G1 arrest of U937 cells by onconase as associated with suppression of cyclin D3 expression, induction of p16INK4A, P21WAF1/CIP1 and p27KIP and decreased pRb phosphorylation

Onconase is a 12 kDa protein homologous to pancreatic RNase A isolated from amphibian oocytes which shows cytostatic and cytotoxic activity in vitro, inhibits growth of tumors in mice and is in phase III clinical trials. The present study was aimed to reveal mechanisms by which onconase perturbs the cell cycle progression. Human histiocytic lymphoma U937 cells were treated with onconase and expression of cyclins D3 and E, as well as of the cyclin-dependent kinase inhibitors (CKIs) p16(INK4A), P21(WAF1/CIP1) and p27(KIP1) (all detected immunocytochemically) was measured by multiparameter flow cytometry, in relation to the cell cycle position. Also monitored was the status of phosphorylation of retinoblastoma protein (pRb) by a novel method utilizing mAb which specifically detects under-phosphorylated pRb in individual cells. Cell incubation with 170 nM onconase for 24 h and longer led to their arrest in G(1) which was accompanied by a decrease in expression of cyclin D3, no change in cyclin E, and enhanced expression of all three CKIs. pRb was underphosphorylated in the onconase arrested G(1) cells but was phosphorylated in the cells that were still progressing through S and G(2)/M in the presence of onconase. The cytostatic effect of onconase thus appears to be mediated by downregulation of cyclin D3 combined with upregulation of p27(KIP1), p16(INK4A) and p21(WAF1/CIP1), the events which may prevent phosphorylation of pRb during G(0/1) and result in cell arrest at the restriction point controlled by Cdk4/6 and D type cyclins.