Promoter hypermethylation of tetraspanin members contributes to their silencing in myeloma cell lines

Abstract

Multiple myeloma (MM) cell interactions with their microenvironment modulate acquired drug resistance and disease progression. Indeed, reported aberrant gene methylation underscores the possible role of epigenetic events in MM's molecular profile. Membranal tetraspanins are often inversely correlated with cancer prognosis and metastasis, however mutations were unidentified hitherto. Their promoter characteristics and frequent down-regulation conform to transcriptional silencing by chromatin remodeling. We delineated the baseline expression of select tetraspanins in MM cell lines (RPMI 8226, U266, ARP1, ARK, CAG and EBV transformed ARH77) and fresh bone marrow samples (n = 9) for the first time and determined reduced expression of CD9, CD81 and absence of CD82. Thus, we aimed to assess their promoter methylation status. Indeed, we established CD9, CD81 and CD82 promoter methylation in MM cell lines employing methyl-specific-PCR of bisulfite modified G-DNA and PCR of G-DNA digested with methylation-sensitive restriction enzyme (Hin6I). Re-transcription of assayed genes in the cell lines following de-methylation [5-aza-2'-deoxycytidine (5-aza-dC)] confirmed the mechanistic significance of methylation to their regulation. Combined de-methylation and de-acetylation [Trichostatin A (TsA)] induced synergistic elevation of CD82 mRNA. We conclude that chromatin remodeling contributes to tetraspanin silencing in MM.

Epigenetic Silencing of the Tetraspanin CD9 during Disease Progression in Multiple Myeloma Cells and Correlation with Survival

“…Indeed, screening of human MM cell lines (n = 6) for tetraspanin expression (by flow cytometry analysis) revealed the predominant absence of CD9, CD81, and CD82 characterized by reduced steady-state mRNA levels and methylation of the promoter regions. Reexpression of these tetraspanins by demethylation in some of the human cell lines used supported the theory that (hyper)methylation plays an active role in regulating the transcription of CD9 (30).…”

Wnt3/RhoA/ROCK signaling pathway is involved in adhesion-mediated drug resistance of multiple myeloma in an autocrine mechanism

“…Human myeloma cell lines RPMI8226, EBV-transformed ARH-77 (27,28), MM1S, and KMS-5 (29,30); lymphoma cell lines Ramos, PEER, and Molt-4; and leukemia cell lines KG-1, HL-60, and U937 were cultured in RPMI 1640 containing 10% heat-inactivated FCS (Life Technologies), 2 mmol/L L-glutamine, 0.1% penicillin (100 units/mL), and streptomycin (100 mg/mL). hTERT gene -transduced human stromal cells (hTERT-stromal cells) were generated as previously described (31,32) and were cultured in longterm culture medium containing MEM-a, 12.5% horse serum (Life Technologies), 12.5% FCS, 1 Â 10 À6 mol/L hydrocortisone (Sigma), 1 Â 10 À4 mol/L h-mercaptoethanol (Sigma), 2 mmol/L L-glutamine, 0.1% penicillin (100 units/mL), and streptomycin (100 mg/mL).…”

Tetraspanin-induced death of myeloma cell lines is autophagic and involves increased UPR signalling

“…Previously, we showed that MM cells and cell lines underexpress tetraspanin members, CD81 and CD82, compared with normal plasma and peripheral blood B cells (Tohami et al, 2004(Tohami et al, , 2007Drucker et al, 2006). Tetraspanin proteins facilitate the spatial organisation and localisation of multi-protein complexes in distinct membranal microdomains that are important to exteriorinterior cell signalling.…”