Roscovitine enhances all-trans retinoic acid (ATRA)-induced nuclear enrichment of an ensemble of activated signaling molecules and augments ATRA-induced myeloid cell differentiation
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Asif Rashid1,2, Xin Duan3, Feng Gao3, Mengsu Yang1 and Andrew Yen2
1 Department of Biomedical Sciences, City University of Hong Kong, Kowloon, Hong Kong SAR, People’s Republic of China
2 Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA
3 The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, People’s Republic of China
Keywords: ATRA; roscovitine; APL; HL-60; Lyn
Received: December 14, 2019 Accepted: February 08, 2020 Published: March 24, 2020
Although ATRA represents a successful differentiation therapy for APL, it is largely ineffective for non-APL AMLs. Hence combination therapies using an agent targeting ATRA-regulated molecules that drive cell differentiation/arrest are of interest. Using the HL-60 human non-APL AML model where ATRA causes nuclear enrichment of c-Raf that drives differentiation/G0-arrest, we now observe that roscovitine enhanced nuclear enrichment of certain traditionally cytoplasmic signaling molecules and enhanced differentiation and cell cycle arrest. Roscovitine upregulated ATRA-induced nuclear c-Raf phosphorylation at S259 and S289/296/301. Nuclear c-Raf interacted with RB protein and specifically with pS608RB, the hinge region phosphorylation controlling E2F binding and cell cycle progression. ATRA-induced loss of pS608RB with cell cycle arrest was associated with loss of RB-sequestered c-Raf, thereby coupling cell cycle arrest and increased availability of c-Raf to promote differentiation. Part of this mechanism reflects promoting cell cycle arrest via ATRA-induced upregulation of the p27 Kip1 CDKI. Roscovitine also enhanced the ATRA-induced nuclear enrichment of other signaling molecules traditionally perceived as cytoplasmic promoters of proliferation, but now known to promote differentiation; in particular: SFKs, Lyn, Fgr; adaptor proteins, c-Cbl, SLP-76; a guanine exchange factor, Vav1; and a transcription factor, IRF-1. Akin to c-Raf, Lyn bound to RB, specifically to pS608RB. Lyn-pS608RB association was greatly diminished by ATRA and essentially lost in ATRA plus roscovitine treated cells. Interestingly Lyn-KD enhanced such ATRA-induced nuclear signaling and differentiation and made roscovitine more effective. ATRA thus mobilized traditionally cytoplasmic signaling molecules to the nucleus where they drove differentiation which were further enhanced by roscovitine.
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