Promoter-hypermethylation is causing functional relevant downregulation of methylthioadenosine phosphorylase ( MTAP ) expression in hepatocellular carcinoma

Abstract

The methylthioadenosine phosphorylase (MTAP) gene is localized in the chromosomal region 9p21. Here, frequently homozygous deletions occur in several kinds of cancer associated with the loss of tumour suppressor genes as p16 and p15. The aim of this study was to analyse MTAP expression in hepatocellular carcinoma (HCC) and to get an insight into the regulation and functional role of MTAP in hepatocancerogenesis. Compared with primary human hepatocytes MTAP expression was markedly downregulated in three different HCC cell lines as determined by real-time PCR and western blotting. This was not due to genomic losses or mutations but to promoter-hypermethylation. Reduced MTAP-expression was confirmed in vivo in HCC compared with non-cancerous liver tissue on both mRNA and protein levels. To study the functional relevance of the downregulated MTAP expression in HCC, MTAP expression was re-induced in HCC cell lines by stable transfection. In these MTAP re-expressing cell clones the invasive potential was strongly reduced, whereas no effects on cell proliferation were observed in comparison with mock transfected cell clones. Furthermore, in MTAP re-expressing cells interferon (IFN)-alpha and IFN-gamma induced a significantly stronger inhibition of cell proliferation than in mock transfected cells. In conclusion, our results suggest a functional role of MTAP inactivation in HCC development and invasiveness. Furthermore, in the light of a recent report revealing an association between MTAP activity and IFN sensitivity, our findings may have clinical significance for therapeutic strategies.

Hepatocellular carcinoma cells surviving doxorubicin treatment exhibit increased migratory potential and resistance to doxorubicin re-treatment in�vitro

“…Cells were seeded in 6-well plates (200,000/well) or 96-well plates (30,000/well), respectively. After 24 h, analysis of lactate dehydrogenase (LDH) secretion into the supernatant (Cytotoxicity Detection Kit PLUS; Roche Diagnostics GmbH, Mannheim, Germany) and a colorimetric XTT assay (Roche Diagnostics GmbH) were used to analyze the viability of HCC cells subsequent to treatment with doxorubicin as described (24). Cell proliferation was assessed using the xCELLigence impedance measurement system (Roche Diagnostics GmbH) according to the manufacturer's protocol.…”

“…Total cellular RNA was isolated from doxorubicin-treated and control HepG2 and Hep3B cells using the RNeasy Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. Reverse transcription was performed as described previously (24). Quantitative polymerase chain reaction was performed using a LightCycler Real-Time PCR System (Roche Diagnostics) (24).…”

GLUT1 Expression Is Increased in Hepatocellular Carcinoma and Promotes Tumorigenesis

“…25 Quantitative real-time polymerase chain reaction (PCR) was performed with primers specific for GLUT1 (forward: 5Ј-AACTCTTCAGCCAGGGTC-CAC; reverse: 5Ј-CACAGTGAAGATGATGAAGAC) using LightCycler technology (Roche, Mannheim, Germany). 26 Expression of MAZ was analyzed applying the QuantiTect primer assay according to the manufacturer's instructions (Qiagen, Hilden, Germany).…”

“…25 Further, cell number was determined by microscopic counting after trypsination of cells seeded in six-well plates (six per condition) at different time points. Migration assays were performed as previously described.…”

Mechanistic principles of chromatin remodeling guided by siRNAs and miRNAs

“…1B). The bestcharacterized gene downstream of RD with potential to generate an RD-overlapping antisense transcript is the MTAP gene encoding methylthioadenosine phosphorylase, 20,21 which generates multiple transcripts including a non-coding transcript, conserved in human and mouse cells, that traverses the entire INK4b-ARF-INK4a locus, including RD, and which we call "extended-MTAP" to distinguish it from the "coding-MTAP" (Fig. 1A).…”