Cellular interactions with microenvironmental components are critical in multiple myeloma (MM) and impede effective disease treatment. Membranal-embedded tetraspanins, associated with metastasis suppression, are underexpressed in MM. We aimed to investigate the consequences of CD81/CD82 tetraspanins over-expression in MM cell lines. CAG and RPMI 8226 were transfected with pEGFP-N1/C1 fusion vectors of CD81/CD82. Employing flow cytometry, immunocytochemistry, and activity assays we assessed transfected cells for: morphology, survival, death, caspases, cell cycle, proliferation, oxidative stress, adhesion, motility and invasion. Overexpressed CD81/CD82 pEGFP-N1 vectors reduced survival without elevation of pre-G1 or AnnexinV+/7AAD- and independently of caspases. Decreased Ki67 and elevated intracellular glutathione were detected. No perturbations in cell cycle distribution were observed. The pEGFP-C1 vectors of CD81/CD82 caused reduction of MM cell adherence with/without fibronectin, insulin-like growth factor (IGF)-I, and matrigel. They also reduced cell motility and attenuated invasion potential, expressed by reduced secreted MMP-9 activity. These novel findings delineate the significance of CD81/CD82 expression to MM cell survival and their negative effects on cell adhesion, motility, and invasion thus, supporting their role as tumor metastasis suppressors.

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.

The role of tetraspanins is undefined, despite their detection in diverse cell types and functions. This study addresses the characterization of tetraspanin expression levels in normal peripheral blood leukocytes (PBL) and in patients with bacterial infection. Membranal and cytoplasmic expression of CD9, CD53, CD63, CD81, CD82 and CD151 in polymorphonuclears (PMN), monocytes, B and T lymphocytes was assessed using flow cytometry. Results suggested that for normal PBL, PMN are distinguished by dominant cytoplasmic CD63; monocytes and B cells prevailingly express CD53; CD82 is primarily expressed on T-cell membranes. However, a major trend of downregulation was demonstrated for the examined tetraspanins, except CD63, in all patients' PBL subtypes. Therefore, tetraspanin modulation in infections may be attributed to elevated leukocyte motility in immune reactions and this is compatible with the previous publications of tetraspanins as metastasis suppressors. This work represents the first comprehensive baseline of tetraspanin expression in normal PBL and in infectious disorders.

No abstract