Differing Effects of Staurosporine and UCN-01 on RB Protein Phosphorylation and Expression of Lung Cancer Cell Lines

Abstract

The retinoblastoma gene product (RB protein) plays a key role in the progression of the cell cycle from G1 to S phase in normal and neoplastic cells. The activity of RB is regulated by phosphorylation and dephosphorylation with cell-cycle-dependent protein kinases. We investigated the effect of the protein kinase inhibitors, staurosporine and 7-hydroxy-staurosporine (UCN-01), on RB protein expression of N417 small cell lung cancer cells (absent RB), H209 small cell lung cancer cells (mutant RB), and Ma-31 non-small cell lung cancer cells (wild-type RB), using immunologic blotting. Staurosporine and UCN-01 each suppressed the growth of N417, H209 and Ma-31 cells in a dose-dependent manner in MTT assay. IC50 values of staurosporine for N417, H209 and Ma-31 cells were 54, 29 and 602 nM, respectively. IC50 values of UCN-01 for N417, H209 and Ma-31 cells were 737,181 and 2,197 nM, respectively. Exposure to staurosporine and UCN-01 for 72 h each suppressed the level of expression and altered the ratio of phosphorylated/dephosphorylated RB protein (ppRB/pRB) of Ma-31 cells. Conversely, these agents increased the expression level of RB protein at concentrations less than IC50, and did not change phosphorylation status of mutant RB protein of H209 cells at the concentrations studied. A time course study demonstrated that exposure to the IC50 concentration of staurosporine for 48–72 h increased the ratio of ppRB/ pRB of Ma-31 cells, while exposure to the IC50 concentration of UCN-01 decreased that ratio. UCN-01 increased % cells in G2+M phase and decreased % cells in S phase, while staurosporine increased % cells in G1 phase and decreased % cells in G2+M phase. UCN-01 did not induce apoptosis (DNA content < 2N) of Ma-31 cells, but staurosporine induced it. These findings suggest that the differing effects of staurosporine and UCN-01 on RB protein expression and cell cycle phases of lung cancer cells may explain their differing in vivo antitumor effect of staurosporine and UCN-01 despite their similar chemical structures.

Small-molecule cyclin-dependent kinase modulators

“…Chen et al (1999a) suggest that Rb, but not p53, function is essential for UCN-01-mediated G1 arrest. However, Shimizu et al (1996) demonstrated that lung carcinoma cell lines with either absent, mutant, or wildtype Rb exposed to UCN-01 displayed G 1 arrest and antiproliferative effects irrespective of Rb function.…”

“…Another interesting property of UCN-01 is its ability to arrest cells in the G 1 phase of the cell cycle Akinaga et al, 1994;Kawakami et al, 1996;Shimizu et al, 1996;Akiyama et al, 1997;Akiyama et al, 1999;Chen et al, 1999a;Usuda et al, 2000). When human epidermoid carcinoma A431 cells (mutated p53) or HN12 head and neck carcinoma cell lines are incubated with UCN-01, these cells were arrested in the G 1 phase with Rb hypophosphorylation and p21 waf1 / p27 kip1 accumulation (Akiyama et al, 1997;Patel et al, 2002a).…”

Small molecule modulators of cyclin-dependent kinases for cancer therapy

“…Another interesting feature of UCN-01 is in the e ects on G1 cell cycle progression (Akinaga et al, 1994;Akiyama et al, 1997Akiyama et al, , 1999Kawakami et al, 1996;Seynaeve et al, 1993;Shimizu et al, 1996). Human epidermoid carcinoma A431 cells exposed to UCN-01 displayed clear evidence of G1 arrest.…”

“…Human epidermoid carcinoma A431 cells exposed to UCN-01 displayed clear evidence of G1 arrest. This arrest was accompanied by hypophosphorylation of the tumor suppressor Rb and the accumulation of p21 waf1 and p27 kip1 and appears to be independent of the p53 and Rb status (Akiyama et al, 1997;Shimizu et al, 1996). In seeking to de®ne the mechanism(s) for cell cycle arrest, UCN-01 displayed moderately potent inhibition of immunoprecipitated cdc2 and cdk2 (IC 50 : 300 ± 600 nM).…”

Imatinib mesylate (STI571) enhances amrubicin-induced cytotoxic activity through inhibition of the phosphatidylinositol 3-kinase/Akt pathway in small cell lung cancer cells

“…In addition, although SCF activated Akt and Erk, and STI571 inhibited the activation of these kinases in SCF-treated H209 cells, neither the growth-promoting effect by SCF nor the growth-inhibitory effect by STI571 was observed. H209 cells harbor loss-of-function retinoblastoma (Rb) protein (16). It was reported that Rb protein inactivation, which is observed in a majority of SCLC cases, makes Erk activation dispensable for the growth of SCLC (17).…”