Comparison of K-ras gene mutations in tumour and sputum DNA of patients with lung cancer

Mutations in the K-ras gene are frequently found in lung tumours and are implicated in the development of lung cancer. In order to investigate the clinical usefulness of these mutations in lung cancer, we applied a sensitive method to compare mutations in codon 12 of the K-ras gene in DNA extracted from lung tumours and the matched sputum samples obtained from 22 lung cancer patients. K-ras mutations were identified in the lung tumours of 12 patients (54.5%) and in the sputum samples of 10 patients (45.5%). Nine patients showed an identical mutation in both the tumour and the matched sputum samples. There was a significant association between the presence of a K-ras mutation in a lung tumour and the detection of an identical mutation in the matched sputum sample of the lung cancer patient (κ = 0.64, 95% confidence interval 0.32-0.95, p <0.01). K-ras mutations were detected in sputum samples from cancer patients with all lung tumour grades, and both in the presence and the absence of lymph node metastasis. Therefore, K-ras mutations may provide useful diagnostic markers for lung cancer.     

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.     

A p53 mutation is required for stable transformation of REF52 cells by themyc andras oncogenes

It is known that neoplastic transformation of rodent primary embryonic fibroblasts culturedin vitro requires coexpression at least of two cooperating oncogenes. In the case of transduction into cells of oncogenesras andmyc, the cell transformation is poorly effective. To study some additional factors necessary for such transformation, c-myc and N-ras ^Asp12 were consecutively introduced into REF52 cells by retroviral infection, and the cell cultures obtained were analyzed. Expression ofmyc broke the regulation of the cell cycle, in particular, canceled the G1 phase arrest for cells with damaged DNA, despite the normal function of protein p53 and induction of the p53-responsive genep21 ^Waf1 in these cells. The subsequent transduction ofras led to morphological transformation of cells and an increase of p53 level. However, reversion of the transformed phenotype to normal morphology took place after less than five passages. On this background, rare clones generated the stable transformed cell lines characterized by accelerated proliferation and having a mutation in thep53 gene. Attempts to obtain stable transformed cell lines by transduction ofras into REF52 cells not expressing exogenousmyc were unsuccessful. Analysis of the stable transformed clones revealed a mutation at codon 271 of thep53 gene, a hot spot of mutations, which led to the replacement of arginine by cysteine. In these clones, p53 is accumulated owing to the increased life time, and has a flexible conformation, being able to interact with monoclonal PAb1620 and PAb240 antibodies recognizing alternative protein conformations. The results obtained suggest that p53 participates in negative regulation of the cell cycle under conditions of oncogenic stimulation, and its inactivation is necessary for full transformation of cells by cooperating oncogenesmyc andras.     

Isolation and characterization of a novel bladder cancer cell line: Inhibition by epidermal growth factor

Summary A novel continuous cell line, designated BC3c, was established from a surgical biopsy of an invasive solid transitional cell carcinoma of the bladder derived from an 82-yr-old Caucasian female. BC3c cells were near-triploid bearing multiple structural and numerical chromosome anomalies. The epithelial origin of the cancer cells was indicated by the expression of cytokeratins 8 and 19 as well as by the absence of mesenchymal markers. Polymerase chain reaction-restriction-fragment length polymorphisms and single-strand conformation polymorphism mutation detection assays did not reveal any mutations in H-ras codon 12 and K-ras codons 12 and 13. In addition, no mutation in specific hot-spot codons of the p53 gene and no accumulation of the p53 protein were observed. BC3c cells grew rapidly in vitro, even in the absence of exogenous growth factors, because they were found to stimulate their growth in an autocrine manner. BC3c cells were found to express the epidermal growth factor-receptor (EGF-r) abundantly, but in contrast to other established bladder cancer cell lines, human recombinant epidermal growth factor inhibited the cells’ proliferation in vitro. These features render the newly established bladder cancer cell line BC3c a useful tool for further experimentation.     

Characterization of mutations and loss of heterozygosity of p53 and K-<Emphasis Type="Italic">ras</Emphasis>2 in pancreatic cancer cell lines by immobilized polymerase chain reaction

Background  

The identification of known mutations in a cell population is important for clinical applications and basic cancer research. In this work an immobilized form of the polymerase chain reaction, referred to as polony technology, was used to detect mutations as well as gene deletions, resulting in loss of heterozygosity (LOH), in cancer cell lines. Specifically, the mutational hotspots in p53, namely codons 175, 245, 248, 249, 273, and 282, and K-ras2, codons 12, 13 and 61, were genotyped in the pancreatic cell line, Panc-1. In addition LOH analysis was also performed for these same two genes in Panc-1 by quantifying the relative gene copy number of p53 and K-ras2.     

Ribozyme as an approach for growth suppression of human pancreatic cancer

Ribozymes (catalytic RNAs, RNA enzymes) are effective modulators of gene expression because of their simple structure, site-specific cleavage activity, and catalytic potential, and have potentially important implications for cancer gene therapy. Point mutations in the K-ras oncogene are found in approx 90% of human pancreatic carcinomas, and can be used as potential targets for specific ribozyme-mediated reversal of the malignant phenotype. In this study, we focused on in vitro manipulation of ribozyme targeting of the mutated K-ras oncogene in a human pancreatic carcinoma cell line. We evaluated the efficacy of an anti-K-ras hammerhead ribozyme targeted against GUU-mutated codon 12 of the K-ras gene in cultured pancreatic carcinoma cell lines. The anti-K-ras ribozyme significantly reduced cellular K-ras mRNA level (GUU-mutated codon 12) when the ribozyme was transfected into the Capan-1 pancreatic carcinoma cells. The ribozyme inhibited proliferation of the transfected Capan-1 cells. These results suggested that this ribozyme is capable of reversing the malignant phenotype in human pancreatic carcinoma cells.     

K-ras Gene Mutation Related to Histological Atypias in Human Colorectal Adenomas

To investigate the relationship of oncogene analysis to morphology, we analyzed K-ras gene mutations by dot-blot hybridization with and without consideration of histological atypias in individual colorectal adenomas. Each of 54 colon polyps were divided into two parts after fixation. One part was used as a mass to assess point mutations; the remaining portion of each polyp was paraffin-embedded, stained with hematoxylin and eosin, and examined for point mutations related to histological atypias. In the first part of our study, K-ras gene mutations at codon 12 were detected in 13 cases (24%). In the second part of our study, 12 cases had distinctly different histological atypias. From each of these 12 cases, two areas, one with higher or one with lower grade atypia in the same polyp were excised to analyze for K-ras gene mutation. Two of these 12 cases (17%) had the mutation in different areas of the same tumor. These two cases contained the mutation only in the areas with higher grade atypia, and only one case added information regarding ras mutation upon microdissection when compared to the entire biopsy. These results suggest that oligonucleotide hybridization can identify the majority of cases containing ras mutations despite regional morphologic variation. Individual cases, however, may contain clonal subpopulations within adenomas with different ras sequences from other regions within the same adenoma.     

Positive and negative modulation of H-ras transforming potential by mutations of phenylalanine-28

Conserved amino-acids of H-ras from residues 25 to 34 were mutated in human H-ras cDNA with a pre-existing valine-12 activating mutation ([V¹²]p21), and built into SV40-driven expression vectors. The influence of the introduced mutations was initially screened by transfection of Rat-1 cells to score foci of transformed cells. Nonconservative mutations of amino-acids 25 (tryptophan for glutamine), 27 (asparagine for histidine) and 34 (alanine for proline) did not abrogate the transforming potential of [V¹²]p21. The conservative mutation of phenylalanine-28 to tryptophan ([V¹²W²⁸]p21) was also still transforming. Significantly, in the absence of the valine-12 activating mutation, tryptophan-28-ras ([W²⁸]p21) was weakly transforming while, in contrast, [V¹²D²⁸]p21 was unable to transform Rat-1 cells and retarded cell growth. Analysis of the binding and dissociation of GTP and GDP to normal and mutated p21 expressed in Escherichia coli showed that [V¹²D²⁸]p21 and [D²⁸]p21 do not bind GTP. The dissociation rate of both GTP and GDP bound to [W²⁸]p21 is increased, suggesting a mechanism for its transforming potential in Rat-1 cells. These studies illustrate the importance of phenylalanine-28 in guanine nucleotide binding by p21 ^h-ras . The mutations described could be valuable tools in investigations of cellular signal transduction involving small GTP-binding proteins.     

Generation of ribozyme-adenoviral vector against K-ras mutant human lung cancer cells

ras mutations represent one of the most common oncogenetic lesions in human non-small cell lung cancer (NSCLC) and adversely affect the survival of patients afflicted with this disease. ras-directed gene therapy in the past employed primarily antisense oligonucleotides (AS-ODN) or expression vectors (such as a viral vector construct) that deliver the antisense sequence to inactivate the mutant oncogene message. These approaches produced minimal toxicity, and yet were limited in efficacy. Ribozymes present a viable alternative in antisense therapy by virtue of their renewable catalytic capability for site-specific RNA cleavage. We recently produced an adenoviral vector with a hammerhead ribozyme transgene (KRbz) that is specific for the K-ras codon 12 mutant sequence GUU, given the considerations that (a) in the United States, approx 30% of human NSCLCs express K-ras oncogene mutations, nearly all of which reside in codon 12; (b) anti-K-ras, anti-H, as well as anti-N-ras hammerhead ribozymes are potent growth inhibitors in various human cancers tested; and (c) in vitro and animal model studies suggest that ribozymes directed at oncogene (K- and H-ras C-fos, BCR-ABL) or human immunodeficiency viral gene messages are more effective than their antisense counterpart. This article describes the techniques involved in the production of the KRbz-adenoviral vector that is specific for the K-ras mutation GTT, and summarizes its in vivo antitumor effect against NSCLC xenografts expressing the relevant K-ras mutation in athymic mice.     

A radioimmunocytological quantitative method for the rapid detection of ras oncogene p21 protein in mammalian cells

In order to provide a sensitive and quantitative detection method of ras p21 at the cytological level, the monoclonal antibody Y 13 259 and iodinated protein A were used to locate theras protein in various mammalian cell lines. The subsequent autoradiograph can be analysed by a computer-assisted system which showed in these reported experiments that the relative levels of p21 detected in these cells corresponded to results obtained earlier using conventional biochemical methods.To whom correspondence should be addressed.     

Mitochondrial DNA mutations in respiratory complex-I in never-smoker lung cancer patients contribute to lung cancer progression and associated with EGFR gene mutation

Mitochondrial DNA (mtDNA) mutations were reported in different cancers. However, the nature and role of mtDNA mutation in never‐smoker lung cancer patients including patients with epidermal growth factor receptor (EGFR) and KRAS gene mutation are unknown. In the present study, we sequenced entire mitochondrial genome (16.5 kb) in matched normal and tumors obtained from 30 never‐smoker and 30 current‐smoker lung cancer patients, and determined the mtDNA content. All the patients' samples were sequenced for KRAS (exon 2) and EGFR (exon 19 and 21) gene mutation. The impact of forced overexpression of a respiratory complex‐I gene mutation was evaluated in a lung cancer cell line. We observed significantly higher (P = 0.006) mtDNA mutation in the never‐smokers compared to the current‐smoker lung cancer patients. MtDNA mutation was significantly higher (P = 0.026) in the never‐smoker Asian compared to the current‐smoker Caucasian patients' population. MtDNA mutation was significantly (P = 0.007) associated with EGFR gene mutation in the never‐smoker patients. We also observed a significant increase (P = 0.037) in mtDNA content among the never‐smoker lung cancer patients. The majority of the coding mtDNA mutations targeted respiratory complex‐I and forced overexpression of one of these mutations resulted in increased in vitro proliferation, invasion, and superoxide production in lung cancer cells. We observed a higher prevalence and new relationship between mtDNA alterations among never‐smoker lung cancer patients and EGFR gene mutation. Moreover, a representative mutation produced strong growth effects after forced overexpression in lung cancer cells. Signature mtDNA mutations provide a basis to develop novel biomarkers and therapeutic strategies for never‐smoker lung cancer patients. J. Cell. Physiol. 227: 2451–2460, 2012. © 2011 Wiley Periodicals, Inc.     

基于微流控芯片的温度梯度毛细管电泳检测大肠癌石蜡标本中K-ras基因突变

K-ras基因突变检测可用于大肠癌的早期筛查与诊断,并有利于筛选出抗表皮生长因子受体靶向药物治疗有效的大肠癌患者,以实现肿瘤的个体化治疗．采用以倾斜式热辐射原理建立的微流控温度梯度毛细管电泳(temperature gradient capillary electrophoresis,TGCE)基因突变检测系统,实现了对98例石蜡包埋大肠癌组织中K-ras基因突变的高灵敏度筛查,突变阳性检出率为47.96%,显著高于PCR产物直接测序的23.47%．克隆测序显示该方法至少能检测到2.08%的K-ras基因突变体．K-ras基因突变与临床病理学参数的关系分析显示,直肠癌中K-ras基因突变率明显高于结肠癌(P < 0.05),而与年龄、性别、组织学类型和肿瘤分期等无显著相关性．该检测方法为肿瘤早期诊断和指导临床用药提供了一种灵敏度高、检测速度快、便于大规模筛查的有效手段.     

Ineffectiveness of the presence of H-ras/p53 combination of mutations in squamous cell carcinoma cells to induce a conversion of a nontumorigenic to a tumorigenic phenotype

Human tumor cells have properties in vitro or in surrogate hosts that are distinct from those of normal cells, such as immortality, anchorage independence, and tumor formation in nude mice. However, different cells from individual tumors may exhibit some, but not all of these features. In previous years, human tumor cell lines derived from different tumor and tissue types have been studied to determine those molecular changes that are associated with the in vitro properties listed above and with tumorigenicity in nude mice. In the present study, seven cell lines derived from human tumors were characterized for p53 and ras mutations that may occur in SCC tumor phenotypes and for tumor formation in nude mice. This investigation was designed to examine whether co-occurrence of mutated ras and p53 lead to a malignant stage in the progression process. None of the seven cell lines contained mutations in the recognized "hot spots" of the p53 tumor suppressor gene, but four had a nonsense/splice mutation in codon 126 and a mutation in codon 12 of the H-ras gene. The remaining three cell lines had p53 mutations in intron 5, in codon 193, and a missense mutation in codon 126, respectively. Four of seven cell lines were nontumorigenic; two of these cell lines contained a nonsense p53-126 mutation and mutated ras; one had a missense mutation at codon 126 but no mutated ras; the the fourth had only a p53 mutation at codon 193. Two of the nontumorigenic cell lines were converted to tumorigenicity after treatment with methyl methanesulfonate or N-methyl-N-nitro-N-nitrosoguanidine with no apparent additional mutations in either gene. Our analysis revealed that there was a high frequency of genetic diversity and mutations in both p53 and H-ras. There was also a lack of a causal relationship in the presence of mutations in p53 and the cells ability to exhibit a malignant potential in nude mice.     

Establishment and characterization of four human pancreatic carcinoma cell lines. Genetic alterations in the TGFBR2 gene but not in the MADH4 gene

We characterized four pancreatic carcinoma cell lines (designated SNU-213, SNU-324, SNU-410, and SNU-494) established from histopathologically varied primary or liver metastatic tumor samples of Korean patients. Three cell lines grew as adherent monolayers and one as adherent and floating cell clumps. All lines had: (1) relatively high viability; (2) an absence of mycoplasma or bacterial contamination; (3) genetic heterogeneity as assessed by DNA-fingerprinting analysis; (4) an absence of MADH4 mutation. Among the lines, three lines had mutations in codon 12 in K- ras, two lines harbored p53 mutations within the DNA-binding domain; two lines had homozygous deletions in both p16 and p15 genes; and one line had a missense mutation. Two lines (SNU-324 and SNU-410) had genetic alterations in the TGFBR2 gene: the SNU-324 line had a -1-bp or +1-bp mutation in 10-bp polydeoxyadenine repeat tracts; the SNU-410 line had a genomic deletion in this gene. Mutation analysis of mismatch repair genes demonstrated that SNU-324 has two heterozygous missense mutations in different exons of the hMLH1 gene. In addition, this line showed microsatellite instability and harbored frameshift mutations in simple repeated sequences of the coding regions of the TGFBR2, BAX, and hMSH3 genes. These defects of microsatellite instability and mismatch repair genes suggest the possibility of a new mutator phenotype for pancreatic carcinogenesis. These cell lines should be very useful for studying the biology of pancreatic carcinoma, particularly those related to mutator phenotype and genetic alterations in the TGFBR2 gene.     

A murine CDC25/ras-GRF-related protein implicated in ras regulation

A partial cDNA encoding a novel putative p2, ras guanine nucleotide release-inducing factor (GRF), GRF2, was amplified from murine embryonic stem cells. The presumptive catalytic region of GRF2 is related to the yeast Ras GRF encoded by CDC25. GRF2 is 80% identical to murine CDC25Mm/ras-GRF, but is more similar to yeast CDC25 than to other ras GRFs related to the Drosophila son of sevenless gene product. A 9-kb GRF2 messenger RNA was highly expressed in brain, but GRF2-specific antibodies recognized apparent GRF2 proteins in various mouse tissues in addition to brain. Thus GRF2 represents a novel widely-expressed protein that is highly related to CDC25Mm/ras-GRF, at least in its catalytic domain. Both GRF2 and CDC25Mm/ras-GRF are expressed in murine embryonic stem cells, suggesting that different Ras activators may regulate ras-dependent proliferation and differentiation in early mouse development. © 1993Wiley-Liss, Inc.     

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.     

Characterization of the cell of origin for small cell lung cancer

Small cell lung carcinoma (SCLC) is a neuroendocrine subtype of lung cancer that affects more than 200,000 people worldwide every year with a very high mortality rate. Here, we used a mouse genetics approach to characterize the cell of origin for SCLC; in this mouse model, tumors are initiated by the deletion of the Rb and p53 tumor suppressor genes in the lung epithelium of adult mice. We found that mouse SCLCs often arise in the lung epithelium, where neuroendocrine cells are located, and that the majority of early lesions were composed of proliferating neuroendocrine cells. In addition, mice in which Rb and p53 are deleted in a variety of non-neuroendocrine lung epithelial cells did not develop SCLC. These data indicate that SCLC likely arises from neuroendocrine cells in the lung.     

ralGDS family members interact with the effector loop of ras p21.

Using a yeast two-hybrid system, we identified a novel protein which interacts with ras p21. This protein shares 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24. We designated this protein RGL, for ralGDS-like. Using the yeast two-hybrid system, we found that an effector loop mutant of ras p21 was defective in interacting with the ras p21-interacting domain of RGL, suggesting that this domain binds to ras p21 through the effector loop of ras p21. Since ralGDS contained a region highly homologous with the ras p21-interacting domain of RGL, we examined whether ralGDS could interact with ras p21. In the yeast two-hybrid system, ralGDS failed to interact with an effector loop mutant of ras p21. In insect cells, ralGDS made a complex with v-ras p21 but not with a dominant negative mutant of ras p21. ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro. ralGDS inhibited both the GTPase-activating activity of the neurofibromatosis gene product (NF1) for ras p21 and the interaction of Raf with ras p21 in vitro. These results demonstrate that ralGDS specifically interacts with the active form of ras p21 and that ralGDS can compete with NF1 and Raf for binding to the effector loop of ras p21. Therefore, ralGDS family members may be effector proteins of ras p21 or may inhibit interactions between ras p21 and its effectors.     

Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function.

Although p21ras is localized to the plasma membrane, proteins it interacts with, such as the GTPase-activating proteins (GAPs) ras GAP and neurofibromin (NF1), are not, suggesting that one function of p21ras GTP may be to target such proteins to the plasma membrane. To investigate the effects of targeting ras GAP to the plasma membrane, ras C-terminal motifs sufficient for plasma membrane localization of p21ras were cloned onto the C terminus of ras GAP. Plasma membrane-targeted ras GAP is growth inhibitory to NIH 3T3 fibroblasts and COS cells. This growth inhibition correlates with GAP catalytic activity, since the plasma membrane-targeted C-terminal catalytic domain or the GAP-related domain of neurofibromin is inhibitory, whereas the similarly targeted N-terminal domain is not. Moreover, the inhibition is abrogated by the inactivating mutation L902I, which abolishes ras GAP catalytic activity. Coexpression of oncogenic mutant ras rescues cell viability, but the majority of rescued colonies are phenotypically untransformed. Furthermore, in focus assays, targeted ras GAP suppresses transformation by oncogenic mutant ras, and in reversion assays, targeted ras GAP can revert cells transformed by oncogenic mutant ras. Neither the targeted or nontargeted N-terminal domain nor the L902I mutant of ras GAP has any transforming activity. These data demonstrate that ras GAP can function as a negative regulator of ras and that plasma membrane localization potentiates this activity. However, if ras GAP is involved in the effector functions of p21ras, it can only be part of the effector complex for cell transformation.     

Development of a simple and sensitive technique for detection of point mutations in the K-ras oncogene

We sought to develop a simple and sensitive method based on mutant allele-specific amplification (MASA) for the detection of point mutations in the k-ras oncogene in blood samples. We used MASA and three nested MASA methods to detect a point mutation (GGT→GAT) in rat DHD cells at codon 12 of exon 1 of the k-ras gene. MASA allowed us to detect one k-ras mutated cell on a background of 10⁷ normal cells. The third nested-MASA (nested-MASA.c) method that we developed allowed us to detect one mutated cell among 10¹⁰ normal cells. Our methods should allow the detection of small amounts of mutant k-ras DNA in tissue, serum, and plasma, combining speed with efficiency and specificity.