Inhibition of PC cell-derived growth factor (PCDGF, epithelin/granulin precursor) expression by antisense PCDGF cDNA transfection inhibits tumorigenicity of the human breast carcinoma cell line MDA-MB-468

PC-cell derived growth factor (PCDGF) is an 88-kDa growth factor originally purified from the highly tumorigenic teratoma PC cell line and corresponds to the epithelin/granulin precursor. In teratoma cells, PCDGF expression was shown to be essential for tumorigenicity. We have reported that PCDGF was expressed in estrogen receptor-positive (ER(+)) human mammary epithelial cells in an estrogen-dependent fashion. In this study, we have investigated PCDGF expression in human mammary epithelial cell lines ranging from immortalized nontumorigenic cells to ER(+) and ER(-) breast carcinoma cells. Northern and Western blot analyses indicated that PCDGF mRNA and protein expression was low in nontumorigenic cells and increased in human breast carcinomas cell lines in a positive correlation with their tumorigenicity. Treatment of the ER(-) MDA-MB-468 cells with anti-PCDGF neutralizing antibody resulted in a dose-dependent inhibition of their proliferation, suggesting that secreted PCDGF acted as an autocrine growth factor for breast carcinoma cells. We then examined the in vitro and in vivo growth properties of MDA-MB-468 cells, where PCDGF expression had been inhibited by antisense PCDGF cDNA transfection. Inhibition of PCDGF expression resulted in a reduced proliferation rate in vitro and a 60-80% reduction in colony formation. Tumor formation in vivo was dramatically inhibited in antisense cells with a 90% inhibition of tumor incidence and tumor weight. These results demonstrate the importance of PCDGF overexpression for the proliferation and tumorigenicity of ER(-) breast carcinomas and suggest that PCDGF overexpression may play an important role in human breast cancer.     

HTRA1 gene expression in gastric epithelial cells

Objective:To explore HtrA1 gene expression aud its regulation in human gastric cancers.Methods:The HtrA1 mRNA levels were examined by QPCR analysis and coufirmed its expression with Northern blot analysis.The HtrA1 protein levels in all six gastric epithelial cell lines were investigated by Western blot analysis.Gene copy number was accessed and then sequenced the coding region from each mRNA in all six cell lines.The HtrA1 promoter region DNA methylation status was detected by using bisulfite sequeucing analysis.Effect of decitabine and TSA on HTRA1 expression in gastric cancer cell line was determined by RTPCR.Results:HIC analysis indicated that HtrA1 was highly expressed in normal epithelium,but dramatically down-regulated in gastric carcinoma tissues and variably expressed in tumor-adjacent tissues.HtrA1 gene expression was dramatically decreased in gastric carcinoma cells compared to nontumorigenic counterparts.The HtrA1 gene loss in any of the 4 breast cancer cell lines was not detected.Total 14 CpGs in this region were all methylated in gastric cancer cells,whereas two normal cells.GES-1 and HFI-145,were having several unmethylated cytosines in this region.HtrA1 showed as~Mr 44,000,Expression of HtrA1 protein was not observed in any of the four gastric caucer cell lines.BGC-823.MKN-45.SGC-7901and MKN-28.HtrA1 expression was observed in the HF1-145and GES-1 cell lines.Conclusions:The epigenetic silencing for HtrA1gene expression could provide a possible strategy for re-activating Htrt1 gene expression in gastric cancer cells.thus facilitating further investigation of HtrA1's role in chemotherapy.     

Autocrine human growth hormone promotes tumor angiogenesis in mammary carcinoma

Accumulating literature implicates pathological angiogenesis and lymphangiogenesis as playing key roles in tumor progression. Autocrine human growth hormone (hGH) is a wild-type orthotopically expressed oncogene for the human mammary epithelial cell. Herein we demonstrate that autocrine hGH expression in the human mammary carcinoma cell line MCF-7 stimulated the survival, proliferation, migration, and invasion of a human microvascular endothelial cell line (HMEC-1). Autocrine/paracrine hGH secreted from mammary carcinoma cells also promoted HMEC-1 in vitro tube formation as a consequence of increased vascular endothelial growth factor-A (VEGF-A) expression. Semiquantitative RT-PCR analysis demonstrated that HMEC-1 cells express both hGH and the hGH receptor (hGHR). Functional antagonism of HMEC-1-derived hGH reduced HMEC-1 survival, proliferation, migration/invasion, and tube formation in vitro. Autocrine/paracrine hGH secreted by mammary carcinoma cells increased tumor blood and lymphatic microvessel density in a xenograft model of human mammary carcinoma. Autocrine hGH is therefore a potential master regulator of tumor neovascularization, coordinating two critical processes in mammary neoplastic progression, angiogenesis and lymphangiogenesis. Consideration of hGH antagonism to inhibit angiogenic processes in mammary carcinoma is therefore warranted.     

Purification and characterization of a newly identified growth factor specific for epithelial cells.

A growth factor specific for epithelial cells was identified in conditioned medium of a human embryonic lung fibroblast cell line. The factor, provisionally termed keratinocyte growth factor (KGF) because of its predominant activity on this cell type, was purified to homogeneity by a combination of ultrafiltration, heparin-Sepharose affinity chromatography, and hydrophobic chromatography on a C4 reversed-phase HPLC column. KGF was both acid and heat labile and consisted of a single polypeptide chain of approximately 28 kDa. Purified KGF was a potent mitogen for epithelial cells, capable of stimulating DNA synthesis in quiescent BALB/MK epidermal keratinocytes by greater than 500-fold with activity detectable at 0.1 nM and maximal at 1.0 nM. Lack of mitogenic activity on either fibroblasts or endothelial cells indicated that KGF possessed a target cell specificity distinct from any previously characterized growth factor. Microsequencing revealed an amino-terminal sequence containing no significant homology to any known protein. The release of this growth factor by human embryonic fibroblasts raises the possibility that KGF may play a role in mesenchymal stimulation of normal epithelial cell proliferation.     

Cloning and sequencing of CATR1.3, a human gene associated with tumorigenic conversion.

The human squamous cell carcinoma cell line SCC83-01-82 (SCC) contains mutations in both the H-ras and p53 genes, but it exhibits a nontumorigenic phenotype in nude mice. This cell line can be converted into a cell line with a tumorigenic phenotype, SCC83-01-82CA (CA), by treatment with the mutagen methyl methanesulfonate (MMS). This indicates that additional genetic events leading to expression of a cooperating tumor susceptibility gene(s) may be required for tumorigenicity. To identify the cooperating gene(s), an expression cDNA library was made from tumorigenic Ca cells. The library DNA was transfected into nontumorigenic SCC cells and the transfected SCC cells were then injected into nude mice for the selection of a tumorigenic phenotype. Tumors developed in 3 of the 18 mice after injection. Several new cell lines were established from these transfected cell-induced tumors and designated as CATR cells. Tumor histology and karyotype analysis of these cells indicated that they were of human epithelial cell origin. All the CATR cells have the library vector sequence integrated in their genome. Cell line CATR1 expressed a single message from the integrated library representing a 1.3-kb cDNA insert that was absent from untransfected SCC cells or MMS-converted CA cells. This 1.3-kb cDNA insert was cloned by PCR amplification of reverse-transcribed CATR1 total RNA and was designated CATR1.3. The nucleotide sequence of CATR1.3 encodes a peptide of 79 amino acids, has a long 3' untranslated region, and represents an unknown gene product that was associated with the tumorigenic conversion due to the transfected expression library.     

Granule membrane protein 140 (GMP140) binds to carcinomas and carcinoma-derived cell lines.

The glycoproteins granule membrane protein 140 (GMP140), endothelial-leukocyte adhesion molecule 1 (ELAM-1), and Leu-8 are members of a family of glycoprotein receptors (selectins or LEC-CAMs) that play an important role in adhesive interactions between circulating leukocytes and vascular endothelium. Recently it has been reported that ELAM-1 is able to mediate the binding of the colon carcinoma cell line HT-29 to cytokine-activated vascular endothelium, suggesting that tumor cell adhesion to vascular endothelium, a prerequisite for tumor extravasation and metastasis, is in part the result of adhesive interactions between blood-borne tumor cells and cell surface proteins expressed by vascular endothelium. Here, using an approach in which soluble immunoglobulin chimeras of the GMP140 and ELAM-1 receptors were prepared and used to carry out immunohistological studies, we establish that GMP140 binds to tumor cells in a variety of human carcinoma tissue sections (colon, lung, and breast), whereas ELAM-1 binds exclusively to tumor cells in colon carcinoma tissue sections. In addition, GMP140 was found to bind to the cell surface of a number of cell lines derived from various carcinomas but not from melanomas, whereas ELAM-1 bound only colon carcinoma cell lines. We further investigated the nature of the ligands of GMP140 and ELAM-1 on the surface of the carcinoma cells and found that the GMP140 ligand on the surface of tumor cells appears to be distinct from that expressed on the myeloid cell line HL-60. Neuraminidase treatment of a breast carcinoma cell line does not affect, or in some instances increases, GMP140 binding, whereas it completely abolishes GMP140 binding to HL-60 cells. On the other hand, the ligand of ELAM-1 on both the colon carcinoma and HL-60 cells is neuraminidase sensitive in accord with its identification as sialyl-CD15. Parallel results were obtained with neuraminidase-treated frozen carcinoma tissue sections. The present findings form the basis for investigating the role of GMP140 in tumor invasiveness and metastasis.     

Production of platelet-derived endothelial cell growth factor by normal and transformed human cells in culture.

Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45-kDa endothelial cell mitogen which has angiogenic properties in vivo. We report here that human foreskin fibroblasts, a human squamous cell carcinoma cell line, and 2 out of the 3 human thyroid carcinoma cell lines investigated produce PD-ECGF, whereas 21 other cell lines examined do not. The positive cell lines contained a 1.8-kilobase PD-ECGF mRNA, and a 45-kDa protein could be demonstrated in lysates of the cell lines by immunoblotting and immunoprecipitation using a specific antiserum against PD-ECGF. Furthermore, the cell lysates contained mitogenic activity for endothelial cells that was neutralized by the PD-ECGF antiserum. PD-ECGF was found to be secreted only slowly from the producer cells, consistent with the previous finding that the primary translation product lacks a signal sequence. The restricted expression and intracellular sequestration of PD-ECGF imply a strictly controlled function in endothelial cell proliferation and angiogenesis. Aberrant production of PD-ECGF may play a role in tumor angiogenesis.     

Angiogenic role for glycodelin in tumorigenesis

Angiogenesis plays an important role in neovascularization in tumors. Glycodelin, a hormone-responsive protein, has been detected in tumors of reproductive organs and is found in high levels in the plasma of subjects with gynecological malignancies. Glycodelin is also found in the endothelial cells of the umbilical cord and in the blood vessels of tumors. In this study, we tested whether glycodelin-rich amniotic fluid and a synthetic peptide derived from the sequence of glycodelin peptide (Gp) might promote angiogenic response by examining the migration and tube formation in human umbilical cord vein endothelial cells (HUVECs). Increased migration and tube formation of HUVECs were found in the presence of amniotic fluid and Gp, and this increase was blocked by antibody to Gp and by an anti-vascular endothelial growth factor (VEGF) antibody, suggesting that the angiogenic effects of glycodelin might be mediated by VEGF. The results also showed that Gp significantly increased the release of VEGF protein and mRNA expression in HUVECs, RL-95 (human endometrial carcinoma cells), OVCAR-3 (human ovarian adenocarcinoma cells), EM42 (human endometrial epithelial cells), THP-1 (human monocyte), and MCF-7 and MDA-MB-231 (human breast adenocarcinoma cells) cell lines. VEGF receptor Fit-1 mRNA expression in HUVECs was also increased in the presence of Gp. These findings, together with the suggestion from the literature that glycodelin may have immunosuppressive properties, suggest that glycodelin might play an important role in neovascularization during embryogenesis and tumor development.     

Comparative analysis of a human pancreatic undifferentiated cell line (MIA PaCa-2) to acinar and ductal cells

Pancreatic ductal cell secretion has not been well characterized due to the difficulty in obtaining sufficient quantities of purified ductal cells. To determine if the MIA PaCa-2 cell line would provide a useful model for in vitro studies of pancreatic ductal cell secretion, the present study was designed to characterize these cells in greater detail. In this investigation, the human pancreatic undifferentiated cell line, MIA PaCa-2, was compared with PANC-1 cells (a human ductal cell line previously characterized), isolated rat and human ducts, acinar cells, and nonpancreatic cell lines. The results indicate that while the morphology of the MIA PaCa-2 cell line is nonpolarized and generally atypical of either ductal or acinar cells, the cell line has retained certain biochemical similarities to ductal cells. Additional morphological studies indicated (a) the presence of intermediate filaments characteristic of epithelial cells, (b) the absence of zymogen granules, and (c) an apparent basolateral plasma membrane localization of Na+, K+-ATPase. Similar to ductal cells, biochemical analyses indicated (a) the presence of Na+, K+-ATPase based on [3H]-ouabain binding assays, (b) high levels of carbonic anhydrase, (c) low levels of gamma-glutamyl transpeptidase, (d) nondetectable levels of amylase, and (e) protein composition and protein synthetic patterns comparable to PANC-1 cells. Finally, as with PANC-1 cells and isolated rat and human ducts, the major sulfated secretory product of MIA PaCa-2 cells was a protein with a molecular weight of approximately 660,000 to 1 million.(ABSTRACT TRUNCATED AT 250 WORDS)     

hnRNPA1在人胃癌细胞株BGC-823中的生物学功能

核不均一核糖核蛋白A1（hnRNPAl）是体内重要的RNA结合蛋白,通过调节pre-mRNA和mRNA参与转录和转录后调控过程,与肿瘤发生、发展密切相关。目前对hnRNPAl与胃癌的关系尚不十分清楚。目的：探讨hnRNPAl在人胃癌细胞中的生物学功能,明确其与胃癌的关系。方法：以蛋白质印迹法检测正常人胃黏膜上皮细胞株GES一1和人胃癌细胞株MKN-28、SGC-7901、BGC-823中的hnRNPAl表达。构建靶向hnRNPAl基因的siRNA重组质粒并稳定转染BGC一823细胞,以稳转空质粒pU6的细胞株作为对照,蛋白质印迹法验证干扰效果。分别以CCK一8实验、细胞划痕实验、Transwell侵袭实验和流式细胞术分析各组BGC-823细胞的增殖、迁移、侵袭能力和细胞凋亡情况。结果：hnRNPA1在3株人胃癌细胞株中均呈高表达,BGC-823细胞表达水平最高。与pU6稳转组相比,siRNAhnRNPAl稳转组BGC．823细胞增殖抑制率为36．3％,细胞迁移[（5．44±0．25）斗In／h对（9．37±0．49）μm／h,P〈0．01]和侵袭（穿膜细胞数146．30±12．56对312．51±9．62,P〈0．01）受抑,细胞凋亡率降低（3．6％±0．3％对12．7％±0．2％,P〈0．01）。结论：hnRNPAl在人胃癌细胞中呈高表达,其可能通过促进肿瘤细胞增殖、迁移、侵袭,在胃癌侵袭和转移过程中发挥作用。     

塞来昔布干预对人胃癌细胞E-钙黏蛋白和基质金属蛋白酶-2表达的影响

目的探讨COX-2与E-cadherin、MMP-2之间的相互关系及其参与胃癌细胞侵袭迁移的可能机制。方法应用塞来昔布(celecoxib)对体外培养的人胃癌细胞SGC7901进行干预,采用实时荧光定量反转录PCR检测COX-2、E-cadherin、MMP-2 mRNA的表达;运用免疫荧光标记法结合激光共聚焦荧光显微镜分析E-cadherin的蛋白表达量;应用Transwell法检测细胞侵袭及迁移能力的变化。结果塞来昔布明显抑制体外培养的人胃癌细胞SGC-7901中COX-2 mRNA的表达,E-cadherin mRNA的表达随着COX-2的表达下降而呈浓度与时间依赖性升高,MMP-2 mRNA的表达随着COX-2的表达下降而呈浓度与时间依赖性降低。塞来昔布30μmol/L干预人胃癌细胞SGC7901 24、36、48 h后,激光共聚焦荧光显微镜检测E-cadherin的蛋白表达量明显升高。塞来昔布干预组细胞穿过Transwell小室的细胞数明显少于对照组。结论 COX-2特异性抑制剂塞来昔布通过抑制COX-2的表达,上调E-cadherin的表达,下调MMP-2的表达,抑制体外培养的胃癌细胞SGC7901的侵袭迁移能力。     

CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth

CYR61 is a secreted, cysteine-rich, heparin-binding protein encoded by a growth factor-inducible immediate–early gene. Acting as an extracellular, matrix-associated signaling molecule, CYR61 promotes the adhesion of endothelial cells through interaction with the integrin α_Vβ₃ and augments growth factor-induced DNA synthesis in the same cell type. In this study, we show that purified CYR61 stimulates directed migration of human microvascular endothelial cells in culture through an α_Vβ₃-dependent pathway and induces neovascularization in rat corneas. Both the chemotactic and angiogenic activities of CYR61 can be blocked by specific anti-CYR61 antibodies. Whereas most human tumor-derived cell lines tested express CYR61, the gastric adenocarcinoma cell line RF-1 does not. Expression of the CYR61 cDNA under the regulation of a constitutive promoter in RF-1 cells significantly enhances the tumorigenicity of these cells as measured by growth in immunodeficient mice, resulting in tumors that are larger and more vascularized than those produced by control RF-1 cells. Taken together, these results identify CYR61 as an angiogenic inducer that can promote tumor growth and vascularization; the results also suggest potential roles for CYR61 in physiologic and pathologic neovascularization.     

H pylori stimulates proliferation of gastric cancer cells through activating mitogen-activated protein kinase cascade

INTRODUCTION H pylori is an important pathogen associated with gastritis and peptic ulcers[1]. It has also been defined as a carcinogen[2]. The mechanisms of pathogenic and carcinogenic effects of H pylori infection are under intensive investigation. Rese…     

PI3K/AKT信号通路在RANKL诱导的SGC-7901胃癌细胞迁移中的作用

目的文献报道RANKL/RANK/OPG途径与肿瘤细胞迁移及骨转移密切相关,但RANKL/RANK途径是否参与胃癌细胞迁移,尚无文献报道。本文拟检测RANK在胃癌细胞系SGC-7901细胞中的表达,并进一步探讨磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶(PI3K/Akt)信号通路在RANKL诱导的胃癌细胞迁移中的作用。方法 West-ern blot检测SGC-7901细胞表面RANK蛋白的表达;RANKL刺激后磷酸化Akt(P-Akt)及Akt的表达;Transwell法测定RANKL及抑制剂刺激后细胞迁移能力的改变。结果 SGC7901细胞表达RANK蛋白。RANKL(1μg/mL)诱导SGC-7901细胞迁移能力增强,迁移增加率为57.2%±5.9%,RANKL抑制剂rOPG(5μg/mL)显著抑制RANKL诱导的细胞迁移(13.88%±3.57%,P<0.05)。RANKL刺激后30 min～3 h,SGC-7901细胞p-Akt表达升高,应用PI3K的抑制剂LY294002(50 mmol/L)显著抑制RANKL诱导的胃癌细胞SGC-7901的迁移(57.28%±5.91%vs23.18%±2.79%,P<0.05)。结论胃癌细胞系SGC-7901细胞表达受体RANK,PI3K/Akt信号通路参与RANKL诱导的SGC-7901细胞迁移。     

Nonhematopoietic tumor cell lines express stem cell factor and display c-kit receptors.

Human stem cell factor (SCF) acts in the presence of other growth factors to stimulate the growth of primitive hematopoietic progenitor cells. These effects are performed by activation of the SCF receptor, c-kit. Because of the potential use of SCF in patients undergoing chemotherapy and bone marrow transplantation, the effect of SCF on nonhematopoietic tumors requires investigation. To determine whether human tumor cell lines display c-kit receptors, we performed binding experiments with 125I-SCF on a breast carcinoma cell line (Du4475), a gastric carcinoma cell line (KATO III), a melanoma cell line (HTT144), as well as two small cell lung carcinoma cell lines (H69 and H128). The biologic effect of SCF on tumor cell lines was assessed by its ability to stimulate tritiated thymidine uptake and to enhance colony growth in methylcellulose. The breast carcinoma cell line, Du4475, as well as two small cell lung carcinoma cell lines, H69 and H128, exhibit high-affinity c-kit receptors with approximate binding affinities of 40, 100, and 90 pmol/L, respectively. The number of high-affinity receptors per cell ranged from 700 to 9,500. The gastric carcinoma cell line, as well as the melanoma cell line, showed trace binding of 125I-SCF. In the presence of SCF alone, or in combination with granulocyte-macrophage colony-stimulating factor or interleukin-3, there was less than a 17% increase in the colony growth of Du4475, H69, or H128 cell lines. Postulating that the lack of growth response could be secondary to endogenous SCF production by the tumor cell lines, we used an RNAse protection assay to determine whether the tumor cell lines contain SCF messenger RNA (mRNA). In addition, we tested tumor cell line supernatants for the presence of secreted SCF protein by enzyme immunoassay, and analyzed the tumor cell lines for membrane-bound SCF by indirect immunofluorescence. Our results show that the Du4475, H69, and H128 cell lines, as well as a melanoma cell line (HTT144), have multiple copies of SCF mRNA. Soluble SCF protein was detected in the cell supernatants in the Du4475 and H69 cell lines and SCF was found on the surface of all four cell lines. These data show that some human solid tumor cell lines display high-affinity c-kit receptors and produce SCF, which can be detected on the cell surface. These results suggest the possibility that autocrine production of SCF by c-kit receptor-bearing tumor cells may enhance cell growth in tumor cell lines.     

Hepatocyte growth factor-induced endothelial cell motility is mediated by the upregulation of inducible nitric oxide synthase expression

OBJECTIVES: Hepatocyte growth factor (HGF) is an angiogenic mitogen which stimulates migration in various cell types and has been shown to induce the production of nitric oxide (NO) in epithelial cells. Conflicting data exist on the effect of NO on endothelial cell migration. The aim of this study was to investigate a possible role for NO in HGF-stimulated endothelial cell motility. METHODS: The study was performed primarily using an endothelial cell line derived from adult human saphenous vein. Transient transfection experiments were additionally performed using an adult human coronary artery endothelial cell line. Nitric oxide synthase expression was examined by western blot analysis. Time-lapse digital image microscopy was used to measure cell motility. A DNA construct was used in transient transfections to over-express inducible nitric oxide synthase (iNOS) as an N-terminal fusion to enhanced green fluorescent protein (EGFP). RESULTS: HGF upregulated the expression of iNOS but not constitutive endothelial nitric oxide synthase (eNOS). Treatment of cells with the specific iNOS inhibitor 1400 W revealed that functional iNOS was required for HGF-stimulated endothelial cell motility. HGF-induced iNOS expression was partially abrogated in the presence of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002, but not the Src kinase inhibitor, PP1. Endothelial cell motility increased significantly (P<0.0001) in the presence of the exogenous NO donor spermine-NO and cells expressing the iNOS-EGFP fusion protein exhibited significantly greater (P=0.0038) motility than those expressing EGFP alone. CONCLUSIONS: These combined data show that elevated NO production is sufficient to stimulate endothelial cell motility and link HGF and NO, both previously implicated in modulating motility, in a common signalling pathway.     

Absence or decreased levels of the hMLH1 protein in human gastric carcinoma cell lines: implication of hMLH1 in alkylation tolerance

Defective hMLH1 function has been increasingly associated with acquired cellular resistance to DNA alkylation damage in human colorectal and endometrial cancer cells. To investigate the relationship between the DNA alkylation tolerance and the hMLH1 status in human gastric carcinoma cells, we determined the cellular response to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), the mutational changes, and the expression of hMLH1 in 11 human gastric carcinoma cell lines. Of 11 cell lines, 4 (SNU-5, -16, -620, and -719) were sensitive, whereas 7 (SNU-1, -216, -484, -520, -601, -638, and -668) were resistant to the cytotoxic effect of MNNG. As determined by Western analysis, it was evident that all the MNNG-resistant cell lines except one (SNU-601) produced very low or undetectable levels of hMLH1 protein compared to the MNNG-sensitive cell lines. A homozygous non-sense mutation that resulted in truncated protein was found in one MNNG-resistant cell line (SNU-1). Therefore, to determine whether the sensitivity of cells to MNNG can be restored by exogenous expression of hMLH1 protein, wild-type hMLH1 cDNA was introduced into the MNNG-resistant cells (SNU-1). The cytotoxicity test showed that expression of exogenous wild-type hMLH1 protein caused an increase in sensitivity to the cytotoxic effect of MNNG. This restoration was confirmed by an increase in the cell population containing less than the G1 amount of DNA (cell death) in the wild-type hMLH1-transfected cells, as determined by flow cytometry analysis. Together our results suggest that (1) the absence or decreased level of wild-type hMLH1 protein may be a frequent event in the human gastric carcinoma cell lines, (2) such alterations in the hMLH1 protein are closely associated with the MNNG tolerance in the human gastric carcinoma cell lines, and (3) the hMLH1 protein participates not only in the repair of DNA mismatches but also in the mechanism of escape from the cytotoxic effects of DNA alkylation damage. Received: 26 January 1998 / Accepted: 18 March 1998     

Cooperation of c-raf-1 and c-myc protooncogenes in the neoplastic transformation of simian virus 40 large tumor antigen-immortalized human bronchial epithelial cells.

Overexpression of c-raf-1 and the myc family of protooncogenes is primarily associated with small cell carcinoma, which accounts for approximately 25% of human lung cancer. To determine the functional significance of the c-raf-1 and/or c-myc gene expression in lung carcinogenesis and to delineate the relationship between protooncogene expression and tumor phenotype, we introduced both protooncogenes, alone or in combination, into human bronchial epithelial cells. Two retroviral recombinants, pZip-raf and pZip-myc, containing the complete coding sequences of the human c-raf-1 and murine c-myc genes, respectively, were constructed and transfected into simian virus 40 large tumor antigen-immortalized bronchial epithelial cells (BEAS-2B); this was followed by selection for G418 resistance. BEAS-2B cells expressing both the transfected c-raf-1 and c-myc sequences formed large cell carcinomas in athymic nude mice with a latency of 4-21 weeks, whereas either pZip-raf- or pZip-myc-transfected cells were nontumorigenic after 12 months. Cell lines established from tumors (designated RMT) revealed the presence of the cotransfected c-raf-1 and c-myc sequences and expressed morphological, chromosomal, and isoenzyme markers, which identified BEAS-2B cells as the progenitor line of the tumors. A significant increase in the mRNA levels of neuron-specific enolase was detected in BEAS-2B cells containing both the c-raf-1 and c-myc genes and derived tumor cell lines. The data demonstrate that the concomitant expression of the c-raf and c-myc protooncogenes causes neoplastic transformation of human bronchial epithelial cells resulting in large cell carcinomas with certain neuroendocrine markers. The presented model system should be useful in studies of molecular events involved in multistage lung carcinogenesis.