Retinoylation (covalent modification by retinoic acid) of Rho-GDIß in the human myeloid leukemia cell line HL60 and its functional significance.

Retinoic acid (RA) has a variety of biological effects in mammalian cells and tissues. It is well known that RA induces differentiation of human acute promyelocytic leukemia (APL) HL60 cells, fresh human APL cells, and clinical remission in patients with APL. Retinoylation (acylation of proteins by RA) is a possible pathway for RA action. However, an understanding of the role that retinoylation plays in the actions of RA is lacking. In the current study, several retinoylated proteins were detected in RA-treated HL60 fractions following Mono Q anion exchange chromatography and analysis using two-dimensional polyacrylamide gel electrophoresis. One of the retinoylated proteins was identified as Rho-GDIß (28kDa) by TOF-MS and co-migration with Rho-GDIß (28kDa). Truncated Rho-GDIß (23kDa, N∆19), a product of cleavage by caspase-3, was not retinoylated. RA covalently bound to the Thr2 residue in Rho-GDIß (5kDa), which is the second product resulting from the cleavage of Rho-GDIß (28kDa) by caspase-3. RA treatment increased the level of Rho-GDIß (28kDa) and decreased the level of Rho-GDIß (23kDa). RA did not induce caspase-3 activity or Rho-GDIß mRNA expression. It is likely that retinoylation of Rho-GDIß increases its metabolic stability.

Reactivation of CDX2 in Gastric Cancer as Mark for Gene Silencing Memory.

To explore the epigenetic mechanism that reactivates CDX2 (a homeobox transcription factor that serves as a tumor-suppressor gene) in intestinal-type gastric cancer during cancer progression, we examined the methylation status of the CDX2 gene promoter and the expression pattern of methyl-CpG binding protein-2 (MeCP2). From archives of the pathology records of surgically excised advanced stomach cancer cases in the Department of Molecular Pathology, Ehime University in a past decate (n=265), 10 cases of intestinal-type tubular adenocarcinoma, well-differentiated type (wel) with minor poorly-differentiated adenocarcinoma (por) components were selected. The expression pattern of CDX2, MUC2 and MeCP2 in these 10 cases was analyzed by immunohistochemistry. The cancerous and non-cancerous areas were selectively obtained by microdissection, and the methylation status of the CDX2 promoter of each area was assessed by methylation-specific polymerase chain reaction (MSP). In all 10 cases, CDX2 expression was clearly observed in the nucleus of the non-cancerous background of the intestinal metaplasic area, where the unmethylation pattern of the CDX2 gene promoter prevailed with reduced MeCP2 expression. In this metaplastic area, CDX2 expression was co-localized with its target gene, MUC2. CDX2 expression then disappeared from the deep invasive wel area. Reflecting the reduced CDX2 expression, microdissected samples from all the wel areas showed hypermethylation of the CDX2 gene promoter by MSP, with prominent MeCP2 expression. Interestingly, while hypermethylation of the CDX2 gene promoter was maintained in the por area in 8 of the 10 cases, CDX2 expression was restored in por areas where MeCP2 expression was markedly and selectively reduced. The other two cases, however, showed a constant MeCP2 expression level comparable to the surrounding deep invasive wel area with negative CDX2 expression. Therefore, gene silencing by hypermethylation may be overcome by the reduction of methyl-CpG binding proteins, resulting in apparent but non-functional reactivation of CDX2 as a mere molecular mark for gene silencing memory.