Constitutive Cyclin A1 Expression Induces Abrogation of Cell Cycle Check Points Associated with Ara-C Resistance in Myeloid Leukemia Cells.

Abstract 4801

Cyclin A1, an alternative A-type cyclin, is frequently over-expressed in acute myeloid leukemia (AML). To explore role(s) of cyclin A1 in leukemia cells, we studied AML cell line, K562.A1, which constitutively express cyclin A1 gene. K562.A1 showed prolonged doubling time in culture (1.3 fold) and decreased colony formation (25.6±6% of those of K562.C, which carries pcDNA3.1Hisc). flow cytometry (FCM) analysis of cells stained with PI showed that decreased cell growth of K562.A1 was accompanied with G1 accumulation and significant decrease in percentage of cells in the G2M (G1:49.5±2.6,S:30.3±2.3,G2M:18.7±1.1% for K562.C and G1:65.7±2.4,S:22.4±2.1,G2M:9.5±0.5% for K562.A1).

Immunoblot analysis showed that k562.A1 constitutively over-expressed checkpoint kinase 1(chk1) protein (3.4±0.2 fold evaluated by densitometric analysis) but not chk2 compared with K562.C. Cdk1/Cdc2 protein, but not Cdk2, was also over-expressed in K562.A1 cells (3.9±0.3 fold). In addition, both chk1 and Cdk1/Cdc2 were constitutively phosphorylated at Ser345 and Tyr15 residues, respectively, while phosphorylation of those proteins was not detected in K562.C. In K562.A1, cell division cycle 20 homolog (Cdc20) was also over-expressed (2.7±0.1 fold), suggesting that de-regulation of spindle checkpoint was also involved in cell cycle inhibition in this cell line. Incubation with Gö6976, a specific chk1 inhibitor, partly restored cell growth of K562.A1 (53±1.2% in medium and 72±3.0% in 1μM Gö6976,% of cell number of K562.C at day3. p<0.01), while Gö6976 showed negligible effect on cell growth of K562.C. Restored cell growth was accompanied with decreased in percentage of cells in the G1 and increased incells in the G2M in the cell cycle. This suggest that a role of inappropriately activated Chk1-Cdk1/Cdc2 pathway in cell growth inhibition of K562.A1.

Small increase of subG1 DNA content portion were observed in K562.A1 compared with K562.C(K562.A1=2.4±0.1% and K562.C=0.7±0.2%), suggesting the presence of low proliferating or apoptosis-prone G1 cells in K562.A1. However, K562.A1, compared with K562.C showed significantly impaired cytosine arabinoside (CA)-induced apoptosis (K562.C=50.9 ±1.5% and K562.A1=29.6±1.2%, p<0.01, Annexin V-FITC/PI stained cells, 1μMCA at day 3). STI571 (1nM-1000nM), a specific abl kinase inhibitor, showed similar cytotoxic effects on both K562.C and K562.A1. It suggests that K562.A1 have an alteration of apoptotic pathway(S) triggered by genotoxic agent. We found significant up-regulation of activated NF-κB in K562.A1 compared to K562.C in response to CA as judged by quantitative enzyme immunoassay. (K562.C=1.0±0.3 and 2.1±0.7 pg/μg (CA) K562.A1=7.9±1.3 and 23.6±0.9pg/μg (CA), P=0.01). Helenalin (NF-κB inhibitor), however, only slightly increased apoptotic cells in k562.A1 in the presence of CA (1μM CA =30.1 ±3.6% and 1μM CA plus 1μM helenalin=34.7±4.2%, p=NS), suggesting a role of activated NF-κB are limited for impaired CA-induced apoptosis in K562.A1. CA induced significant increase of cell surface expression of CD95 in K562.C (53.5±2.9 fold, mean signal intensity), while the increase in K562.A1 was only 1.7±0.1 fold, suggesting that activation of the FAS apoptotic pathway are impaired in K562.A1. Incubation with 1μM CA for 3 days further augmented protein expression of Cdc20 in K562.A1 cells (2.7±0.5 fold compared to those without CA), while the change in K562. C cells was negligible.

The present study shows that de-regulated expression of molecule(s) responsible for transition through G2 and spindle checkpoint is evident in myeloid leukemia cells which constitutively express cyclin A1. The de-regulated activation of Chk1-Cdk1/Cdc2 pathway and abrogation of G2/M checkpoint in K562.A1 cells not only resulted in inhibition of CA-induced erythroid differentiation and cell cycle arrest, but also possible inhibition of Fas/CD95-mediated apoptosis. Association between ATM (ataxia teleangiectasia mutated) DNA damage response pathway and Fas/CD95-mediated apoptotic pathway is recently reported. Furthermore over-expressed Cdc20protein, a potential cancer therapeutic target, may be associated mitotic abnormalities and induction of aneuploidy in leukemia cells. It suggests that, in addition to activated NF-κB, abrogation of cell cycle checkpoint(s) are involved in chemo-resistant myeloid leukemia with over-expression of cyclin A1.