Constitutive Cyclin A1 Expression Impairs Retinoic Acid-Induced Growth Arrest and Differentiation of Myeloid Leukemia Cells

Abstract 2894

Acute promyelocytic leukemia (APL) differentiation syndrome (DS) in all-trans retinoic acid (ATRA) therapy is often associated with increase of leukocyte count (hyperleukocytosis). It suggests deregulated cell proliferation of ATRA-induced APL cells is possibly involved in the development of DS. The molecular mechanism(s) of hyperleukocytosis are unknown. We previously found increased expression of cyclin A1 mRNA were associated with both increase of initial leukocyte count and development of DS in the therapy with ATRA in APL. To clarify role(s) of cyclin A1 in the proliferation and differentiation of myeloid leukemia cells, we generated U937.A1 which is stably transfected with cyclin A1 gene. U937.A1 showed increased expression of cyclin A1 protein compared with U937.C, which carries pCDNA3.1Hisc (2.5 fold, immunoblot). U937.A1 showed increased in vitro colony formation compared with U937.C (1.3 fold).

Flow cytometry (FCM) analysis of cells stained with propidium iodide(PI) showed that U937.A1 was characterized with significant decrease in cells in G1 and increase in G2M (P<0.01)(U937.C=G1:70.1±1.1,S:26.5±1.1,G2M:13.8±1.0% and U937.A1=G1:55.5±0.9,S:30.1±2.9,G2M:21.1±0.8%). U937.A1 showed constitutive higher Cdk1 kinase activities compared with U937.C more than 100 times, as measured by ELISA which quantitate enzyme activities by detecting phospholyrated synthetic peptide using monoclonal antibody 4A4. It suggests that inappropriate activation of cyclin A1/Cdk1 complex is associated with altered cell cycle progression of U937.A1 cells. Immunoblot analysis showed that U937.A1 over-expressed checkpoint kinase 1(Chk1) protein compared with U937.C.(1.4 fold)suggesting deregulated G2M checkpoint in U937.A1. Anti-apoptotic protein, bcl2, was increased in U937.A1 cells(1.5 fold).

Incubation with 1μ M ATRA for 4 days significantly inhibited cell growth in U937.C (U937.C=44.9±1% and U937.A1=70.8±3%, p<0.01, cell No. with ATRA/without ATRA).

ATRA-induced growth inhibition in U937.C was accompanied with significant increase of cells in G1 (U937.C=88.9±0.9% and U937.A1=68.2±0.8%) and decreased in the G2M(U937.C=4.9±0.5% and U937.A1=19.7±1.2%). Expression of both cyclin A2 and cyclin B proteins were markedly down-regulated at days 3 and 4 in U937.C in the culture (U937.C=69.1±1.3%(cyclin A2) and 52.1±2.1%(cyclin B), U937.A1=90.2±1.7%(cyclin A2)and 94.5±2.4%(cyclin B),day4, ATRA/without ATRA) Cdk1 protein expression was also significantly down-regulated in U937.C(U937.C=53.5±2.5% and U937.A1=115±5%, p<0.01, ATRA/without ATRA). In both of U937.C and U937.A1 cells, Cdk1 kinase activities were significantly suppressed by 4 days' culture with 1μ M ATRA,however, the activities in U937.A1 still remained 10 times higher than those in U937.C (U937.C=0.06±0.01/0.25±0.02 and U937.A1=0.19±0.01/1.04±0.04,day4, O.D., ATRA/without ATRA). Incubated with ATRA, expression of Chk1 protein in U937.C cells time-dependently decreased (U937.C=44.3±0.1% and U937.A1=86.1±0.1%, p<0.01, day4. with ATRA/without ATRA) Checkpoint kinase activities, as measured by ELISA using anti-phospho-Cdc25C serine 216 specific antibody, was significantly suppressed by ATRA in U937.C (U937.C=1.25±0.02/2.69±0.09 and U937.A1=3.42±0.06/2.79±0.22,day4, O.D., ATRA/without ATRA). Incubation with Go6976 at 10 μ M, a specific chk1 inhibitor, produced significant cell growth inhibition in U937.A1 in combination with ATRA. The growth inhibition by ATRA plus Go6976 was accompanied with accumulation of cells in G1(79.2±0.9%) and decrease of cells in G2M (7.7%±0.3%)compared with those incubated with ATRA alone, suggesting deregulated chk1 activities are involved in impaired ATRA-induced growth inhibition in U937.A1 cells. ATRA-induced down-regulation of expression of bcl2 protein was significant in U937.C (U937.C=41.8±0.1% and U937.A1=77.8±0.2%, day4, with ATRA/without ATRA). Decreased bcl2 protein was inversely co-related with up-regulation of CD11b induced by ATRA (U937.C=4.3±0.2% and U937.A1=3.9±0.1%, FCM, day4).

The present results showed that forced expression of cyclin A1 resulted in impaired ATRA-induced growth suppression and differentiation of U937 cells. It was related with inappropriately activated Chk1-Cdk1 pathway. Deregulated G2M cell cycle checkpoint(S)is a candidate for the therapeutic target in ATRA-induced hyperleukocytois in APL.