| Description |
The nucleotide 5-methyl-2'-deoxycytidine (mdC) has been known since the late 1940s; however, its epigenetic role in human disease has been the subject of increasingly intense research over the last decade. Genomic methylation changes are early though not initial events in carcinogenesis. Methylation utilizes a family of proteins known collectively as methyl-CpG binding domain or MBD proteins. Mutations in the methyl-CpG binding protein 2 (MeCP2) gene, involved in methylation-mediated silencing, have been linked to the developmental disorder Rett Syndrome. Mutation in MBD4, or loss of expression of MBD2, is also associated with disease phenotypes. MBD2 has two isoforms of interest based on their unique binding activities and varied protein partners. Little is known, however, regarding the sites of activity for MBD2. Only two gene promoter sites have been identified to date as specific target loci for MBD2 binding, p16 and the pi class of glutathione S-transferase (GSTP1). Interest in both the normal and disease aspects of methylation as well as the histone modification and chromatin remodeling associated with the MBD proteins has prompted discoveries into new potential chemotherapeutics. Methylation can be inhibited using nucleotide analogs such as 5-aza-2'-deoxycytidine, however this type of inhibition generates serious side effects. We hypothesized that reduction of the expression of MBD2 could act as an alternative to reducing or eliminating methylation of the genome in that it might allow re-expression of methylation-silenced genes. We have identified a small interfering RNA (siRNA), named M4, which reduces the messenger RNA and protein levels of methyl-CpG binding domain protein 2 (MBD2) in RKO colon cancer cells. Reduction of MBD2 mRNA and protein levels by M4 siRNA introduction did not produce gross morphological changes, alter cellular viability or reduce heterochromatin condensation after 48 h. M4 siRNA against MBD2 however, caused changes in gene transcription in RKO and other cancer cells. Some of the gene pathways induced by M4 siRNA in RKO cells are the similar to gene pathways induced by a colon cancer cell line undergoing differentiation. The M4 induced transcriptional changes are not identical to the CaCo2 differentiation model however, and the transcriptional effects in other colon and cervical cancer cells suggest that MBD2 may play distinct roles in cells. This variation in MBD2 affected genes may be dependent upon differentiation levels, genetic background or tissue type. The genes induced by M4 were not induced by an unrelated siRNA against a transcriptional activator the caudal-related homeobox 2 (Cdx2) gene. In fact, M4 and Cdx2-1 siRNA treatments appeared to produce opposing results. MBD2 failed to induce expression from well-known methylation silenced tumor suppressor genes. This is in direct contrast to treatment with 5-aza-2'-deoxycytidine. The importance of this work is that it indicates that MBD2 may play a unique role affecting gene transcription in specific cancers, and that reduction of MBD2 does not mimic treatment with 5-aza-2'-deoxycytidine. MBD2 may therefore not provide a novel target for therapeutic intervention in lieu of current de-methylating treatments. Further study is needed to ascertain a more complete list of the genes affected by MBD2 activity. |
