS: Fifteen tumors (14%) were positive for either H-ras or K-ras mutation: 9 (13%) of 70 Korean patients and 6 (18%) of 34 US patients. Seven (78%) of the 9 mutated tumors from Korean patients and all 6 (100%) from the US patients were intestinal-type lesions. Either ERK1 and/or ERK2 was overexpressed in 68 samples (65%). No association was established between ras mutations and overexpression of ERK1/2. However, the correlation between ERK1/2 and progression (early vs late) was statistically significant (P =.007). CONCLUSIONS: These data suggest that ras mutations are uncommon in gastric adenocarcinomas and that differing racial and/or geographic mechanisms may not underlie ras gene alteration. Most ras mutations were, however, observed in the group of intestinal-type samples, supporting the different genetic mechanisms of carcinogenesis between the intestinal- and diffuse-type tumors. It is noteworthy that enhanced ERK1/2 activity could be one of the characteristics of tumor invasiveness in gastric cancers.

Adenocarcinoma↗
PubMed1995

Regulatory elements in the first intron of the mouse Ha-ras gene.

The Ha-ras gene is one of the three oncogenes (Ha-ras, Ki-ras, and N-ras) of the ras superfamily of small G proteins. The p21ras proteins encoded by the ras genes are key proteins involved in the transduction of signals from membrane receptor-tyrosine kinases to downstream targets. The ras genes seem to play a ubiquitous role in the control of cell proliferation and cell differentiation. At the same time, ras genes may perform specific differentiated functions in certain cell types. Little is known about the regulation of expression of the Ha-ras gene. The first intron of the Ha-ras gene has been reported to be highly conserved between human and rodent. We investigated the role that this intron may play in the regulation of expression of Ha-ras. The promoter region of the Ha-ras gene exhibits characteristics of a housekeeping gene. Deletion analysis shows the existence of an enhancer-type element in the 5' region of the first intron (intron 0). DNase 1 footprinting experiments reveal five sites that interact with nuclear proteins from fibroblast and epithelial cell lines. Deletion and site-directed mutagenesis of three of these sites show that two are involved in a positive effect and one in a negative effect on the regulation of expression of the mouse Ha-ras gene.

3T3 Cells↗
PubMedDate not supplied

In contrast to EGFr gene overexpression, H-ras gene expression decreases in human gliomas.

We have examined the expression of the epidermal growth factor receptor (EGFr) gene and 14 other oncogenes in several human gliomas. EGFr overexpression was apparent in 17 of 18 tumors. Nevertheless, remarkable amounts of aberrantly-sized EGFr mRNAs were not found in the 11 tumors examined. Among the 14 other oncogene probes employed, only the H-ras probe revealed changes in the expression level. The expression of this oncogene was 2-3-fold reduced in 13 of 18 tumors. In neural tissue the H-ras gene expression can be correlated with cellular differentiation. A correlation between H-ras protein expression and a favourable clinical prognosis was found in neuroblastomas. The decreased amounts of H-ras transcripts in gliomas may be a sign of a lower state of differentiation of the cells.

Brain↗
PubMed1993

Correlation of intrinsic chemoresistance of non-small-cell lung cancer cell lines with HER-2/neu gene expression but not with ras gene mutations.

BACKGROUND: At diagnosis, most small-cell lung cancers (SCLCs) are chemosensitive, whereas non-small-cell lung cancers (NSCLCs) are usually chemoresistant. Activation of ras genes and HER-2/neu genes (also known as ERBB2) is encountered in subpopulations of NSCLC but not in SCLC and has been linked to shortened survival. Therefore, activation of these genes may be associated with intrinsic chemoresistance in NSCLC. Studies have also suggested that the multidrug-resistant phenotype expressed by the MDR1 gene (also known as PGY1) does not correlate with the in vitro chemosensitivity of NSCLC cells or with clinical response to therapy and does not explain the spectrum of cross-resistance to drugs. PURPOSE: The purpose of this study was to investigate the relationships between chemoresistance and the presence of ras gene point mutations and overexpression of the HER-2/neu gene in NSCLC cell lines, which indicates gene activation. METHODS: Using a panel of 20 NSCLC cell lines established from untreated patients, we assessed the differences in HER-2/neu messenger RNA (mRNA) expression in the cell lines with or without ras mutations. We performed in vitro drug sensitivity testing by the tetrazolium-based MTT [i.e., 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H- tetrazolium bromide] assay with doxorubicin, carmustine, cisplatin, melphalan, mitomycin, and etoposide, and we determined the differences in IC50 values (i.e., the drug concentrations required to inhibit cell growth by 50%) for the cell lines. RESULTS: We found a statistically significant correlation between the IC50 values for all six drugs and the degree of HER-2/neu gene expression in all 20 cell lines (r = .67-.86; P < .005) as well as in the subpopulation of eight cell lines with ras mutations (r = .83-.98; P < .05). The IC50 values for doxorubicin, carmustine, cisplatin, and melphalan were not significantly different in the cell lines with or without ras mutations, but the values for mitomycin and etoposide in lines with ras mutations were slightly lower than in those without ras mutations (borderline significance, P = .031). Levels of HER-2/neu expression in cell lines with ras mutations were lower than those without ras mutations, but the difference was not statistically significant. CONCLUSION: Our findings indicate that overexpression of HER-2/neu is a marker for intrinsic multidrug resistance in NSCLC cell lines. IMPLICATIONS: If the clinical relevance of our findings is confirmed, HER-2/neu gene expression can be used as a predictor of therapeutic failure in NSCLCs. The relationships between HER-2/neu gene expression, cell proliferation, and chemoresistance in NSCLC require further investigation.

Antineoplastic Agents↗
PubMed1999

Estrogen inducibility of c-Ha-ras transcription in breast cancer cells. Identification of functional estrogen-responsive transcriptional regulatory elements in exon 1/intron 1 of the c-Ha-ras gene.

Although mutation of ras gene is rare in human breast cancer, overexpression of normal c-Ha-ras gene is frequently observed. Using a mouse mammary metastasis model consisting of genetically related mammary tumor sublines with variant metastatic potential, we have previously (i) demonstrated a direct correlation between c-Ha-ras mRNA and protein levels and metastatic potential and (ii) identified a novel hormone-responsive transcriptional regulatory element in intron 1 of the mouse c-Ha-ras gene that contains the consensus half-site of a glucocorticoid response element and flanking consensus half-sites for estrogen response element. Here, we have examined the functionality of intron 1 sequence in context of upstream sequences by using transient transfection assays with plasmids expressing chloramphenicol acetyltransferase. Intron 1 sequence and sequences similar to intron 1 element located in exon 1 function as transcriptional regulatory elements that confer hormonal inducibility to chloramphenicol acetyltransferase gene expression both independently and in context of 5'-flanking sequences. Measurement of c-Ha-ras transcription rates and protein expression by nuclear run-on and metabolic labeling assays showed a 5-12-fold enhancement, respectively, following treatment with 17beta-estradiol that was blunted by ICI 182,780 in the nonmetastatic variant. In contrast, constitutive overexpression of c-Ha-ras transcripts and protein in the metastatic subline was unaffected by estrogen and ICI 182,780. Gel shift assays demonstrated specific interaction of c-Ha-ras exon 1 sequence with nuclear proteins of human breast cancer MCF-7 cells with formation of two complexes, one of which contains estrogen receptor. Our data demonstrate a direct (i) interaction of c-Ha-ras sequence with estrogen receptor and (ii) stimulatory effect of estrogen on c-Ha-ras gene transcription and suggest that alteration in transcriptional regulation of c-Ha-ras gene by estrogen may play an important role in progression of breast cancer.

Animals↗
PubMed1998

Tumourigenicity of MTG8, a leukaemia-related gene, in concert with v-Ha-ras gene in BALB/3T3 cells.

The MTG8 (ETO) gene has been identified as the translocation partner of AML1 (PEBP2alphaB or CBFalpha2) gene in the AML1/MTG8 (ETO) fused gene caused by t(8;21) translocation in human acute myeloid leukaemia, M2 type. Although AML1/MTG8 chimaeric protein is known to inhibit the functioning of AML1 protein, the precise function of MTG8 gene itself is not known yet. We studied the significance of MTG8 gene in the oncogenicity of AML1/MTG8 fused gene, by introducing full-length MTG8 cDNA into both BALB/3T3 cells containing v-Ha-ras gene (Bhas 42 cells) and BALB/3T3 cells without v-Ha-ras gene. Irrespective of the overexpression of MTG8 gene in both groups of cells, Bhas-MTG8 clones which contained v-Ha-ras gene and expressed the MTG8 gene at a level more than twice that of parental Bhas 42 cells induced cell transformation, whereas BALB-MTG8 clones without v-Ha-ras gene did not. Furthermore, injection of the transformed Bhas-MTG8 clones into the subcutaneous tissue of nude mice induced tumours, whereas that of BALB-MTG8 clones did not. These results suggest that MTG8 gene product, in cooperation with viral Ras protein, resulted in tumour formation. We provide the first evidence that MTG8 gene by itself has a carcinogenic property within the AML1/MTG8 (ETO) fused gene.

3T3 Cells↗
PubMed1989

Detection of point mutations in N-ras and K-ras genes of human embryonal rhabdomyosarcomas using oligonucleotide probes and the polymerase chain reaction.

Previous studies have demonstrated that genes of the ras family (H, K, and N) can be activated by point mutations at codons 12, 13, and 61. In the present study we have used oligonucleotide probes corresponding to these regions to assess the role of ras gene mutations in the genesis of human rhabdomyosarcoma. To increase the sensitivity of this method the appropriate regions of the three ras genes were first amplified using the polymerase chain reaction. The results show that 35% (5/14) embryonal rhabdomyosarcomas investigated contain mutations in the N-ras or K-ras genes. Thus ras gene mutation is implicated in the development of mesenchymal and embryonal tumors in addition to its previously documented role in epithelial and hematological neoplasia.

Adolescent↗
PubMed1995

Introduction of wild-type p53 gene downregulates the expression of H-ras gene and suppresses the growth of bladder cancer cells.

Retroviral vectors were used to introduce the wild-type p53 gene into human bladder cancer cell lines BIU-87 and EJ, which express endogenous wt-p53 gene and have a mutation in H-ras gene. The expression of the exogenous wt-p53 gene in cells suppresses the growth of the bladder cancer cells in standard culture and in soft agar and blocks the cell cycle progression in G1. The BIU-87 and EJ cells developed tumors with average volumes of 6.53 cm3 and 6.61 cm3 in nude mice in 9 weeks after inoculation, while the cells transduced with wt-p53 gene failed to form tumors. The expression of H-ras gene in bladder cancer cells was reduced at mRNA level. These results suggest that the overexpression of the wt-p53 gene suppresses the expression of mutant H-ras gene and inhibits the tumor cell growth in vivo and in vitro.

Animals↗
PubMed2002

Decreased susceptibility to NMU-induced mammary carcinogenesis in transgenic rats carrying multiple copies of a rat ras gene driven by the rat Harvey ras promoter.

Ras protein over-expression has been observed in human breast cancers although the significance of Ras over-expression in the etiology of breast cancer is unknown and its contribution to breast cancer prognosis is still debated. In this study, the over-expression of both wild-type Harvey and Kirsten Ras proteins as contributors to rat mammary carcinogenesis were examined using a transgenic rat model. Three rat transgenic lines (designated HrHr transgenics) carrying three to six copies of wild-type rat Harvey ras driven by the wild-type rat Harvey ras promoter were produced. In addition, transgenic lines carrying either three or seven copies of the Kirsten ras gene under the same promoter (HrKr) were produced. No pathological changes in the mammary gland were observed in any of the HrHr or HrKr transgenic rat line heterozygotes. Two of the Ras transgenic lines, HrHr (R8) and HrKr (4334), had a significant reduction in NMU-induced rat mammary cancer when compared to their non-transgenic littermates. All five Ras transgenic lines developed fewer carcinomas than their non-transgenic littermates following NMU exposure. The percentage of NMU-induced G35 to A35 activating mutations in the endogenous Harvey ras gene in mammary carcinomas from the HrHr, HrKr transgenic rats and their non-transgenic littermates was similar ( approximately 50%). In contrast, less than 1% of the NMU-induced carcinomas in these Ras transgenic rats had an activating ras mutation in their transgenes. These findings highlight the potential of Ras to function as a modifier gene in repressing mammary carcinogenesis.

Animals↗
PubMed1992

Inactivation of the murine N-ras gene by gene targeting.

The mammalian ras genes have been implicated in the genesis of a wide variety of tumors. Although it is likely that they play an essential role in signal transduction, their specific function is still unknown. To initiate a genetic analysis of the ras genes in mammals we inactivated the N-ras gene in murine embryonic stem cells by gene targeting. The frequency of integration at the N-ras locus of our targeting vector being low (of the order of 1/5000 transfectants), we used a positive/negative selection followed by an analysis of individual colonies in order to minimize the in vitro manipulation of the embryonic stem cells. Using this approach, we isolated two clones of ES cells with one inactivated N-ras allele. These cells have no distinctive phenotype either in vitro or in vivo in chimeric mice.

Alleles↗
PubMed2003

Preferential carcinogen-DNA adduct formation at codons 12 and 14 in the human K-ras gene and their possible mechanisms.

In the ras gene superfamily, codon 12 (-TGGTG-) of the K-ras gene is the most frequently mutated codon in human cancers. Recently, we have found that bulky chemical carcinogens preferentially form DNA adducts at codons 12 and 14 (-CGTAG-) in the K-ras gene in normal human bronchial epithelial (NHBE) cells. Furthermore, DNA adducts formed at codon 12 of the K-ras gene are poorly repaired compared with those at other codons including codon 14. These results suggest that targeted carcinogen-DNA adduct formation is a major reason for the observed high mutation frequency at codon 12 of the K-ras gene in human cancers. This preferential carcinogen-DNA adduct formation at codons 12 and 14 could result from effects of (1) primary sequences of these codons and their surrounding codons in the K-ras gene, (2) the chromatin structure, and/or (3) epigenetic factors such as C5 cytosine methylation or other DNA modifications at these codons and their surrounding codons. To distinguish these possibilities, we have introduced modifications with benzo[a]pyrene diol epoxide, N-hydroxy-2-aminofluorene, and aflatoxin B1 8,9-epoxide in (1) naked intact genomic DNA isolated from NHBE cells, (2) fragmented genomic DNA digested by restriction enzymes, and (3) in vitro synthesized DNA fragments containing the K-ras gene exon 1 sequence with or without methylation of the cytosines at CpG sites and the cytosines pairing with the guanines of codons 12 and 14. The distribution of carcinogen-DNA adducts in the K-ras gene was mapped at the nucleotide sequence level using the UvrABC nuclease incision method with or without the ligation-mediated polymerase chain reaction technique. We have found that carcinogens preferentially form adducts at codons 12 and 14 in the K-ras gene exon 1 in intact as well as in fragmented genomic DNA. In contrast, this preferential DNA adduct formation at codons 12 and 14 was not observed in PCR-amplified DNA fragments containing the K-ras gene exon 1 sequence. Methylation of the cytosine at the CpG site of codon 14, or the cytosine pairing with guanine of codon 14, greatly enhanced carcinogen-DNA adduct formation at codon 14 but did not affect carcinogen-DNA adduct formation at codon 12. Methylation of the cytosine pairing with the guanine of codon 12 also did not enhance carcinogen-DNA adduct formation at codon 12. Furthermore, we found that the cytosine at the CpG site of codon 14 is highly methylated in NHBE cells. These results suggest that cytosine methylation at the CpG site is the major reason for the preferential DNA damage at codon 14 and that epigenetic modification(s) other than cytosine methylation may contribute to the preferential DNA damage at codon 12 of the K-ras gene.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1993

Investigation of the mechanism underlying the inhibitory effect of heterologous ras genes in plant cells.

The ras genes from yeast and mammalian cells were fused to plant expression promoters, and introduced into plant cells via Agrobacterium, to study their effect on cell growth and development. All introduced ras genes had a strong inhibitory effect on callus and shoot regeneration from plant tissues. This is consistent with earlier findings that heterologous ras genes were highly lethal to protoplasts following direct DNA uptake. These effects could not be reversed by increasing exogenous or endogenous cytokinin levels. These effects were also independent of the v-Ha-ras mutations in functionally important regions of Ras proteins such as effector-binding and membrane-binding sites. Similarly, co-transformation with the genes encoding the Ras-negative regulators, GTPase-activating protein and neurofibromin did not affect the ras inhibitory effect, indicating that the mechanism of ras inhibition of plant cells is not related to normal ras cellular functions. This conclusion was supported by further studies in which ras gene expression was modified using various promoters and antisense constructs. The introduced ras sequences remained fully inhibitory regardless of which promoters (inducible or tissue-specific) or which orientations (sense or antisense) were tested. This strongly suggests that the ras DNA sequence itself, rather than the Ras protein or ras mRNA, is directly involved in the inhibitory effect. The mechanism underlying this novel phenomenon remains unknown. Introduced ras genes may inhibit plant cell growth by inducing co-suppression of unknown endogenous ras or ras-related genes, thereby leading to the arrest of cell growth.

Animals↗
PubMed2002

Frequent polymorphism of peroxisome proliferator activated receptor gamma gene in colorectal cancer containing wild-type K-ras gene.

We analyzed the K-ras gene mutation and the polymorphism of peroxisome proliferator activated receptor gamma (PPARgamma) gene, which is known as a master gene for adipocyte differentiation and a tumor suppressor gene, in patients with colorectal cancer by polymerase chain reaction-restriction fragment length polymorphism. We detected a mutation of K-ras gene at codon 12 in 8 of 39 colorectal cancers. We also detected a polymorphism of PPARgamma gene at codon 12 in 10 of 39 patients with colorectal cancer. Homozygous polymorphism (Ala12Ala) in PPARgamma gene was found only in one patient, but heterozygous polymorphism (Pro12Ala) was found in 9 of 29 patients. Surprisingly, all 10 colorectal cancers developed in the patients with polymorphism of PPARgamma gene carrying the wild-type K-ras gene. These findings suggest that PPARgamma gene polymorphism may be implicated with the development of colorectal cancers, in which K-ras gene is not mutated.

Adult↗
PubMed1983

Structure and activation of the human N-ras gene.

The normal human N-ras gene has been cloned. In structure and sequence it closely resembles the human H-ras and K-ras genes. The three genes share regions of nucleotide homology and nucleotide divergence within coding sequences and have a common intron/exon structure, indicating that they have evolved from a similarly spliced ancestral gene. The N-ras gene of SK-N-SH neuroblastoma cells has transforming activity, while the normal N-ras gene does not, the result of a single nucleotide change substituting lysine for glutamine in position 61 of the N-ras gene product. From previous studies we conclude that amino acid substitutions in two distinct regions can activate the transforming potential of ras gene products.

Amino Acid Sequence↗