Increased inosine-5'-phosphate dehydrogenase gene expression in solid tumor tissues and tumor cell lines.

Inosine-5'-phosphate (IMP) dehydrogenase, a regulatory enzyme of guanine nucleotide biosynthesis, may play a role in cell proliferation and malignancy. To assess this role we examined IMP dehydrogenase expression in a series of human solid tumor tissues and tumor cell lines in comparison with their normal counterparts. Increased IMP dehydrogenase gene expression was observed in brain tumors relative to normal brain tissue and in sarcoma cells relative to normal fibroblasts. Similarly, in several B- and T-lymphoid leukemia cell lines, elevated levels of IMP dehydrogenase mRNA and cellular enzyme were observed in comparison with the levels in peripheral blood lymphocytes. These results are consistent with an association between increased IMP dehydrogenase expression and either enhanced cell proliferation or malignant transformation.     

Amplification, expression and localization of the c-myc gene in BSp73 rat tumor cell lines

Metastatic (ASML 14-1, ASmv) and non metastatic (AS17-4) cell lines of the rat BSp73 pancreatic adenocarcinoma were investigated for amplification and expression of oncogene DNA. The c-myc gene was amplified, but only in one metastatic variant, ASML. The degree of amplification (3.5-fold) increased after prolongued in vitro cultivation (17.5-fold). All three tumor cell lines expressed c-myc and ras mRNA. Ras expression was at the same level as in rat liver. C-myc expression was considerably above the level in rat liver, but also differed considerably between the metastatic variants. In the metastatic ASML cells the c-myc gene was localized by in situ hybridization on a marker chromosome derived from chromosome 7. The karyotypes of the metastatic variants are different and have no common marker chromosomes. Our results obtained with the BSp73 tumor model do not support a role of the c-myc gene in the metastatic process.     

Patterns of DNA methylation and gene expression in human tumor cell lines

Restriction endonuclease analysis was used to determine the methylation status of collagen, c-Ha-ras, and thymidine kinase genes in human fibroblasts and tumor cell lines. When digested with the methylation sensitive enzymes HpaII or HhaI, the DNA of each cell line generated a unique banding pattern for each gene examined. No generalized trend of gene hypomethylation or decreases in overall cytosine methylation were observed in tumor cell lines when compared to fibroblasts. Collagen biosynthetic profiles were also determined, and no correlations could be made between patterns of type I and type III collagen gene methylation and expression. Our findings support those of a previous report in which methylation and expression of the chick alpha 2(I) collagen gene were examined, and this represents the first such analysis of the pro-alpha 1 (III) collagen gene. MspI restriction fragment length polymorphisms were detected within c-Ha-ras, pro-alpha 2(I) collagen, pro-alpha 1(III) collagen, and thymidine kinase genes. The ras gene polymorphisms can be attributed to variation in the number of tandem repeats within a MspI fragment at the 3' end of the gene. The other gene polymorphisms may be due to base pair mutations at methylated cytosine residues within CCGG sequences.     

Structure and expression of the beta-platelet-derived growth factor receptor gene in human tumor cell lines.

Southern digests of DNA from 15 human connective tissue tumor-derived cell lines (and cell strains) and from peripheral blood lymphocytes from normal volunteers were prepared to compare structure of the beta-platelet-derived growth factor receptor gene in normal and tumor-derived cells. The tumor-derived samples had no alterations in gene structure, nor was gene amplification evident. Two restriction fragment length polymorphisms were detected within the beta-receptor gene. Expression of the alpha- and beta-platelet-derived growth factor receptor was quantified using Northern blots. Expression was not tumor-type specific. For example, one rhabdomyosarcoma cell line expressed only the alpha-receptor, whereas two others expressed the beta. In contrast, a human fibroblast cell strain expressed both receptor types. Alterations in receptor expression may be a result of the tumorigenic process.     

Amplification of the c-myc gene in human medulloblastoma cell lines and xenografts

Cultured cell lines and xenografts derived from 7 human medulloblastomas were evaluated for amplification of the c-myc, N-myc, epidermal growth factor receptor, and gli genes by Southern blot analysis. Karyotypes of the original biopsies and early passaged cells demonstrated double minute chromosomes in 4 of the 7 cases. All 7 samples (3 cell lines and 4 xenografts) from the 4 tumors with double minute chromosomes contained amplification of the c-myc gene. Cell lines and xenografts derived from the 3 biopsies without double minute chromosomes failed to demonstrate amplification of the 4 genes which were tested, but a rearrangement of the c-myc gene occurred in 1 of the 3 tumors. These observations demonstrate that the c-myc gene is often amplified and/or rearranged in human medulloblastomas and suggest that amplification of this gene provides a growth advantage for medulloblastoma cells in vitro and in vivo.     

Production of neuromedin B and neuromedin B gene expression in human lung tumor cell lines.

Gastrin-releasing peptide (GRP), a mammalian bombesin-like peptide, has been shown to be an important autocrine growth factor for small cell lung cancer (SCLC). However, not all SCLC cell lines express the GRP gene or respond mitogenically to GRP stimulation, suggesting the existence of other autocrine pathways in this tumor. Neuromedin B (NMB), the mammalian counterpart of amphibian ranatensin, has been shown to be a mitogen for SCLC cell lines in vitro. To determine whether NMB is a potential autocrine growth factor for lung tumors, NMB gene expression, peptide synthesis, and secretion have been investigated in a panel of SCLC and non-SCLC (NSCLC) cell lines. Northern blot analysis and enzymatic amplification from mRNA by polymerase chain reaction showed that the NMB gene was expressed in all SCLC and NSCLC cell lines examined. In contrast, the GRP gene was expressed in four of six classic SCLC cell lines but not in variant SCLC or NSCLC cell lines. Immunoreactive NMB was detected by radioimmunoassay in the majority of classic SCLC, in one of three variant SCLC and in one of three NSCLC cell lines, and secreted NMB was detected in medium conditioned by a SCLC and a NSCLC cell line. The present study also demonstrated the presence of immunoreactive GRP in the absence of detectable GRP gene expression. The antiserum used in the GRP radioimmunoassay failed to cross-react with NMB but showed some cross-reactivity with amphibian phyllolitorin raising the possibility that certain SCLC cell lines may produce a phyllolitorin-like peptide.     

Intrinsic MDR-1 gene and P-glycoprotein expression in human melanoma cell lines

Metastatic malignant melanoma is considered a chemotherapy-refractory malignancy. A few previous studies have delivered contradictory results regarding the presence and functionality of P-glycoprotein (P-gp), a transmembranous protein associated with the classical multidrug resistance (cMDR), in malignant melanoma. Therefore we have investigated this issue on 33 cell lines established from primary and metastatic lesions of human malignant melanoma, comparing different cMDR detection methods. Immunocytochemically 33% of the cell lines stained positive for P-gp. The data correlated with those of a P-gp-radioimmunometric (antibody-binding) assay. When RT-PCR was used for MDR-I mRNA determination, 76% of the melanoma cell lines scored positive. Slot-blot analysis was seen to be less sensitive than RT-PCR. Results from the functional P-gp assays, using daunomycin (DM) as MDR-substrate, showed no influence of P-gp expression on drug accumulation and cytotoxicity. However, the cMDR-modifier verapamil (VP) significantly increased both parameters in those melanoma cells with the highest P-gp levels. We conclude that cMDR is apparently not the decisive but probably a complementary protective mechanism against toxic agents in malignant melanoma.     

六氧甲基鸟嘌呤DNA甲基转移酶基因表达与肿瘤耐药

六氧甲基鸟嘌呤ＤＮＡ甲基转移酶能阻止由氮芥类导致的ＤＮＡ交连形成，因而降低其细胞毒性。本研究探讨肿瘤细胞对氯乙基亚硝脲耐药与其ＭＧＭＴ基因表达之间的关系。方法：采用ＲＮＡ印迹试验和蛋白印迹试验对１３株人肿瘤细胞的ＭＧＭＴ基因表达进行了检测，并与采用改良的Ｓｕｌｆｏｒｈｏｄａｍｉｎａ Ｂ比色抗癌药筛选法测定的肿瘤细胞对ＥＣＥＮＵ的耐药性进行比较。     

Tumor-suppressive activity of the growth arrest-specific gene GAS1 in human tumor cell lines

The GAS1 gene product induces growth arrest through a p53-dependent mechanism. To investigate whether GAS1 is a tumor suppressor gene, we transfected GAS1-negative human tumor cells with GAS1 plasmids and analyzed growth characteristics of stable transfectants. When a constitutively expressing GAS1 plasmid was transfected into A549 cells, no stable colonies expressing GAS1 were isolated. When A549 cells were transfected with a dexamethasone-inducible GAS1 plasmid, expression of GAS1 inhibited growth in vitro, and fewer slow-growing tumors arose in nude mice. GAS1 also inhibited proliferation of an HT1080 subline with wild-type (wt) p53 and normal MDM2. However, when the HT1080 subline HTD114 was transfected with the constitutive GAS1 plasmid, there was no reduction in colony number. GAS1-transfectant clones had unaltered growth in vitro, were morphologically unchanged and showed no difference in their ability to form tumors in nude mice. Although HTD114 cells contain wt p53, levels of MDM2 were elevated by 10–15 fold. The HT1080-6TGc5 subline with mutant p53 and normal MDM2 was also refractory to GAS1. Our results show that GAS1 suppresses the growth and tumorigenicity of human tumor cells and overexpression of MDM2 or p53 mutation inhibits the GAS1-mediated growth-suppressing pathway. Int. J. Cancer 75:568–577, 1998. © 1998 Wiley-Liss, Inc.     

Loss of caspase-1 gene expression in human gastric carcinomas and cell lines

The caspases are a family of aspartic acid-specific proteases that fulfill varied and often critical roles in mammalian apoptosis or in the proteolytic activation of cytokines. Caspase-1 (interleukin-1beta-converting enzyme) is a member of the cysteine protease family, which cleaves target proteins following aspartic acid residues. We investigated caspase-1 expression in stomach cancer, tissues and cell lines. Of 301 consecutive gastric carcinomas, 58 cases (19.3%) showed the expressional loss of caspase-1. Loss of caspase-1 expression was significantly associated with pTNM stage (p=0.03), lymph node metastasis (p=0.01) and patient survival (p<0.01). Caspase-1 expression was also significantly correlated in an inverse manner with p53 expression (p<0.01). Among the 11 gastric cancer cell lines examined, three cell lines showed loss of expression at the protein and mRNA levels. On treatment with 5-aza-2'-deoxycytidine (5-aza-C), and/or trichostatin A (TSA), all three cell lines re-expressed caspase-1 mRNA. The above findings suggest that epigenetic events such as DNA methylation and histone deacetylation play important roles in the regulation of caspase-1, and that loss of caspase-1 expression is associated with poor survival in gastric carcinoma.     

Isolation of two human tumor epithelial cell lines from solid breast carcinomas.

Most of the available human breast tumor cell lines have been derived from pleural effusions. The two cell lines herein described, BT-474 and BT-483, were derived from solid, invasive ductal breast carcinomas. Both are epithelial and neoplastic as judged by their general morphology, their fine structure, and their ability to produce growing nodules in nude mice and colonies in soft agar and methocel. BT-474 and BT-483 are human as expressed by chromosome morphology and aneuploid with a modal number of 55 and 72 chromosomes, respectively. Trypsin-Giemsa banding did not reveal the presence of obvious HeLa markers, and the glucose 6-phosphate dehydrogenase electrophoretic migration pattern was of the B-type. Furthermore, the migration of lactic dehydrogenase, malic dehydrogenase, and 6-phosphogluconate dehydrogenase isoenzymes was consistent with a human pattern and different from that of the mouse, rat, or hamster. Quarterly tests to detect the presence of aerobic and anaerobic mycoplasmas were repeatedly negative. A culture medium containing insulin, increased amounts of amino acids, vitamins, and glucose facilitated the isolation of the tumor cells. Cell replication was maintained with 10% fetal calf serum absorbed with activated charcoal and dextran. No production of alpha-lactalbumin was detected by radioimmunoassays, but high levels of progesterone receptors were found in both cell lines.     

神经元外单胺转运蛋白在人肿瘤细胞系的表达

我们先前发现的一个新的氯乙基亚硝脲的类似物２－氯乙基－３－肌氨酸酰胺－１－亚硝基脲, 是一种通过单胺递质的神经元外转运蛋白（ＥＭＴ）进入细胞内的选择性的细胞毒素。此药已进入１期临床试验。在本研究中,我们检测ＥＭＴ在２３个人类肿瘤细胞系里的表达水平,以证实ＥＭＴ的表达是否与ＳａｒＣＮＵ的细胞毒性有关。方法应用反转录多聚酶链反应（ＲＴ－ＰＣＲ）检测ＥＭＴ在肿瘤细胞系的表达。同时也用蛋白质印迹技术检测Ｄ     

Expression and methylation of the blym gene in human tumor cell lines

We have examined Blym expression in 11 human tumor cell lines. Increased Blym expression was observed in one of three osteosarcoma cell lines relative to nontransformed human foreskins fibroblasts. In addition, enhanced Blym expression was observed in a melanoma cell line and in 2 of 6 squamous carcinoma cell lines relative to nontransformed, low passage human epithelial cells. We found no evidence of gene amplification or rearrangements of Blym sequences in any of the cell lines we have examined. We further analyzed the state of methylation of the Blym gene in several of the tumor cell lines by Msp I/Hpa 11 restriction endonuclease digestion. All cell lines examined had similar Msp I digestion patterns. However, the different tumor cell lines had different Hpa 11 digestion patterns. Therefore, our results indicate that the Blym gene is differentially expressed and methylated in human tumor cell lines.     

Galectin-1 gene expression and methylation state in human T leukemia cell lines

Galectin-1 has been demonstrated to be a mediator of T-cell apoptosis acting on activated T-cells and, in a selective manner, on different T leukemia cell lines. Here we show that the sensitivity to galectin-1 is associated with repression of the endogenous galectin-1 gene whereas non-sensitive cells express high levels of galectin-1. Repression of galectin-1 gene in sensitive cells is associated with hyper-methylation of the promoter region. Transient treatment of non-expressing cells with the demethylating agent 5-azacytidine led to irreversible demethylation and subsequent reactivation of galectin-1 gene.     

Effect of retinoblastoma tumor suppressor gene expression on chemosensitivity of human osteosarcoma cell lines

We examined the effects of inactivation of the RB gene on chemosensitivity of human osteosarcoma cell lines, using the MTT assay and calculating the inhibition index. Although the human osteosarcoma cell lines HOS and MG63 have a wild-type RB gene, SaOS-2 and OSrb (established from retinoblastoma patient) have no active RB gene. We used these 4 cell lines in growth inhibition assays for doxorubicin, cisplatin and methotrexate, and assessed the chemosensitivity. In the growth inhibition assay for methotrexate, cell lines lacking an active RB gene were more resistant than cell lines with an active RB gene.     

Differential gene expression in cisplatin-resistant and -sensitive testicular germ cell tumor cell lines

Testicular germ cell tumors (TGCTs) represent a well curable malignity due to their exceptional response to cisplatin (CDDP). Despite remarkable treatment results, approximately 5% of TGCT patients develop CDDP resistance and die. Exceptional curability makes TGCTs a highly valuable model system for studying the molecular mechanisms of CDDP sensitivity. Our study was aimed at revealing difference in gene expression between the CDDP-resistant and -sensitive TGCT cell lines, and hence at identifying candidate genes that could serve as potential biomarkers of CDDP response. Using gene expression array, we identified 281 genes that are differentially expressed in CDDP-resistant compared to -sensitive TGCT cell lines. The expression of 25 genes with the highest fold change was validated by RT-qPCR. Of them, DNMT3L, GAL, IGFBP2, IGFBP7, L1TD1, NANOG, NTF3, POU5F1, SOX2, WNT6, ZFP42, ID2, PCP4, SLC40A1 and TRIB3, displayed comparable expression change in gene expression array and RT-qPCR, when all CDDP-resistant TGCT cell lines were pairwise combined with all -sensitive ones. Products of the identified genes are pluripotency factors, or are involved in processes, such as cell metabolism, proliferation or migration. We propose that, after clinical validation, these genes could serve as prognostic biomarkers for early detection of CDDP response in TGCT patients.     

酪氨酸蛋白激酶受体EphA2及其配体EFNA1在23种肿瘤细胞系中的表达及其意义

背景与目的：酪氯酸蛋白激酶受体EphA2及其配体EFNA1可能在肿瘤的发生发展过程中起着重要的作用。本研究探讨EphA2／EFNA1在不同肿瘤细胞中的表达及其意义。方法：用RT-PCR方法，检测了23种人类肿瘤细胞系中EphA2／EFNA1在mRNA水平上的表达，、并用Western blot方法检测了EphA2蛋白在不同肿瘤细胞系中的表达水平。结果：RT—PCR结果显示．除白血病细胞K562和HL60，以及视网膜母细胞瘤RB几乎检测不到EphA2／EFNA1外．EphA2／EFNA1高表达于肝癌、肺癌、卵巢癌、官颈癌、前列腺癌、胃腺癌、脑胶质瘤、膀胱癌、成骨肉瘤及恶性黑色素瘤等20种细胞系中。Western blot结果显示，除K562外，EphA2蛋白在不同肿瘤细胞系中均有表达。结论：EphA2／EFNA1高表达于人类多种肿瘤细胞中，并且EFNA1的表达水平相对低于EphA2的表达、EphA2．／EFNA1可作为一个较为广潜的肿瘤标记，并有可能成为肿瘤治疗的靶点。     

Differential expression of antiapoptotic gene BAG-1 in human breast normal and cancer cell lines and tissues.

BAG-1 is an antiapoptotic protein that binds to and enhances the antiapoptotic activity of Bcl-2. It binds several growth factor and hormone receptors and modulates their function. BAG-1 was also shown recently to be expressed as four protein isoforms, p50, p46, p33, and p29, through alternative translation initiation. Although many apoptosis-associated genes have been linked to oncogenesis of human breast cancer, the role of BAG-1 has not been fully elucidated. In this study, we examined the expression of BAG-1 RNA or protein isoforms and its interacting antiapoptotic proteins, Bcl-2 and BcI-X(L), in breast normal and tumor cell lines and tissues by Northern or Western blot analysis. We provide convincing evidence that both BAG-1 RNA and protein are overexpressed in human breast cancer cell lines. More importantly, we found that the expression of two isoforms of BAG-1, p46 and p33, was also much higher in breast primary tumors. The expression of Bcl-2 and Bcl-X(L) correlated with that of BAG-1 in breast normal and carcinoma cell lines but not tissues. Our study suggests that BAG-1 isoforms may serve as a molecular marker, independent of Bcl-2 and Bcl-X(L), for human breast cancer.