p27Kip1: a key mediator of retinoic acid induced growth arrest in the SMS–KCNR human neuroblastoma cell line

Abstract

Retinoic acid (RA) treatment of SMS ± KCNR neuroblastoma (NB) cells leads to G1 growth arrest and neuronal di erentiation. To investigate the molecular mechanisms by which RA alters cell growth, we analysed the expression and activity of components of the cell cycle machinery after culture in RA. Within 2 days of RA treatment and prior to the arrest of NB cells in the G1 phase of the cell cycle, there is a complete downregulation of G1 cyclin/Cdk activities. Protein levels for the G1 cyclin/Cdks were essentially unchanged during this time although there was a decrease in the steady-state levels of p67 N-Myc and hyperphosphorylated Rb proteins. The Cdk inhibitors, p21 Cip1 and p27 Kip1 were constitutively expressed in KCNR while p15 INK4B and p16 INK4A were not detected. RA induced an increase in the expression of p27 Kip1 but not p21 Cip1 . Furthermore, coincident with the decrease in kinase activity there was an increase in G1 cyclin/Cdk bound p27 Kip1 . These results indicate that changes in the level of p27 Kip1 and its binding to G1 cyclin/Cdks may play a key role in RA induced growth arrest of NB cells.

Retinoic acid decreases targeting of p27 for degradation via an N-myc-dependent decrease in p27 phosphorylation and an N-myc-independent decrease in Skp2

“…The cyclin E-dependent kinase activities in cultures treated with RA were reduced to less than 20% of the control while cyclin D-dependent kinase activity was reduced to 50% of the control (Figure 1e). Consistent with our previous results 6 the increases in p27 steady-state levels stimulated with RA were accompanied by increases in p27 bound to cyclin E (Figure 1f). These results raise the possibility that decreases in N-myc and Cdk6 and an increase in p27 play important roles in RA induced arrest of NB cell growth.…”

“…6 In this study, we found that there was a five-fold decrease in the levels of Cdk6 protein in KCNR cells treated for 72 h with RA ( Figure 1c). The steadystate levels of Cdk6 were decreased in time-dependent manner in RA-treated KCNR cells (data not shown).…”

“…To examine the effects of RA on regulation of cell cycle machinery, KCNR cells were treated with 5 mM of RA for 72 h since the accumulation of cells in G1 reached a plateau after 72 h of RA treatment. 6 After a 72 h RA treatment, flattening and disaggregation of cells was observed in RA-treated cultures of NB cells with cells showing neurite extensions (Figure 1a). The RA-induced differentiation is accompanied by inhibition of DNA synthesis, as a bromodeoxyuridine (BrdU) incorporation assay (Figure 1b) shows that incorporation of BrdU into DNA in RA-treated cells was significantly less than the control (Po0.01).…”

“…KCNR cells do not express p15 and p16. 6 Since the decrease in cyclin/Cdk-dependent kinase activity was coincident with an increase in p27 bound to G1 cyclin/Cdk in culture, we investigated whether RA could affect the G1 cyclin/Cdk kinase activity. The cyclin E-dependent kinase activities in cultures treated with RA were reduced to less than 20% of the control while cyclin D-dependent kinase activity was reduced to 50% of the control (Figure 1e).…”

“…6 This led us to hypothesize that these were important components in the ability of RA to arrest NB cell growth and raised the possibility that N-myc may regulate p27 levels. Studies on another member of the myc family indicate that c-myc acts upstream of Cdks in stimulating cell proliferation 7 and increased c-myc expression in arrested fibroblasts results in the activation of Cdk activity and decreases in p27 levels.…”