CDK4 depletion inhibits cell cycle progression of insulinoma cells with suppressed Rb phosphorylation
To obtain insight into the mechanism underlying the requirement for CDK4 in neuroendocrine tumorigenesis, we examined INS-1 insulinoma cells, which express very low levels of menin and have been widely used for functional studies by forced expression of wild-type and mutant menin proteins 27, 28. At 24 hrs after transfection with siRNA against CDK2 or CDK4, cells were treated with medium containing a low level of glucose (0.1 mM) and incubated for 48 more hours to suppress proliferation. Cell cycle progression was then stimulated by switching to medium with a high glucose concentration (11 mM). Immunoblotting (Fig. 5A) demonstrated that both CDK2 and CDK4 siRNAs successfully downregulated the expression of the target proteins. The stimulation of cells with 11 mM glucose resulted in marked upregulation of Cyclin D1 and substantial increases in Cyclin D3 expression, for which the knockdown of Cdk2 or Cdk4 had minimal impact. The expression levels of Cyclin E and CDK6 did not significantly alter during glucose starvation and stimulation. It was noted that CDK2 knockdown resulted in modest increases in CDK4 levels (2nd panel, C vs. K2 at 0, 6 and 24h). Immunoblots for total RB showed accumulation of the slower migrating hyperphosphorylated forms of RB after the switch to 11 mM glucose, and CDK4 knockdown clearly inhibited the hyperphosphorylation of RB. In contrast, CDK2 knockdown resulted in slightly accelerated kinetics of RB phosphorylation, compared with control cells. Immunoblots using phospho-specific RB antibodies showed that phosphorylation of RB at Ser780 was delayed in cells with CDK4 knockdown (8th panel, K4-6 h vs. C-6 h and K4-24 h vs. C-24 h), whereas phosphorylation at Ser807/811 was minimally affected by CDK2 or CDK4 siRNA. We did not observe major effects of CDK2 or CDK4 knockdown in phosphorylation of p130 at Ser952. Consistently with the suppression of hyperphosphorylated forms of RB, CDK4-depleted cells exhibited a substantial delay in glucose-mediated cell cycle stimulation relative to control or CDK2 depleted cells, as shown as partially inhibited G1-S transition in flow cytometric analysis using DNA staining with propidium iodide and immunodetection of incorporated bromodeoxyuridine (BrdU)(Fig. 5B). Using another insulinoma cell line, Min6 cells, we confirmed that siRNA-mediated silencing of CDK4 significantly decreased Ser780 phosphorylation of RB, as well as hyperphosphorylated forms of the protein (Fig. 5C). Thus, these data using insulinoma cells demonstrate that CDK4 depletion inhibits site-specific RB phosphorylation and cell cycle stimulation in this cell type, whereas CDK2 depletion has minimal effect on these molecular processes.