1. ¹Immunovirology and Biotherapy Unit, Centro di Riferimento Oncologico, IRCCS – National Cancer Institute, Aviano, PN, Italy
2. ²Mechanisms of Neoplastic Progression Unit, Centro di Riferimento Oncologico, IRCCS – National Cancer Institute, Aviano, PN, Italy
3. ³Department of Pre-clinical and Epidemiological Research, Centro di Riferimento Oncologico, IRCCS – National Cancer Institute, Aviano, PN, Italy

Correspondence: R Dolcetti, Immunovirology and Biotherapy Unit, Department of Pre-clinical and Epidemiological Research, Centro di Riferimento Oncologico, CRO-IRCCS, National Cancer Institute, via Pedemontana Occidentale 12, 33081 Aviano, PN, Italy. E-mail: rdolcetti@cro.it

Received 9 April 2003; Revised 1 June 2004; Accepted 9 August 2004; Published online 14 February 2005.

Abstract

Retinoic acid (RA) arrests the growth of EBV-immortalized lymphoblastoid B cell lines (LCLs) by upregulating the cyclin-dependent kinase inhibitor p27^Kip1. Here, we show that in LCLs, RA inhibits ubiquitination and proteasome-dependent degradation of p27^Kip1, a phenomenon that is associated with downregulation of Thr187 phosphorylation of the protein, whereas the phosphorylation on Ser10 is unaffected. Furthermore, we demonstrate that RA downregulates the expression of the p45^Skp2 and Cks1 proteins, two essential components of the SCF^Skp2 ubiquitin ligase complex that target p27^Kip1 for degradation. Downregulation of p45^Skp2 and Cks1 occurs before the onset of growth arrest and is due to enhanced proteasome-mediated proteolysis of these proteins. Moreover, overexpression of p45^Skp2 in DG75 cells prevents p27^Kip1 protein accumulation and promotes resistance to the antiproliferative effects of RA. Treatment with Leptomycin B (LMB) blocked the translocation of p27^Kip1 to the cytoplasm and prevented its degradation, indicating that CRM1-dependent nuclear export is required for p27^Kip1 degradation. The shuttle protein p38^Jab1, however, does not accumulate in the nucleus upon LMB treatment, nor does it interact with p27^Kip1. Conversely, p45^Skp2 is associated with p27^Kip1 both in the nucleus and in the cytoplasm, accumulating within the nuclei after exposure to LMB and co-localizing with the exportin CRM1, suggesting a possible involvement of p45^Skp2 in CRM1-dependent nuclear export of p27^Kip1. These results indicate that downregulation of p45^Skp2 is a key element underlying RA-induced p27^Kip1 stabilization in B cells, resulting in an impaired targeting of the protein to the ubiquitin–proteasome pathway and probably contributing to the nuclear accumulation of p27^Kip1.