Sains Malaysiana 49(1)(2020): 161-168

http://dx.doi.org/10.17576/jsm-2020-4901-20

 

DNA Methylation of Human Choline Kinase Alpha Gene

(Metilasi DNA Gen Alfa Kolina Kinase Manusia)

 

SITI AISYAH FATEN MOHAMED SA'DOM, NUR FARAH HANI AZEMI, MOHAMMAD SHAHRIZAL MOHAMMAD UMAR, YOKE HIANG YEE, WEI CUN SEE TOO & LING LING FEW*

 

School of Health Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan Darul Naim, Malaysia

 

Received: 12 August 2018/Accepted: 17 October 2019

 

Abstract

Increased level of choline kinase (CK) is a common feature in cancers and inhibition of this enzyme has been applied as anticancer strategy. DNA methylation of gene promoter especially at CpG island is associated with suppression of gene expression. Despite the importance of CK especially the alpha isoform in cancer pathogenesis, epigenetic regulation of cka expression has not been investigated. Hence, this study aimed to determine the effect of DNA methylation on cka promoter activity and gene expression by using hypomethylating (5-aza) and methylating (budesonide) agents. The level of DNA methylation in the second CpG island of cka promoter was determined by PCR-based method. 5-aza and budesonide increased the methylation of the selected CpG island compared to untreated control. Treatment with the drugs produced opposite effect, with 5-aza induced cka promoter activity and gene expression while budesonide suppressed the promoter activity and mRNA level of this gene. Deletion of a region containing the second CpG island on cka promoter resulted in significantly lower promoter activity. In conclusion, this study showed that DNA methylation could be one of the mechanisms that regulate the expression of cka gene.

 

Keywords: Choline kinase; DNA methylation; epigenetics; gene expression

 

Abstrak

Peningkatan tahap kolina kinase (CK) adalah ciri umum kanser dan perencatan enzim ini telah diguna sebagai strategi antikanser. Metilasi DNA promoter gen terutamanya pada pulau CpG telah dikaitkan dengan perencatan pengekspresan gen. Walaupun CK, terutamanya isoforma alfa, penting dalam patogenesis kanser, pengawalaturan epigenetik pengekspresan cka masih belum pernah dikaji. Maka, kajian ini bertujuan menentukan kesan metilasi DNA terhadap aktiviti promoter dan pengekspresan gen cka dengan menggunakan agen hipometilasi (5-aza) dan metilasi (budesonida). Tahap metilasi DNA pada pulau CpG kedua pada promoter cka telah ditentukan dengan kaedah berdasarkan PCR. 5-aza dan budesonida meningkatkan tahap metilasi pulau CpG yang dikaji berbanding kawalan tanpa rawatan. Rawatan dengan agen ini menghasilkan kesan berlawanan dengan 5-aza merangsang aktiviti promoter dan pengekspresan gen cka manakala budesonida merencat aktiviti promoter dan tahap mRNA gen ini. Pemadaman salah satu kawasan yang mengandungi pulau CpG kedua pada promoter cka menyebabkan penurunan secara signifikan aktiviti promoter. Kesimpulannya, kajian ini menunjukkan metilasi DNA mungkin merupakan salah satu mekanisme yang mengawalatur pengekspresan gen cka.

 

Kata kunci: Epigenetik; kolina kinase; metilasi DNA; pengekspresan gen

 

REFERENCES

Alyaqoub, F.S., Tao, L., Kramer, P.M., Steele, V.E., Lubet, R.A., Gunning, W.T. & Pereira, M.A. 2007. Prevention of mouse lung tumors and modulation of DNA methylation by combined treatment with budesonide and R115777 (ZarnestraMT). Carcinogenesis 28(1): 124-129.

Bird, A.P. 1986. CpG-rich islands and the function of DNA methylation. Nature 321(6067): 209-213.

Bird, A. 2007. Perceptions of epigenetics. Nature 447(7143): 396-398.

Christman, J.K. 2002. 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: Mechanistic studies and their implications for cancer therapy. Oncogene 21(35): 5483-5495.

Ciechomska, M., Roszkowski, L. & Maslinski, W. 2019. DNA methylation as a future therapeutic and diagnostic target in rheumatoid arthritis. Cells8(9): E953.

Comb, M. & Goodman, K.M. 1990. CpG methylation inhibits proenkephalin gene expression and binding of the transcription factors AP-2. Nucleic Acids Research 18(13): 3975-3982.

Das, P.M. & Singal, R. 2004. DNA methylation and cancer. Journal Clinical Oncology 22(22): 4632-4642.

Deaton, A.M. & Bird, A. 2011. CpG islands and regulation of transcription. Genes & Development 25(10): 1010-1022.

Du, X., Han, L., Guo, A.Y. & Zhao, Z. 2012. Features of methylation and gene expression in the promoter-associated CpG islands using human methylome data. Comparative and Functional Genomics 2(6): 775-780.

Gailhouse, L., Liew, L.C., Hatada, I., Nakagama, H. & Ochiya, T. 2018. Epigenetic reprogramming using 5-azacytidine promotes an anti-cancer response in pancreatic adenocarcinoma cells. Cell Death and Disease 9: 468. doi: 10.1038/s41419-018-0487-z.

Gallego-Ortega, D., Gómez del Pulgar, T., Valdés-Mora, F., Cebrián, A. & Lacal, J.C. 2011. Involvement of human choline kinase alpha and beta in carcinogenesis: A different role in lipid metabolism and biological functions. Advances in Enzyme Regulation 51(1): 183-194.

Gómez-Perez, V., McSorley, T., See Too, W.C., Konrad, M. & Campos, J.M. 2012. Novel 4-amino bis-pyridinium and bis-quinolinium derivatives as choline kinase inhibitors with antiproliferative activity against the human breast cancer SKBR-3 cell line. ChemMedChem 7(4): 663-669.

Gruber, J., See Too, W.C., Wong, M.T., Lavie, A., McSorley, T. & Konrad, M. 2012. Balance of human choline kinase isoforms is critical for cell cycle regulation: Implications for the development of choline kinase-targeted cancer therapy. FEBS Journal 279(11): 1915-1928.

Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77(1): 51-59.

Hon, G.C., Hawkins, R.D., Caballero, O.L., Lo, C., Lister, R., Pelizzola, M., Valsesia, A., Ye, Z., Kuan, S., Edsall, L.E., Camargo, A.A., Stevenson, B.J., Ecker, J.R., Bafna, V., Strausberg, R.L., Simpson, A.J. & Ren, B. 2012. Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer. Genome Research 22(2): 246-258.

Kholod, N., Boniver, J. & Delvenne, P. 2007. A new dimethyl sulfoxide-based method for gene promoter methylation detection. The Journal of Molecular Diagnostics 9(5): 574-581.

Kurdyukov, S. & Bullock, M. 2016. DNA methylation analysis: Choosing the right method. Biology 9: 3. doi: 10.3390/biology5010003.

Lacal, J.C. 2015. Choline kinase as a precision medicine target for therapy in cancer, autoimmune diseases and malaria. Precision Medicine 2: e980. doi: 10.14800/pm.980.

Liang, H., Kowalczyk, P., Junco, J.J., Santiago, K.D., Heather, L., Malik, G., Sung-Jen, W. & Slaga, T.J. 2014. Differential effects on lung cancer cell proliferation by agonists of glucocorticoid and PPARα receptors. Molecular Carcinogenesis 53(9): 753-763.

Lykidis, A., Wang, J., Karim, M.A. & Jackowski, S. 2001. Overexpression of a mammalian ethanolamine-specific kinase accelerates the CDP-ethanolamine pathway. Journal of Biological Chemistry 276(3): 2174-2179.

Malito, E., Sekulic, N., See Too, W.C., Konrad, M. & Arnon, L. 2006. Elucidation of human choline kinase crystal structures in complex with the products ADP or phosphocholine. Journal of Molecular Biology 364(2): 136-151.

Nan, X., Ng, H.H., Johnson, C.A., Laherty, C.D., Turner, B.M., Eisenmen, R.N. & Bird, A. 1998. Transcriptional repression by methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 393(6683): 386-389.

Pereira, M.A., Tao, L., Liu, Y., Li, L., Steele, V.E. & Lubet, R.A. 2006. Modulation by budesonide of DNA methylation and mRNA expression in mouse lung tumors. International Journal of Cancer 120: 1150-1153.

Prokhortchouk, E. & Hendrich, B. 2002. Methyl-CpG binding proteins and cancer: Are MeCpGs more important than MBDs? Oncogene 21(35): 5394-5399.

Ramirez de Molina, A., Gallego-Ortega, D., Sarmentero-Estrada, J., Lagares, D., Gómez Del Pulgar, T., Bandrés, E., García-Foncillas, J. & Lacal, J.C. 2008. Choline kinase as a link connecting phospholipid metabolism and cell cycle regulation: Implications in cancer therapy. The International Journal of Biochemistry & Cell Biology 40(9): 1753-1763.

Ramirez de Molina, A., Gutierrez, R., Ramos, M.A., Silva, J.M., Silva, J., Bonilla, F., Sanchez, J.J. & Lacal, J.C. 2002. Increased choline kinase activity in human breast carcinomas: Clinical evidence for a potential novel antitumor strategy. Oncogene 21(27): 4317-4322.

Rodriguez-Gonzalez, A., Ramirez de Molina, A., Fernandez, F. & Lacal, J.C. 2004. Choline kinase inhibition induces the increase in ceramides resulting in a highly specific and selective cytotoxic antitumoral strategy as a potential mechanism of action. Oncogene 23(50): 8247-8259.

Sasai, N., Nakao, M. & Defossez, P. 2010. Sequence-specific recognition of methylated DNA by human zinc-finger proteins. Nucleic Acids Research 38(15): 5015-5022.

Shah, T., Wildes, F., Penet, M.F., Winnard Jr., P.T., Glunde, K., Artemov, T., Ackerstaff, E., Gimi, B., Kakkad, S., Raman, V. & Bhujwalla, Z.M. 2010. Choline kinase overexpression increases invasiveness and drug resistance of human breast cancer cells. NMR in Biomedicine 23(6): 633-642.

Singal, R., Wang, S.Z., Sargent, T., Zhu, S.Z. & Ginder, G.D. 2002. Methylation of promoter proximal-transcribed sequences of an embryonic globin gene inhibits transcription in primary erythroid cells and promotes formation of a cell type-specific methyl cytosine binding complex. Journal of Biological Chemistry 277(3): 1897-1905.

Veronesi, G., Lazzeroni, M., Szabo, E., Brown, P.H., DeCensi, A., Guerrieri-Gonzaga, A., Bellomi, M., Radice, D., Grimaldi, M.C., Spaggiari, L. & Bonanni, B. 2015. Long-term effects of inhaled budesonide on screening-detected lung nodules. Annals of Oncology 26(5): 1025-1030. doi: 10.1093/annonc/mdv064.

Watt, F. & Molloy, P.L. 1988. Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter. Genes & Development 2(9): 1136-1143.

Wu, G. & Vance, D.E. 2010. Choline kinase and its function. Biochemistry and Cell Biology 88(4): 559-564.

 

*Corresponding author; email: fewling@usm.my

 

 

 

previous