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.     

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.     

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.     

Overexpression of N-Myc Downstream-Regulated Gene 2 (NDRG2) Regulates the Proliferation and Invasion of Bladder Cancer Cells In Vitro and In Vivo

N-Myc downstream-regulated gene 2 (NDRG2) is a candidate tumor suppressor gene, which plays an important role in controlling tumor growth. The aim of this study was to investigate the expression of NDRG2 gene in bladder cancer (BC) tissues and several bladder cancer cell lines, and to seek its clinical and pathological significance. Ninety-seven bladder carcinoma and 15 normal bladder tissue sections were analyzed retrospectively with immunohistochemistry. The human bladder cancer cell line T24 was infected with LEN-NDRG2 or LEN-LacZ. The effects of NDRG2 overexpression on T24 cells and T24 nude mouse xenografts were measured via cell growth curves, tumor growth curves, flow cytometric analysis, western blot and Transwell assay. NDRG2 was highly expressed in normal bladder tissue, but absent or rarely expressed in cacinomatous tissues (χ²=8.761, p < 0.01). The NDRG2 level was negatively correlated with tumor grade and pathologic stage(r=-0.248, p < 0.05), as well as increased c-myc level (r=-0.454, p< 0.001). The expression of NDRG2 was low in the three BC cell lines. T24 cells infected with LEN-NDRG2 showed inhibition of proliferation both in vitro and in vivo, and NDRG2 overexpression can inhibit tumor growth and invasion in vitro.     

Significant Differences in the Development of Acquired Resistance to the MDM2 Inhibitor SAR405838 between In Vitro and In Vivo Drug Treatment

SAR405838 is a potent and specific MDM2 inhibitor currently being evaluated in Phase I clinical trials for the treatment of human cancer. Using the SJSA-1 osteosarcoma cell line which harbors an amplified MDM2 gene and wild-type p53, we have investigated the acquired resistance mechanisms both in vitro and in vivo to SAR405838. Treatment of SJSA-1 cells with SAR405838 in vitro leads to dose-dependent cell growth inhibition, cell cycle arrest and robust apoptosis. However, prolonged treatment of SJSA-1 cells in vitro with SAR405838 results in profound acquired resistance to the drug. Analysis of in vitro-derived resistant cell lines showed that p53 is mutated in the DNA binding domain and can no longer be activated by SAR405838. Treatment of the parental SJSA-1 xenograft tumors with SAR405838 in mice yields rapid tumor regression but the tumors eventually regrow. Culturing the regrown tumors established a number of sublines, which showed only modest (3–5 times) loss of sensitivity to SAR405838 in vitro. Sequencing of the p53 showed that it retains its wild-type status in these in vivo sublines, with the exception of one subline, which harbors a single heterozygous C176F p53 mutation. Using xenograft models of two in vivo derived sublines, which has either wild-type p53 or p53 containing a single heterozygous C176F mutation, we showed that while SAR405838 effectively achieves partial tumor regression in these models, it no longer induces complete tumor regression and tumors resume growth once the treatment is stopped. Harvesting and culturing tumors obtained from a prolonged treatment with SAR405838 in mice established additional in vivo sublines, which all contain a single heterozygous C176F mutation with no additional p53 mutation detected. Interestingly, SAR405838 can still effectively activate p53 in all sublines containing a single heterozygous C176F mutation, with a moderately reduced potency as compared to that in the parental cell line. Consistently, SAR405838 is 3–5 times less effective in all the in vivo derived sublines containing a single heterozygous C176F p53 mutation than in the SJSA-1 parental cell line in assays of cell growth and apoptosis. Computational modeling suggested that a p53 tetramer containing two wild-type p53 molecules and two C176F mutated molecules can maintain the structural stability and interactions with DNA by formation of additional hydrophobic and cation-π interactions which compensate for the loss of sulphur-zinc coordination. Our data thus show that SJSA-1 tumor cells acquire very different levels of resistance in vitro and in vivo to the MDM2 inhibitor SAR405838. Our present study may have a significant implication for the investigation of resistant mechanisms for other classes of anticancer drugs.     

Expression of genes related to apoptosis,cell cycle and signaling pathways are independent of <Emphasis Type="Italic">TP53</Emphasis> status in urinary bladder cancer cells

Urinary bladder cancer is the fourth most common malignancy in the Western world. Transitional cell carcinoma (TCC) is the most common subtype, accounting for about 90% of all bladder cancers. The TP53 gene plays an essential role in the regulation of the cell cycle and apoptosis and therefore contributes to cellular transformation and malignancy; however, little is known about the differential gene expression patterns in human tumors that present with the wild-type or mutated TP53 gene. Therefore, because gene profiling can provide new insights into the molecular biology of bladder cancer, the present study aimed to compare the molecular profiles of bladder cancer cell lines with different TP53 alleles, including the wild type (RT4) and two mutants (5637, with mutations in codons 280 and 72; and T24, a TP53 allele encoding an in-frame deletion of tyrosine 126). Unsupervised hierarchical clustering and gene networks were constructed based on data generated by cDNA microarrays using mRNA from the three cell lines. Differentially expressed genes related to the cell cycle, cell division, cell death, and cell proliferation were observed in the three cell lines. However, the cDNA microarray data did not cluster cell lines based on their TP53 allele. The gene profiles of the RT4 cells were more similar to those of T24 than to those of the 5637 cells. While the deregulation of both the cell cycle and the apoptotic pathways was particularly related to TCC, these alterations were not associated with the TP53 status.     

Galectin-3 mRNA level depends on transformation phenotype in ras-transformed NIH 3T3 cells

Summary— The increase in galectin-3 lectin content observed in tumours or in in vitro transformed cells suggests that this lectin is important in the transformation process. In the present study, we investigated the mRNA expression level of the galectin-3, galectin-I and macrophage mannose receptor in normal and ras-transformed NIH 3T3 cells in relation to their transformation state. The galectin3 mRNA content in ras-transformed cells is increased in fully transformed cells, with a maximum in ras-transformed cells that have lost their growth anchorage-dependence. Under the same conditions, the galectin-1 mRNA level which was high in normal cells, increased slightly in transformed cells. The mRNA for the macrophage mannose receptor was not detected in 3T3 cells or in their ras-transformed counterparts.     

The detection of mutations induced in vitro in the human p53 gene by hydrogen peroxide with the restriction site mutation (RSM) assay

We have analysed five mutation hotspots within the p53 gene (codons 175, 213, 248, 249, and 282) for mutations induced by hydrogen peroxide (H₂O₂), employing the restriction site mutation (RSM) assay. In addition, four other restriction sites covering non-hotspot codons of exons 5–9 of the p53 gene (codons 126, 153/54, 189 and the 3′ splice site of exon 9) were analysed by the RSM assay for H₂O₂-induced mutations. Two cell types were concurrently analysed in this study, i.e. primary fibroblast cells and a gastric cancer cell line. Using the RSM assay, H₂O₂-induced mutations were only detected in exon 7 of the p53 gene. This was true for both cell types. These mutations were mainly induced in the Msp I restriction site (codon 247/248) and were predominantly GC to AT transitions (71%). Hence these GC to AT mutations were presumably due to H₂O₂ exposure, possibly implicating the 5OHdC adduct, which is known to induce C to T mutations upon misreplication. Importantly, this study demonstrates that the RSM methodology is capable of detecting rare oxidative mutations within the hotspot codons of the p53 tumour suppressor gene. Hence, this methodology may allow the detection of early p53 mutations in pre-malignant tissues.     

Isolation and characterization of a novel human bladder cancer cell line: BK10

Summary Molecular studies of bladder carcinomas have aided in determining causative genetic events and the prognosis of cancers endowed with certain abnormalities. In vitro bladder cancer characterization of key cytogenetic alterations is useful for study of molecular changes that may promote oncogenic events. In our laboratory, a novel human bladder cancer cell line, BK10, has been established in vitro and passaged for more than 20 mo. This new bladder cancer cell line (BK10) was derived from bladder tissue containing grade III-IV/IV transitional cell carcinoma. Bladder cancer tissue was obtained at the time of radical cystoprostatectomy extirpation. Cell cultures derived from this surgical sample exhibited an epithelial morphology and expressed epithelial cytokeratins. Immunostains of BK10 were negative for prostate specific antigen (PSA), fibronectin, smooth muscle actin alpha, and desmin. Karyotypic analysis revealed an aneuploid chromosomal content 〈4n〉 with many numerical and structural abnormalities previously linked to bladder oncogenesis. Translocations occurred in chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 13, 14, 15, 16, 17, 19, 20, 21, 22, X and Y. G-banding analysis revealed rearrangements involving chromosomes 9q and 17p, and the location of the ab11 oncogene and the p53 gene, respectively. The availability of this bladder cancer cell line will provide a useful too for the further study of bladder carcinoma oncogenesis and gene therapy.     

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.     

Transformation of NIH/3T3 to Anchorage Independence by H-Ras Is Accompanied by Loss of Suppressor Activity

Despite their familiar sensitivity to transformation by dominant-acting ras oncogenes, NIH/3T3 cells carry a ras suppressor. When tested by cell fusion they were able to suppress the anchorage-independent phenotype of both mouse and human cells transformed by activated H-ras or N-ras. This suppression occurred without a decrease in expression of the activated ras oncogene. Ras-transformed NIH/3T3 clones cured of their oncogene by benzamide treatment reverted to a non-transformed phenotype, but had lost the ability to suppress other ras transformants, indicating that their initial transformation was accompanied by suppressor loss. In hamster cells an active ras oncogene increased the rate of chromosome segregation by >100-fold. These results suggest that in vitro transformation of NIH/3T3 cells by ras may be more similar to multistep in vivo tumor development than previously suspected, involving not only expression of an active oncogene but also loss of a suppressor activity, perhaps induced by the clastogenic oncogene.     

A cell culture model of chemically and spontaneously derived mouse lung alveologenic carcinoma

Malignant cell lines related to mouse lung alveologenic carcinoma have been established from urethane-induced tumors and after in vitro spontaneous transformation of preneoplastic cell lines. Both the chemically and spontaneously transformed cell lines formed invasive, poorly differentiated carcinomas with secondary lung deposits when implanted subcutaneously in immune-suppressed mice. They differed from the related preneoplastic cell line in coordinately exhibiting anchorage-independent growth, reduced epidermal growth factor receptor activity and absence of pericellular fibronectin. These data suggest that similar molecular events may occur in type 2 pneumocyte-related cells in order to generate mouse lung alveologenic adenomas and carcinomas by both spontaneous and chemical carcinogen induction mechanisms. A reduced level of pericellular fibronectin was also demonstrated in an in situ compressive urethane-induced mouse lung adenoma. Loss of pericellular fibronectin may therefore be an early and persistent phenotypic alteration during transformation to the alveologenic adenoma and carcinoma.Abbreviations EGF epidermal growth factor - TGF transforming growth factor     

Effect of growth factors and antitumor drugs on normal and Vall2Ha-ras transformed C3H 10T1/2 cell transplasma membrane electron transport and growth

Summary Bothras and the transplasma membrane electron transport system have been implicated in the control of cell growth. Since the loss of growth regulation is at the heart of the cancerous phenotype, we have investigated the effect of factors like antitumor drugs, which inhibit cell growth, and growth factors which either cause or prepare the cells for growth. We have found that growth factors increase the plasma membrane electron transport of the normal cells but not that of the cells which have been transfected with oncogenicras (which have a 2–5-fold faster basal rate). In the presence of adriamycin the activity of the electron transport system is and cell numbers are decreased more in normal cells than in the cells which have been transfected with the oncogenic version of the Harveyras gene. Cisplatin inhibits cell growth but does not inhibit ferricyanide reduction. Ferricyanide alone or in conjunction with EGF or TPA does not stimulate cell proliferation with either cell line in the absence of fetal calf serum, so simple activation of plasma membrane electron transport is not sufficient to activate a complete growth response.Abbreviations EGF epidermal growth factor - TPA phorbol myristate acetate - PDGF platelet derived growth factor - MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromides - SDS sodium dodecyl sulfate     

Genetic analysis of the catalytic domain of the GAP gene in human lung cancer cell lines

Cell lines of non-small cell lung cancer (nonSCLC) have been shown to contain activating mutation of the K-ras oncogene in about 30% of cases, whereas no small cell lung cancer (SCLC) cell lines displayed these mutations. Biochemically, these mutations result in the ras gene product (p21) being constitutively activated in its GTP-bound form and insensitive to the hydrolytic action of the ras-specific GTPase-activating protein (ras GAP). We hypothesized that, if tumor development is related to the p21 ras being in the active GTP-bound state, then a similar malignant phenotype may result from an inactivating mutation in the ras GAP gene in the region that interacts with ras p21 (so-called catalytic domain). To test this hypothesis, we screened a panel of SCLC and non-SCLC cell lines for major genetic alterations in the catalytic domain of the GAP gene with the Sothern blot technique, and for minor genetic abnormalities (e.g., point mutations) with denaturing gradient gel electrophoresis and single-strand conformation polymorphism. Mutations in the catalytic domain of the GAP gene could not be demonstrated by any technique in any cell line examined. We conclude that mutational inactivation of the catalytic domain of the GAP gene probably does not contribute to the development of lung cancer.     

Rasfonin,a novel 2-pyrone derivative,induces ras-mutated Panc-1 pancreatic tumor cell death in nude mice

Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR–Ras–Raf–MEK–ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [³H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC₅₀=5.5 μM) than BxPC-3 cells (IC₅₀=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras–MAPK activity could be important in its anticancer activity.     

c-Ha-rasEJ transfection of rat aortic smooth muscle cells induces epidermal growth factor responsiveness and characteristics of a malignant phenotype

Although the role of several protooncogenes, including sis, myc, and myb in the regulation of growth and differentiation of vascular cells has been examined in some detail, limited information is available on the contribution of ras genes to these processes. In the present studies the influence of oncogenic ras transfection on the phenotypic expression of rat aortic smooth muscle cells (SMCs) was examined. Cultured rat aortic SMCs during early passage (P₄) were transfected by lipofection with c-Ha-ras^EJ in a pSV2 neo vector or with pSV2 neo vector alone. Stable transfectants were selected in G418 over a 6-week period. Oncogene-transfected cells (ras-LF-1) exhibited differences in morphology and growth pattern relative to vector controls (neo-LF-1), or naive SMCs, including the development of prominent processes and the appearance of focal cellular arrangements giving rise to latticelike structures. Southern analysis revealed multiple integration of oncogenic ras in ras LF-1 cells. Transfection of c-Ha-ras^EJ was associated with a twofold increase in p21 levels relative to pSV2 vector controls demonstrating that exogenous ras was expressed in these cells. Overexpression of ras p21 afforded SMCs a lower serum requirement for growth compared to vector controls, anchor-age independent growth on soft agar, and acquisition of epidermal growth factor (EGF) responsiveness. Stimulation of serum-deprived SMCs with 5% fetal bovine serum (FBS) increased steady-state levels of c-Ha-ras mRNA in both ras-LF-1 and neo-LF-1 but ras induction was more pronounced in ras-transfected cells. α-smooth muscle (SM) actin gene expression was markedly reduced in ras-transfected cells relative to vector controls. These results show that transfection of c-Ha-ras^EJ into aortic SMCs induces an altered phenotypic state characterized by alterations in growth factor-related signal transduction and tumorigenic potential. © 1994 Wiley-Liss, Inc.     

Transformation of mouse mammary epithelial cells with the Ha-ras but not with the neu oncogene results in a gene dosage-dependent increase in transforming growth factor-α production

An enhanced expression of transforming growth factor-α (TGFα) was demonstrated in two clones of NOG-8 mouse mammary epithelial cells, NOG-8 SR1 and NOG-8 SR2, that have been transformed by a v-Ha-ras oncogene. The amount of TGFα production in NOG-8 SR1 and NOG-8 SR2 cells was dependent on the level of p21^ras expression in these clones, which directly correlated with their cloning efficiency in soft agar. There was also a decrease in the number of epidermal growth factor (EGF) receptors on the NOG-8 SR1 and NOG-8 SR2 cells that is proportional to the amount of TGFα secreted. These effects were specific for ras because neu-transformed NOG-8 cells grew in soft agar at a comparable level to NOG-8 SR2 cells yet did not show any increase in TGFα production or change in EGF receptor expression.     

In VitroModulation of Implantation and Intraepithelial Expansion of Bladder Tumor Cells by Epidermal Growth Factor

A major problem in the management of bladder cancer is the high risk for recurrence of bladder tumors after transurethral resection. This has generally been attributed to the attachment and subsequent expansion of exfoliated tumor cells to the traumatized bladder wall. Anin vitrococultivation model was used to study the implantation and growth of human tumor cells in traumatized murine urothelium. Furthermore, we investigated in a time-course experiment whether stimulation of the regenerative activity of the normal urothelium by a growth factor could affect implantation and subsequent growth of bladder tumor cells. After inoculation on injured confluent cultures of murine urothelium, human T24 and SD bladder carcinoma cells preferentially attached to the denuded areas. SD cells expanded into the normal urothelium as a sharply demarcated tumor, while T24 cells infiltrated as single cells. Treatment of the primary urothelium with epidermal growth factor (EGF) stimulated the proliferation of the primary urothelium and reduced the implantation and growth of T24 considerably. EGF reduced the implantation of the SD tumor cells but could not prevent the further expansion at the expense of surrounding normal urothelium. Since EGF had no effect on migration or proliferation of SD or T24 cells, its modulation of expansive growth is most probably due to an increase in the regeneration of normal urothelium. This study suggests that recurrence of transitional cell carcinomas might in some instances be inhibited by stimulation of the regeneration of traumatized urothelium. The reportedin vitrococultivation model may be useful for studying additional factors involved in intraepithelial expansion of carcinoma cells.