Cancer/testis (CT) antigens exhibit highly tissue-restricted expression and are considered promising targets for cancer vaccines. Here we identified a novel CT gene ZNF645 which restrictively expresses in normal human testes and lung cancer patients (68.3%). To investigate the promoter methylation s...
Cancer/testis (CT) antigens exhibit highly tissue-restricted expression and are considered promising targets for cancer vaccines. Here we identified a novel CT gene ZNF645 which restrictively expresses in normal human testes and lung cancer patients (68.3%). To investigate the promoter methylation status of ZNF645, we carried out bisulfite genomic sequencing and found that the CpG island in its promoter was heavily methylated in normal lung tissues without the expression of ZNF645, whereas there was high demethylation in normal human testes and lung carcinoma tissues with its expression. Also ZNF645 could be remarkably activated in A549 and HEK293T cells treated by DNA demethylation agent 5'-aza-2'-deoxycytidine. And the dual luciferase assay revealed that the promoter activity of the ZNF645 was inhibited by methylation of the CpG island region. Therefore, we proposed that ZNF645 is a CT gene and activated in human testis and lung cancers by demethylation of its promoter region.
Cancer/testis (CT) antigens exhibit highly tissue-restricted expression and are considered promising targets for cancer vaccines. Here we identified a novel CT gene ZNF645 which restrictively expresses in normal human testes and lung cancer patients (68.3%). To investigate the promoter methylation status of ZNF645, we carried out bisulfite genomic sequencing and found that the CpG island in its promoter was heavily methylated in normal lung tissues without the expression of ZNF645, whereas there was high demethylation in normal human testes and lung carcinoma tissues with its expression. Also ZNF645 could be remarkably activated in A549 and HEK293T cells treated by DNA demethylation agent 5'-aza-2'-deoxycytidine. And the dual luciferase assay revealed that the promoter activity of the ZNF645 was inhibited by methylation of the CpG island region. Therefore, we proposed that ZNF645 is a CT gene and activated in human testis and lung cancers by demethylation of its promoter region.
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제안 방법
Then we performed in vitro methylation analysis to confirm whether the promoter activities of ZNF645 gene were inhibited by CGI methylation. The HepG2 cells were transiently transfected with the P1 and P3 promoter constructs methylated with SssI (CpG) methylase and the untreated P1 and P3 mock construct vectors, and the following luciferase activities were measured respectively. As shown in Fig.
The PCR amplification was carried out with the following modifications: Semi-nested PCR amplified the CpG island region of the ZNF645 gene promoter; the first PCR primer set was NESTF: 5’-TAGGGTTTGTTGGGAATTTATT-3’ and NESTR:5’-CACATTCTTATTCACCAACAAAC-3’; then 1 μl of the PCR product was used for the second PCR amplification.
Total RNA was isolated from cell lines and tissues using Trizol reagent (Invitrogen, CA) according to the manufacturer’s protocols and 2 μg RNA was reverse transcribed into single-strand cDNA in 20 μl of reaction buffer using Molony murine leukemia virus reverse transcriptase (Promega, USA) and oligo (dT)15 (Promega, USA) as a primer. Then RT-PCR was performed as follows: 94℃, 15 s; 60℃, 30 s; and 72℃, 30 s; 30 cycles, followed by 5 min at 72℃. Visualization of target bands on a 1.
2A) (14, 15). Then we performed bisulfite genomic sequencing to examine the methylation status of selected ZNF645 CGI in genomic DNA extracted from normal human testis and lung cancer tumors respectively. It was found that the 13 CpGs are highly unmethylated in the normal human testis tissue (Fig.
Then we performed in vitro methylation analysis to confirm whether the promoter activities of ZNF645 gene were inhibited by CGI methylation. The HepG2 cells were transiently transfected with the P1 and P3 promoter constructs methylated with SssI (CpG) methylase and the untreated P1 and P3 mock construct vectors, and the following luciferase activities were measured respectively.
To examine the effect of methylation status in 5' upstream CGI region of ZNF645 gene in the regulation of gene expression in vitro, we cloned several 5' upstream sequences of ZNF645 and subcloned them into the pGL3 basic reporter vector (Fig. 3A).
대상 데이터
Strong demethylation was observed in testis and two cancer tissues while heavy methylation was shown in paired noncancerous tissues. Five clones of each sample were sequenced. Solid circle: methylated CpG site; hollowed circle: unmethylated CpG site.
Normal tissue cDNA panels (multiple tissue cDNA panels I and II) composed of human brain, colon, heart, kidney, leukocytes, liver, lung, ovary, pancreas, placenta, prostate, skeletal muscle, small intestine, spleen, thymus, and testis were purchased from Clontech (Palo Alto, USA). Total RNA was isolated from cell lines and tissues using Trizol reagent (Invitrogen, CA) according to the manufacturer’s protocols and 2 μg RNA was reverse transcribed into single-strand cDNA in 20 μl of reaction buffer using Molony murine leukemia virus reverse transcriptase (Promega, USA) and oligo (dT)15 (Promega, USA) as a primer.
이론/모형
Genomic DNA was extracted from the tissues and cell lines by standard phenol-chloroform methods (31). Bisulfite treatment was carried out by using the DNA Methylation-Gold kit (Zymo Research, USA) according to the manufacturer’s instructions.
성능/효과
All of these results indicated that the expression of ZNF645 in the two cell lines was regulated by the methylation of CGI in the 5' upstream region.
It was observed that there existed DNA hypomethylation of the 5' flanking CGI region of ZNF645 in testis and some lung cancer cases whereas heavy DNA methylation was observed in the same region of the corresponding adjacent non-cancer tissue without ZNF645 expression.
Immunohistochemistry (IHC) was carried out to analyze the location of the ZNF645 protein by using sections of formalin-fixed, paraffin-embedded sections of lung cancer tissues and paired adjacent non-cancerous tissues. The results showed that ZNF645 protein was predominantly present in the cytoplasm of lung cancer cells (Fig. 1C).
And the similar results were also found when transfecting A549 cells (data not shown). These results further confirmed the hypothesis that the promoter activity of ZNF645 could be repressed by DNA methylation and the expression of ZNF645 was mediated by CGI methylation.
To further confirm that hypermethylation is responsible for the silencing of the ZNF645, a DNA methyl-transferase inhibitor 5'-aza-CdR was used to treat human embryo kidney cell line 293T and lung cancer cell line A549 cells in which ZNF645 expression is negative.
후속연구
(16-18), which play an important role in carcinomagenesis as CT antigen. We will carry out further investigation on the biological function of ZNF645 in tumor and testis.
참고문헌 (31)
Scanlan, M., Gure, A., Jungbluth, A., Old, L. and Chen, Y. (2002) Cancer/testis antigens: an expanding family of targets for cancer immunotherapy. Immunol Rev. 188, 22-32.
Scanlan, M., Simpson, A. and Old, L. (2004) The cancer/testis genes: review, standardization, and commentary. Cancer Immun. 4, 1.
van der Bruggen, P., Traversari, C., Chomez, P., Lurquin, C., De Plaen, E., Van den Eynde, B., Knuth, A. and Boon, T. (1991) A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 254, 1643-1647.
Tajima, K., Obata, Y., Tamaki, H., Yoshida, M., Chen, Y., Scanlan, M., Old, L., Kuwano, H., Takahashi, T. and Mitsudomi, T. (2003) Expression of cancer/testis (CT) antigens in lung cancer. Lung Cancer 42, 23-33.
De Smet, C., Lurquin, C., Lethe, B., Martelange, V. and Boon, T. (1999) DNA methylation is the primary silencing mechanism for a set of germ line- and tumor-specific genes with a CpG-rich promoter. Mol. Cell. Biol. 19, 7327-7335.
Grunwald, C., Koslowski, M., Arsiray, T., Dhaene, K., Praet, M., Victor, A., Morresi-Hauf, A., Lindner, M., Passlick, B., Lehr, H., Schafer, S., Seitz, G., Huber, C., Sahin, U. and Tureci, O. (2006) Expression of multiple epigenetically regulated cancer/germline genes in nonsmall cell lung cancer. Int. J. Cancer 118, 2522-2528.
Sigalotti, L., Fratta, E., Coral, S., Tanzarella, S., Danielli, R., Colizzi, F., Fonsatti, E., Traversari, C., Altomonte, M. and Maio, M. (2004) Intratumor heterogeneity of cancer/testis antigens expression in human cutaneous melanoma is methylation-regulated and functionally reverted by 5-aza-2'-deoxycytidine. Cancer Res. 64, 9167-9171.
Dong, X., Yang, X., Wang, Y. and Chen, W. (2004) Zincfinger protein ZNF165 is a novel cancer-testis antigen capable of eliciting antibody response in hepatocellular carcinoma patients. Br. J. Cancer 91, 1566-1570.
Loukinov, D., Pugacheva, E., Vatolin, S., Pack, S., Moon, H., Chernukhin, I., Mannan, P., Larsson, E., Kanduri, C., Vostrov, A., Cui, H., Niemitz, E., Rasko, J., Docquier, F., Kistler, M., Breen, J., Zhuang, Z., Quitschke, W., Renkawitz, R., Klenova, E., Feinberg, A., Ohlsson, R., Morse, H. R. and Lobanenkov, V. (2002) BORIS, a novel male germline-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma. Proc. Natl. Acad. Sci. U.S.A. 99, 6806-6811.
Lim, F., Soulez, M., Koczan, D., Thiesen, H. and Knight, J. (1998) A KRAB-related domain and a novel transcription repression domain in proteins encoded by SSX genes that are disrupted in human sarcomas. Oncogene. 17, 2013-2018.
Gama-Sosa, M., Slagel, V., Trewyn, R., Oxenhandler, R., Kuo, K., Gehrke, C. and Ehrlich, M. (1983) The 5-methylcytosine content of DNA from human tumors. Nucleic. Acids. Res. 11, 6883-6894.
De Smet, C., De Backer, O., Faraoni, I., Lurquin, C., Brasseur, F. and Boon, T. (1996) The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation. Proc. Natl. Acad. Sci. U.S.A. 93, 7149-7153.
Iguchi-Ariga, S. and Schaffner, W. (1989) CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation. Genes Dev. 3, 612-619.
Coral, S., Sigalotti, L., Altomonte, M., Engelsberg, A., Colizzi, F., Cattarossi, I., Maraskovsky, E., Jager, E., Seliger, B. and Maio, M. (2002) 5-aza-2'-deoxycytidine-induced expression of functional cancer testis antigens in human renal cell carcinoma: immunotherapeutic implications. Clin. Cancer. Res. 8, 2690-2695.
Christman, J. (2002) 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene. 21, 5483-5495.
Coral, S., Sigalotti, L., Gasparollo, A., Cattarossi, I., Visintin, A., Cattelan, A., Altomonte, M. and Maio, M. (1999) Prolonged upregulation of the expression of HLA class I antigens and costimulatory molecules on melanoma cells treated with 5-aza-2'-deoxycytidine (5-AZA-CdR). J. Immunother 22, 16-24.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York, USA.
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