Further analyses of the mechanisms that regulate CD138 expression and related biological processes including cell adhesion or drug sensitivity should contribute not only to a better understanding of the disease, but also to an improvement of the prognosis of myeloma. Acknowledgments This study was supported by a grant from your Amyloidosis Research Committee for Research on Intractable Diseases from your fallotein Ministry of Health, Labour and Welfare, Japan.. those of CD20, CXCR4 and B cell-specific transcription factors increased compared with those under normoxic conditions. Stem cell-specific transcription factors were upregulated under hypoxic conditions, while no difference was observed in ALDH activity. The reduced CD138 expression under hypoxic conditions recovered when cells were treated with ATRA, even under hypoxic conditions, along with decreases in the expression of stem cell-specific transcription factor. Interestingly, ATRA treatment sensitized MM cells to bortezomib under hypoxia. We propose that hypoxia induces immature and stem cell-like transcription phenotypes in myeloma cells. Taken together with our previous observation that decreased CD138 expression is usually correlated with disease progression, the present data suggest that a hypoxic microenvironment affects the phenotype of MM cells, which may correlate with disease progression. (3) reported that myeloma stem cells are enriched in the CD138-negative populace. During normal B-cell development, abundant CD138 (also known as syndecan-1: SDC1) expression is highly specific for terminally differentiated plasma cells in the bone marrow (4). Since CD138 expression is also a hallmark of malignant plasma cells (myeloma cells), it has been utilized for myeloma cell purification (5) and is considered to be a target for treatment (6). While the majority of myeloma cells express CD138, decreased expression of CD138 is occasionally found in clinical practice (7C9). Even though association between CD138 expression and myeloma stem cells remains a matter of argument (10), several reports have shown that CD138-low or -unfavorable myeloma cells may contribute to drug resistance or relapse of the disease (9,11,12). Therefore, analysis of CD138 downregulation in myeloma cells is required for a better understanding of myeloma biology. Previous reports Gemfibrozil (Lopid) have indicated that this bone marrow microenvironment may contribute to CD138 downregulation (13C16). Among numerous factors in the tumor microenvironment, hypoxia is one of the important factors associated with tumor progression, poor clinical outcomes, dedifferentiation, and formation of malignancy stem cell niches in solid tumors (17). Based on recent findings showing a correlation of MM at the advanced stage with hypoxic conditions in the microenvironment within the bone marrow (18), we hypothesized that CD138 expression may be influenced by hypoxia. In the present study, we compared the changes in CD138 and various transcription factor expressions in myeloma cells under hypoxic or normoxic conditions. We also attempted to Gemfibrozil (Lopid) revert CD138 expression in cells under hypoxia by treatment with all-trans retinoic acid (ATRA). The influence of ATRA around the sensitivity to bortezomib under hypoxic conditions was also examined. Materials and methods Cell culture Human myeloma cell lines, KMS-12BM (19) and RPMI 8226 (20), were Gemfibrozil (Lopid) obtained from the Health Science Research Resources Lender (Osaka, Japan) and managed in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37C under 5% CO2. The two myeloma cell lines were cultured under normoxic (21% O2) and hypoxic (1% O2) conditions for up to 30 days, with new medium provided every 3 days. Experiments under hypoxic conditions were performed in a Personal CO2 Multigas Incubator (ASTEC, Fukuoka, Japan). Circulation cytometric analysis of surface antigens MM cell lines cultured under normoxic and hypoxic conditions were stained with the following fluorescently-labeled antibodies: FITCCD138 (clone MI15), FITC-CD38 (clone HIT2), PE-CD44 (clone 515), PE-CD45 (clone HI30), FITC-CD49d (clone gf10) (BD Biosciences, Franklin Lakes, NJ, Gemfibrozil (Lopid) USA); PE-CD54 (clone HCD54), PE-CXCR4 (clone 12G5), PE-MDR-1 (clone UIC2), APC-ABCG2 (clone 5D3) (Biolegend, San Diego, CA, USA); FITC-CD19 (clone HD37), FITC-CD20 (clone B-Ly1) (Dako, Glostrup, Denmark); and Alexa 647-CS1 (clone 162) (AbD Serotec, Oxford, UK). Density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden), the forward/side scatter profile and 7-amino-actinomycin D (7-AAD) (BD Biosciences).