Development of resistance to chemotherapeutic drugs in human osteosarcoma cell lines largely depends on up-regulation of Clusterin/Apolipoprotein J. Clusterin/Apolipoprotein J (CLU) is differentially regulated during in vivo cancer progression. We have addressed the role of CLU during the acquisition and maintenance of human cancer cells resistance to chemotherapeutic drugs. We used two osteosarcoma (OS) cell lines, namely U-2 OS and KH OS, and selected three generations of doxorubicin (DXR)-resistant cells (R1, R2 and R3; resistant to 0.0035, 0.035 and 0.35 microM DXR, respectively) by continuous exposure to increasing, clinically relevant, DXR concentrations. Our studies showed that the DXR-resistant OS cell lines were cross-resistant to a variety of unrelated cytotoxic agents. Analysis of the CLU mRNA and protein expression levels revealed a minimal CLU up-regulation in the U-2 OS R2 cells and a significant, more than 4-fold, induction in the KH OS R2 and R3 cells. Antibody-mediated neutralization of the extracellular CLU, or silencing of CLU gene expression via small interfering RNA (siRNA) partially sensitized KH OS R2 cells to the drugs assayed. Moreover, siRNA-mediated CLU knock down in the absence of DXR induced high levels of endogenous spontaneous apoptosis in both the parental and R2 OS cell lines. This effect was enhanced by more than 60% in the KH OS R2 cells as compared to their parental counterparts, indicating that the high CLU levels in the KH OS R2 cells are essential for survival. Overall, we suggest that CLU up-regulation in the multi-drug resistant OS cells relates to enhanced drug resistance. Therefore, CLU may represent a predictive marker, which correlates to response of cancer cells to chemotherapy.