Microarray Analysis of Epigenetic Silencing of Gene Expression in the KAS-6/1 Multiple Myeloma Cell Line

The epigenetic control of gene transcription in cancer has been the theme of many recent studies and therapeutic approaches. Carcinogenesis is frequently associated with hypermethylation and consequent down-regulation of genes that prevent cancer, e.g., those that control cell proliferation and apoptosis. We used the demethylating drug zebularine to induce changes in DNA methylation, then examined patterns of gene expression using cDNA array analysis and Restriction Landmark Genomic Scanning followed by RNase protection assay and reverse transcription-PCR to confirm the results. Microarray studies revealed that many genes were epigenetically regulated by methylation. We concluded that methylation decreased the expression of, or silenced, several genes, contributing to the growth and survival of multiple myeloma cells. For example, a number of genes (BAD, BAK, BIK, and BAX) involved in apoptosis were found to be suppressed by methylation. Sequenced methylation-regulated DNA fragments identified by Restriction Landmark Genomic Scanning were found to contain CpG islands, and some corresponded to promoters of genes that were regulated by methylation. We also observed that after the removal of the demethylating drug, the addition of interleukin 6 restored CpG methylation and re-established previously silenced gene patterns, thus implicating a novel role of interleukin 6 in processes regulating epigenetic gene repression and carcinogenesis.

[1]  N. Callander,et al.  AML-1A and AML-1B regulation of MIP-1alpha expression in multiple myeloma. , 2003, Blood.

[2]  W. Farrar,et al.  IL-6 induces expression of the Fli-1 proto-oncogene via STAT3. , 2002, Biochemical and biophysical research communications.

[3]  T. Kinoshita,et al.  Production and effects of interleukin-6 and other cytokines in patients with non-Hodgkin's lymphoma. , 1998, Leukemia & lymphoma.

[4]  D. Hornby,et al.  Zebularine: a novel DNA methylation inhibitor that forms a covalent complex with DNA methyltransferases. , 2002, Journal of Molecular Biology.

[5]  J. Ward,et al.  IL-6 transgenic mouse model for extraosseous plasmacytoma , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[6]  G. Chenevix-Trench,et al.  Reduced expression of intercellular adhesion molecule-1 in ovarian adenocarcinomas , 2001, British Journal of Cancer.

[7]  K. Robertson,et al.  Chromatin remodeling, histone modifications, and DNA methylation—how does it all fit together? , 2002, Journal of cellular biochemistry.

[8]  M. Szyf,et al.  Interleukin-6 Regulation of the Human DNA Methyltransferase (HDNMT) Gene in Human Erythroleukemia Cells* , 2001, The Journal of Biological Chemistry.

[9]  B. Ryan,et al.  Aminosalicylates and colorectal cancer in IBD: a not-so bitter pill to swallow , 2003, American Journal of Gastroenterology.

[10]  K. Kinzler,et al.  Analysis of the 5' region of PMS2 reveals heterogeneous transcripts and a novel overlapping gene. , 1995, Genomics.

[11]  Chun Xing Li,et al.  Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3 , 2000, Nature Genetics.

[12]  T. Sawada,et al.  Expression of intercellular adhesion molecule-1 and prognosis in colorectal cancer. , 2002, Oncology reports.

[13]  H. Mitsuya,et al.  Down-Regulation of CD98 in Melphalan-Resistant Myeloma Cells with Reduced Drug Uptake , 2000, Acta Haematologica.

[14]  J. Minna,et al.  Aberrant methylation of TMS1 in small cell, non small cell lung cancer and breast cancer , 2003, International journal of cancer.

[15]  D. Tindall,et al.  The role of PTEN in the progression and survival of prostate cancer. , 2003, Minerva endocrinologica.

[16]  A. Blann,et al.  Increased soluble intercellular adhesion molecule-1, breast cancer and the acute phase response , 2002, Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis.

[17]  Peter A. Jones,et al.  Analysis of gene induction in human fibroblasts and bladder cancer cells exposed to the methylation inhibitor 5-aza-2'-deoxycytidine. , 2002, Cancer research.

[18]  C. Huff,et al.  Characterization of clonogenic multiple myeloma cells. , 2004, Blood.

[19]  R. Rintoul,et al.  Cross-linking CD98 promotes integrin-like signaling and anchorage-independent growth. , 2002, Molecular biology of the cell.

[20]  J. Plowman,et al.  Antitumor properties of 2(1H)-pyrimidinone riboside (zebularine) and its fluorinated analogues. , 1991, Journal of medicinal chemistry.

[21]  N. Callander,et al.  AML-1A and AML-1B regulation of MIP-1α expression in multiple myeloma , 2003 .

[22]  En Li,et al.  De novo methylation of MMLV provirus in embryonic stem cells: CpG versus non-CpG methylation. , 2002, Gene.

[23]  M. Younes,et al.  Wide expression of the human erythrocyte glucose transporter Glut1 in human cancers. , 1996, Cancer research.

[24]  Y. Tomizawa,et al.  Epigenetic inactivation of RASSF1A candidate tumor suppressor gene at 3p21.3 in brain tumors , 2003, Oncogene.

[25]  E. S. Yang,et al.  Human sensitive to apoptosis gene protein inhibits peroxynitrite-induced DNA damage. , 2003, Biochemical and biophysical research communications.

[26]  M. Lin,et al.  cDNA Cloning and Expression of a Human Aldehyde Dehydrogenase (ALDH) Active with 9-cis-Retinal and Identification of a Rat Ortholog, ALDH12* , 2000, The Journal of Biological Chemistry.

[27]  E. Eisenhauer,et al.  DNA methyltransferase inhibitors-state of the art. , 2002, Annals of oncology : official journal of the European Society for Medical Oncology.

[28]  Dominique Mouchiroud,et al.  CpGProD: identifying CpG islands associated with transcription start sites in large genomic mammalian sequences , 2002, Bioinform..

[29]  P. Musiani,et al.  Coexpression of IL‐6 and soluble IL‐6R causes nodular regenerative hyperplasia and adenomas of the liver , 1998, The EMBO journal.

[30]  B. Halliwell,et al.  Free radicals in biology and medicine , 1985 .

[31]  A. Hamburger,et al.  Repression of E2F1-mediated transcription by the ErbB3 binding protein Ebp1 involves histone deacetylases. , 2003, Nucleic acids research.

[32]  Y. Tomita,et al.  Hypermethylation of death‐associated protein (DAP) kinase CpG island is frequent not only in B‐cell but also in T‐ and natural killer (NK)/T‐cell malignancies , 2003, Cancer science.

[33]  M. Frommer,et al.  CpG islands in vertebrate genomes. , 1987, Journal of molecular biology.

[34]  M. Hung,et al.  Systemic tumor suppression by the proapoptotic gene bik. , 2002, Cancer research.

[35]  Yilun Sun,et al.  SAG/ROC/Rbx/Hrt, a zinc RING finger gene family: molecular cloning, biochemical properties, and biological functions. , 2001, Antioxidants & redox signaling.

[36]  P. Koistinen,et al.  All-trans retinoic acid induces apoptosis in acute myeloblastic leukaemia cells , 2004, Apoptosis.

[37]  J. Tong,et al.  Alterations of RAS signalling in Chinese multiple myeloma patients: absent BRAF and rare RAS mutations, but frequent inactivation of RASSF1A by transcriptional silencing or expression of a non‐functional variant transcript , 2003, British journal of haematology.

[38]  C. Plass,et al.  A new tool for the rapid cloning of amplified and hypermethylated human DNA sequences from restriction landmark genome scanning gels. , 1999, Genomics.

[39]  P. Schwartsburd.  Chronic inflammation as inductor of pro-cancer microenvironment: Pathogenesis of dysregulated feedback control , 2003, Cancer and Metastasis Reviews.

[40]  E. Jost,et al.  Molecular cloning of a murine cDNA encoding a novel protein, p38-2G4, which varies with the cell cycle. , 1995, Experimental cell research.

[41]  G. Ahmann,et al.  Establishment and characterization of three myeloma cell lines that demonstrate variable cytokine responses and abilities to produce autocrine interleukin-6. , 1996, Leukemia.

[42]  A. Bird,et al.  Number of CpG islands and genes in human and mouse. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Y. Hayashizaki,et al.  A genomic scanning method for higher organisms using restriction sites as landmarks. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[44]  M. Giphart-Gassler,et al.  Identification of RUNX1/AML1 as a classical tumor suppressor gene , 2003, Oncogene.

[45]  P. Heinrich,et al.  Principles of interleukin (IL)-6-type cytokine signalling and its regulation. , 2003, The Biochemical journal.

[46]  L. Zacharski.  Anticoagulants in cancer treatment: malignancy as a solid phase coagulopathy. , 2002, Cancer letters.

[47]  R. Genta.  The Gastritis Connection: Prevention and Early Detection of Gastric Neoplasms , 2003, Journal of clinical gastroenterology.

[48]  B. Dörken,et al.  Macrophage inflammatory protein 1-alpha (MIP-1 alpha ) triggers migration and signaling cascades mediating survival and proliferation in multiple myeloma (MM) cells. , 2003, Blood.

[49]  J. Blake,et al.  Creating the Gene Ontology Resource : Design and Implementation The Gene Ontology Consortium 2 , 2001 .

[50]  David A Jones,et al.  Reactivating the expression of methylation silenced genes in human cancer , 2002, Oncogene.

[51]  J. Minna,et al.  The RASSF1A Tumor Suppressor Blocks Cell Cycle Progression and Inhibits Cyclin D1 Accumulation , 2002, Molecular and Cellular Biology.

[52]  K. Nephew,et al.  Epigenetic gene silencing in cancer initiation and progression. , 2003, Cancer letters.