Nutlin Induces Apoptosis in Multiple Myeloma Cells through Negative Regulation of CKS1
Manujendra N Saha, Yan Chen, Hua Jiang, Hong Chang. University Health Network, Toronto, Canada; University of Toronto, Toronto, Canada; First Affiliated Hospital of Nanchang University, Nanchang, China
Background: CKS1, a member of the cyclin-dependent kinase subunit family, contributes to cell cycle control in all eukaryotes. CKS1 protein is found over-expressed in a number of tumors including multiple myeloma (MM). MM patients with CKS1B over-expression (∼40%) have shown a poor prognosis and resistant to current therapies. Therefore, improved therapeutic strategies are needed for this subgroup of patients. We and others have previously shown that nutlin, an MDM2 antagonist, induces apoptosis in MM cells. However, molecular mechanism of nutlin-mediated apoptosis is not fully elucidated and its relation to CKS1 is not known. Here we examined the involvement of CKS1 in nutlin-induced apoptosis of MM cells.
Design: Two human MM cell lines, MM.1S and H929 with wild type 53 and CKS1B amplification were treated with nutlin and were assessed for the expression of p53 and its downstream targets by Western blot analysis. Cell cycle arrest and apoptosis were analysed by Flow cytometry. Regulation of CKS1 and S-phase kinase-associated protein 2 (SKP2) in relation to p53 was examined by silencing the expression of p53 or inhibiting the p53 transcription by pifithrin α (PFT-α) in MM cells. The role of CKS1 in nutlin-induced apoptosis was examined in OCI-MY5 cell line (harbouring both wild type and mutant p53) with forced expression of CKS1B by lentivirus vector-mediated CKS1B cDNA transfection.
Results: Nutlin induced a time- and dose-dependent up-regulation of p53 and its two immediate downstream targets, p21 and p27, and down-regulation of CKS1B and SKP2 resulting in apoptosis in both MM cell lines. In addition, nutlin caused a cellular arrest in G1-S or G2/M-S phase in these cells. However, inhibition of p53 transcription by PFT-α or selective knockdown of p53 expression by p53 siRNA in MM cells did not significantly affect nutlin-induced down-regulation of CKS1B, suggesting that CKS1B modulation is not fully dependent on activation of p53. On the other hand, nutlin treatment induced significantly less growth inhibition and cell death in CKS1B over-expressed OCI-MY5 cells compared with empty virus-transfected controls (p<0.05).
Conclusions: Our study for the first time demonstrates that nutlin-induced apoptosis in MM cells is mediated, at least in part, by down-regulation of CKS1 and SKP2, and provides the rationale for clinical evaluation of nutlin in MM patients especially those with CKS1B over-expression.
Wednesday, March 6, 2013 1:00 PM
Poster Session VI # 230, Wednesday Afternoon