DNA mismatch binding in human lung tumor cell lines

Defects in mismatch repair (MMR) genes have been involved in several types of sporadic and hereditary cancers. In order to elucidate the role of MMR in human lung carcinogenesis we examined DNA mismatch binding in cell-free extracts of seven lung tumor cell lines and five corresponding lymphoblastoid cell lines from lung cancer patients. Using the technique of bandshift assay we have demonstrated that 2/7 of the tumor cell lines are aberrant in binding to specific DNA mismatches while all lymphoblastoid cell lines were proficient in binding to all tested mismatches. Both extracts were aberrant in binding to G/T mismatch whereas one of the cell lines showed deficiency in binding to the C:A mismatches as well. Immunoblotting analysis showed that all known DNA mismatch repair (MMR) proteins were present in these extracts. The cell line deficient in binding to both G:T and C:A mismatches showed microsatellite instability (MSI) in tumor DNA and higher resistance to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This report indicates that DNA mismatch binding deficiencies may be implicated in at least a subgroup of human lung cancer.     

PRIMARY NEUROENDOCRINE CARCINOMA OF THE SKIN (NERKEL CELL TUMOR)

ＰＲＩＭＡＲＹＮＥＵＲＯＥＮＤＯＣＲＩＮＥＣＡＲＣＩＮＯＭＡＯＦＴＨＥＳＫＩＮ（ＭＥＲＫＥＬＣＥＬＬＴＵＭＯＲ）ＬｕＮｉｎｇ；吕宁；ＬｉＪｉｎｇｘｉａｎ；李竞贤（ＤｅｐａｒｔｍｅｎｔｏｆＰａｔｈｏｌｏｇｙ，ＣａｎｃｅｒＩｎｓｔｉｔｕｔｅ，Ｃｈｉｎｅｓ...     

Frequent occurrence of RASSF1A promoter hypermethylation and merkel cell polyomavirus in merkel cell carcinoma

Merkel cell carcinoma (MCC) is one of the most aggressive cancers of the skin. It has recently been reported that integration of a Merkel cell polyomavirus (MCPyV) in receptor tyrosine phosphates type G (PTPRG) gene occurs in MCC, and that viral infections are associated with epigenetic silencing of tumor suppressor genes (TSG) in cancer. To examine whether a correlation between TSG inactivation and viral infection can be found in MCC, we investigated the promoter hypermethylation of RASSF1A, TP73, PTPRG, FHIT, and CDKN2A and the presence of MCPyV and SV40 in 98 MCC by PCR. Hypermethylation of RASSF1A was frequently found in 42 of 83 (51%) of MCC. Methylation of CDKN2A was present in 9 of 41 (22%) of MCC. Hypermethylation of TP73 (0%), PTPRG (4%), and FHIT (0%) was infrequent in MCC. Interestingly, MCPyV was found in 90 of 98 (92%) MCC, however, no SV40 signal was detected. No correlation between TSG hypermethylation and viral infection was found. Our results show frequent hypermethylation of RASSF1A and the presence of MCPyV in primary MCC, and that these events may contribute to the pathogenesis of MCC. © 2009 Wiley‐Liss, Inc.     

Nuclear matrix protein composition of human lung carcinoma cell lines

The nuclear matrix is the RNA-protein skeleton within the nucleus that contributes to the structural and functional organization of DNA. Differences in the nuclear matrix protein composition between cancer and normal cells have been reported in various cell lines and tissues, suggesting altered gene expression. This study examined the nuclear matrix protein composition of various human lung cell lines. Using high resolution two-dimensional electrophoresis, at least ten common proteins, as well as specific differences, were identified in each category of lung cell lines. These protein differences may be responsible, at least in part, for the different phenotypes of human lung cancer.     

Binding of gastrin(17) to human gastric carcinoma cell lines

The hormone gastrin stimulates acid secretion by gastric parietal cells and acts as a growth factor for the gastric mucosa. Gastrin receptors with dissociation constants of approximately 0.5 nM have been detected on isolated gastric parietal cells, and on some cell lines derived from colon carcinomas. We now report that gastrin is also bound by five cell lines derived from human gastric carcinomas, but that the affinities of these lines for gastrin range from 0.2 to 1.3 microM. Cholecystokinin8 binds to the cell line Okajima with an affinity similar to gastrin17, while shorter gastrin analogues bind with reduced affinity. Binding of gastrin is unaffected by acetylcholine, histamine, or a number of other hormones with the exception of insulin which inhibits binding with an IC50 value of 0.5 microM. The ability to bind gastrin with affinities in the microM range appears to be a property widespread among other tumor cell lines.     

Estramustine binding protein and anti-proliferative effect of estramustine in human glioma cell lines

Four human cell lines derived from malignant gliomas were immunohistochemically examined for their content of estramustine-binding protein (EMBP). EMBP was detected in a large amount in all glioma cells during the entire cell cycle. EMBP has previously been demonstrated to be the major receptor protein in prostatic cancers for the cytostatic drug estramustine-phosphate (EMP). EMP caused a dose-dependent inhibition of exponentially growing cells by increasing the number of cells in G2/M stage of the cell cycle as monitored by flow cytofluorometry. The effect may be coupled to arrest of the glioma cells at metaphase. The presence of EMBP may suggest a selective binding and effect of EMP in glioma cells.     

DNA damage inducible-gene expression following platinum treatment in human ovarian carcinoma cell lines

 Repair of cisplatin-damaged DNA was investigated in a human ovarian carcinoma cell line (2008) and its cisplatin-resistant variant (C13^*) using a host-cell reactivation (HCR) assay. The HCR of cisplatin-damaged adenovirus (Ad) was not significantly different in C13^* cells compared to 2008 cells. The cisplatin concentrations required to reduce the amount of viral DNA replicated to 50% were 0.12±0.02 μM and 0.10±0.01 μM after 48 h of repair in 2008 and C13^* cells respectively. Similarly, the cisplatin concentration required to reduce the expression of a reporter gene inserted in the viral DNA was not significantly altered in C13^* cells compared to the parental line (IC₅₀ values were 0.28±0.04 μM in 2008 cells and 0.17±0.06 μM in C13^* cells after 48 h of repair). Pretreatment of the cells with cisplatin, immediately prior to Ad infection, did not significantly alter the HCR of cisplatin-damaged Ad in either cell type. In addition, a cisplatin-sensitive variant derived from the C13^* cells, namely the RH4 cells, did not differ significantly from either the 2008 or C13^* cells in their ability to reactivate cisplatin-damaged Ad. Furthermore, a component of the nucleotide excision repair (NER) pathway, DNA polymerase α, was investigated using the competitive inhibitor aphidicolin. The combination of cisplatin and aphidicolin resulted in similar synergistic growth inhibition in both the 2008 and C13^* cells providing additional support to the HCR results which suggest that enhanced NER is not responsible for the cisplatin resistance in C13^* cells. Received: 21 April 1995/Accepted: 9 October 1995     

上皮来源的肿瘤细胞表达免疫球蛋白A

目的 证实上皮来源的肿瘤细胞表达免疫球蛋白,确定所表达的免疫球蛋白的类别。方法 采用免疫组化、Western blot及ELISA等方法检测MCF－7（人乳腺癌细胞系）、SW480（人结肠癌细胞系）、MGC（人胃癌细胞系）、HeLa(人宫颈癌细胞系）、CNE1－LMP1（稳定表达LMP1的鼻咽癌细胞系）、HNE2（鼻咽癌细胞系）和Tet-on-LMP1-HNE2(受四环素诱导可调控的鼻咽癌细胞系）等7种上皮来源的细胞系的细胞蛋白提取液及培养上清中的免疫球蛋白。结果 在7种上皮来源的瘤细胞系的蛋白提取液及培养上清中均检测到了人免疫球蛋白A。结论 上皮来源的肿瘤细胞表达免疫球蛋白A。     

Detection of human papillomavirus (HPV) in laryngeal carcinoma cell lines provides evidence for a heterogeneic cell population

The role of human papillomavirus (HPV) has been studied in laryngeal carcinomas with contradictory results. To evaluate the causal relationship between HPV infection and epithelial malignancies of the larynx, 27 laryngeal carcinoma cell lines from 22 patients were studied. Also, paraffin-embedded biopsy samples of the original tumours were available from 12 patients. First, Southern blot hybridisation (SBH) was used for the analysis of 18 cell lines and 12 original tumour sections were studied by in situ hybridisation (ISH) to detect HPV. Further, cell lines and tumour biopsy samples were investigated with polymerase chain reaction (PCR) using three sets of consensus primers directed to L1 and E1 ORFs (open reading frames) and type-specific primers to HPV 16 E6 region. The adjacent apparently normal epithelium of one original biopsy sample showed positive signals for HPV by ISH. All other samples were HPV negative with these methods. The study was then extended to 27 laryngeal carcinoma cell lines, including the 18 cell lines studied earlier. A new nested PCR method was used with MY as external and general primers (GP) as internal primers for the cell lines and original tumour samples to achieve a maximal sensitivity. Subsequent SBH was performed to confirm the specificity of PCR products with both low- and high-risk HPV oligonucleotide probe mixtures and also with the HPV 16 oligoprobe. With this method, seven of 27 (26%) cell lines and seven of 12 (58%) tumour samples were found to harbour high-risk HPV. In two cases both the original tumour sample and the derived cell line showed HPV positivity. These results indicate that HPV copy numbers are low and only a minority of tumour cells harbour HPV DNA, explaining partly the controversial results reported earlier.     

Elastases in human breast carcinoma cell lines

Elastosis, the deposition of large amounts of elastin, is characteristic of the desmoplastic reaction to human breast carcinoma. Dissolution of the elastin often occurs following treatment regimens that involve steroid hormones or their antagonists. Elastinolytic activities must be invoked to account for the loss of this elastin-cotaining stroma. We have utilized a tissue culture model to explore the molecular aspects of this phenomenon. The elastases of several human fibroblast and breast carcinoma cell lines were examined. The tumor cells had 10- to 30-fold higher elastase activity than did the fibroblasts. Three separate elastinolytic activities were observed in the tumor cell lines, and partial purification was achieved. The effect of steroid hormones on these elastases was examined. No stimulation of activity was found with any of the hormones, in any combination. However, there was marked inhibition of elastase with estradiol, progesterone, and dexamethasone of the ZR75-1 cell line. This is the estrogen receptor positive line that is estrogen responsive. The corticosteroids also inhibited the elastases of the estrogen receptor positive, non-responder cell line ZR75-30. No effect was seen on the elastases of receptor negative cells ZR75-31A with any of the steroid hormones. Stimulation of elastinolytic activities in these tumor cells must occur by some as yet unidentified pathway.     

Collagenases in human breast carcinoma cell lines

The intense stromal response to some human tumors is termed the desmoplastic reaction. It is found with most human breast carcinomas. Dissolution of this response, containing predominantly fibrous proteins such as collagen and elastin, can occur with treatment. We have undertaken a study of the collagenases of the breast tumor desmoplastic reaction using a tissue culture model composed of human breast tumor cell lines and various human fibroblasts. The breast tumor cells had the higher collagenase activity, particularly the ZR75-31A cell line. Activity was 10-fold higher than that of the stromal cells. The enzyme was secreted into the media and required trypsin pretreatment for activity to be manifest. Partial purification was achieved of the major collagenase species. The protein was a metalloprotease and, like other mammalian collagenases, had a relative molecular weight of 60,000. Classical 3/4 and 1/4 cleavage products of the triple helical collagen substrate were demonstrated, typical of most mammalian collagenases. Only types I and III collagens were suitable substrates for this enzyme, with no apparent preference between the two. The breast tumor collagenases were not responsive to hormones; however, stimulation of activity was apparent in the absence of proteolytic pretreatment. This may represent conversion of the procollagenases of the breast tumor cells to the active form by an estrogen-sensitive plasminogen activator secreted by the same tumor cells.     

Endocrine-responsive pancreatic carcinoma: steroid binding and cytotoxicity studies in human tumor cell lines

We have begun to investigate the steroid responsiveness of pancreatic cancer by comparing human (MiaPaCa, Colo-357, RWP-1, RWP-2) and rodent (AR42j) pancreatic tumor cell lines with cultured estrogen receptor-positive breast cancer cells (MCF-7, T47-D). The four human pancreatic tumors contain measurable levels of specific estradiol binding sites with dissociation constants (Kd) that range from 1 to 9 nM, in contrast to the higher-affinity binding sites measured in the breast cancer cells (Kd less than or equal to 1 nM). Growth of one pancreatic tumor line (MiaPaCa) is stimulated 40% above control by exposure to nanomolar concentrations of estradiol, suggesting that the estrogen receptor in these cells is functioning like that in MCF-7 and T47-D cells. Glucocorticoids (dexamethasone, hydrocortisone) and androgen (fluoxymesterone) stimulate proliferation of Colo-357 cells by as much as 30%. Paradoxically, glucocorticoids inhibit AR42j cells to less than 50% of control growth. Micromolar exposures of estrogen (17 beta-estradiol), antiestrogen (tamoxifen), antiandrogen (dehydroxyflutamide), progestins (progesterone, R5020, medroxyprogesterone acetate), and inhibitors of steroid-metabolizing enzymes (17 beta-N,N-diethylcarbamyl-4-methyl-4-aza-5 alpha-androstan-3-one, danazol) impair growth of these pancreatic tumors to varying degrees, and with little relationship to estrogen receptor content. In general, progestins are slightly more growth inhibiting to these pancreatic tumor lines than the other endocrine agents tested, including tamoxifen. Only the RWP-2 cells appear completely resistant to steroidal therapy, showing less than 25% growth inhibition with exposure to therapeutic concentrations (less than or equal to 2.5 microM) of these agents. Colo-357, MiaPaCa, and AR42j cells are most responsive to these endocrine agents, and their overall pattern of sensitivity suggests that the steroid-dependent growth-inhibitory mechanisms of some pancreatic carcinomas may involve both receptor antagonism and direct inhibition of steroidal oxidoreductases. 17 beta-N,N-Diethylcarbamyl-4-methyl-4-aza-5 alpha-androstan-3-one, a potent inhibitor of 5 alpha-reductase with minimal affinity for androgen receptor, inhibits growth of Colo-357 cells to less than 40% of control and also inhibits AR42j and MiaPaCa cells. Dehydroxyflutamide, a potent androgen receptor antagonist with no direct influence on 5 alpha-reductase activity, inhibits growth of MiaPaCa and AR42j cells but has no affect on Colo-357 growth.(ABSTRACT TRUNCATED AT 400 WORDS)     

一个具有肿瘤转移抑制基因活性的人DNA序列

目的 分离鉴定人类肿瘤转移抑制基因或相应ＤＮＡ序列,探讨转移表型逆转的分子生物学机制。方法 应用Ｉｎｔｅｒ Ａｌｕ ＰＣＲ技术,将导入正常人肺基因组ＤＮＡ片段后转移表型抑制细胞株中的人源性ＤＮＡ片段扩增出来,对其中的７００ｂｐ左右ＤＮＡ片段进行核苷酸序列分析,并在ＧｅｎＢａｎｋ序列数据库进行类似性检索。将该ＤＮＡ再次转染高转移小鼠瘤细胞株,通过体内、体外转移相关性实验,验证其是否具有抑制高转移小鼠     

A novel human DNA sequence with tumor metastasis suppressive activity

　Objective To isolate human tumor metastasis suppressive DNA sequence and to study the molecular mechanisms regulating tumor metastasis. Methods A mouse lung adenocarcinoma cell clone 12 derived from its parent cell line LM2, which had been transduced with normal human genomic DNA, was previously reported. Compared with LM2, the metastatic potential of clone 12 was very much decreased. Clone 12 was used in this study to amplify the human DNA fragments by Inter Alu PCR technique. The human DNA fragments obtained were then transfected into LM2 cells and their malignant phenotype was tested in vitro and in vivo, and compared with that of the untransfected LM2 cells. Results Three human DNA fragments of 700, 500 and 300 bp were isolated. DNA sequencing revealed that the 700bp fragment does not show homology with hitherto reported genes and was accepted by the Genbank (pt712 U67835). In vitro proliferation and colony formation in soft agar of the 700 bp fragment-transfected LM2 cells were significantly inhibited as compared to the untransfected LM2 cells. Upon subcutaneous inoculation to syngeneic T739 mice, the 700bp–transfected LM2 cells grew more slowly and smaller tumors developed compared to the untransfected ones. Moreover, lung metastasis was not found in 6 of 10 mice inoculated with the 700bp-transfected LM2 cells, while it was found in 9 of 10 mice inoculated with the untransfected LM2 cells. The difference was statistically significant (P<0.001). The frequency of lymph node metastasis was also statistically different between the 2 groups of mice. Conclusion The newly isolated 700bp human DNA fragment may be a metastasis suppressor gene of malignant tumor.     

Genome-wide analysis of DNA methylation identifies novel cancer-related genes in hepatocellular carcinoma

Aberrant DNA methylation has been implicated in the development of hepatocellular carcinoma (HCC). Our aim was to clarify its molecular mechanism and to identify useful biomarkers by screening for DNA methylation in HCC. Methylated CpG island amplification coupled with CpG island microarray (MCAM) analysis was carried out to screen for methylated genes in primary HCC specimens [hepatitis B virus (HBV)-positive, n?=?4; hepatitis C virus (HCV)-positive, n?=?5; HBV/HCV-negative, n?=?7]. Bisulfite pyrosequencing was used to analyze the methylation of selected genes and long interspersed nuclear element (LINE)-1 in HCC tissue (n?=?57) and noncancerous liver tissue (n?=?50) from HCC patients and in HCC cell lines (n?=?10). MCAM analysis identified 332, 342, and 259 genes that were methylated in HBV-positive, HCV-positive, and HBV/HCV-negative HCC tissues, respectively. Among these genes, methylation of KLHL35, PAX5, PENK, and SPDYA was significantly higher in HCC tissue than in noncancerous liver tissue, irrespective of the hepatitis virus status. LINE-1 hypomethylation was also prevalent in HCC and correlated positively with KLHL35 and SPDYA methylation. Receiver operating characteristic curve analysis revealed that methylation of the four genes and LINE-1 strongly discriminated between HCC tissue and noncancerous liver tissue. Our data suggest that aberrant hyper- and hypomethylation may contribute to a common pathogenesis mechanism in HCC. Hypermethylation of KLHL35, PAX, PENK, and SDPYA and hypomethylation of LINE-1 could be useful biomarkers for the detection of HCC.     

A systematic profile of DNA methylation in human cancer cell lines

Human cancer cell lines are commonly used in basic cancer research to understand the behavior of primary tumors. Aberrations in the DNA methylation patterns are nowadays recognized as a hallmark of the cancer cell. However, no comprehensive study defines the DNA methylation environment present in the established cancer cell lines used in everyday laboratory-based research. To address this matter, we have analyzed 70 widely used human cancer cell lines of 12 different tumor types for CpG island promoter hypermethylation of 15 tumor suppressor genes, global 5-methylcytosine genomic content, chemical response to the demethylating agent 5-aza-2'-deoxycytidine, and their genetic haplotype for methyl-group metabolism genes. Several conclusions arise from our study: (a) a specific profile of CpG island hypermethylation exists for each tumor type, allowing its classification within hierarchical clusters according to the originating tissue; (b) cancer cell lines generally have higher levels of CpG island hypermethylation than primary tumors, because of the contribution of particular CpG islands and tumor types; and (c) there are no major differences between cell lines in their 5-methylcytosine DNA content, efficacy of 5-aza-2'-deoxycytidine treatment, and distribution of allelotypes of methyl-group metabolism genes. Our data provide a basis for a better use of human cancer cell lines in basic and translational research with respect to their DNA methylation environment.     

Cross-contamination of human esophageal squamous carcinoma cell lines detected by DNA fingerprint analysis

DNA "fingerprint" analysis has recently become known as a valuable technique for positive identification of any given individual. The chances for mistaken identity have been estimated to be 10(-6) for close siblings or as little as 10(-23) for randomly selected individuals. This methodology thus represents a significant improvement over previously established identification tests using protein or enzyme analysis techniques and has already found application in forensic medicine. One of the chief problems in tissue culture studies is the question of the unequivocal identity of the cultured cells used and the very real possibility of their being contaminated by cells of a similar morphological appearance. We report here the application of the DNA "fingerprint" technique to the genotypic analysis of cultured human squamous carcinoma cells. The results show that a number of lines, designation HCu, have become cross-contaminated. Lines SNO, HCu 10, and HCu 13 are genetically distinct, however lines HCu 10, 18, 33, 37, and 39 are genetically identical and are in fact subcultures of the same cells. In addition, a myocardial line known as Girardi is shown to be identical to HeLa cells. The introduction of this technique to tissue culture laboratories could therefore prevent contaminated cultures from being disseminated or used in research studies.