Abstract 887 Hematology-Oncology I Platform, Saturday, 5/1

Transforming growth factor-β1 (TGFβ1) protects normal (non-cancerous) cells from cell cycle-acting chemotherapeutic agents including etoposide (VP-16). VP-16 binds to the topoisomerase IIα (topo IIα) enzyme which causes DNA damage and initiates programmed cell death (apoptosis). Thus, expression of topo IIα is necessary for the function of VP-16. It has been speculated that the protective effect of TGFβ1 is primarily due to the ability of TGFβ1 to reversibly arrest cell cycle progression (growth) in the G1 phase. Because peak expression of topo IIα occurs during the G2 phase of the cell cycle, we hypothesized that TGFβ1 would inhibit topo IIα expression. We further hypothesized that the inhibition of topo IIα by TGFβ1 would be dependent on cell cycle arrest. We used the MvlLu cell line as a model system because they are normal cells derived from primary cultures and their responsiveness to TGFβ1 is well characterized. To test the hypothesis that TGFβ1 inhibits topo IIα expression we determined topo IIα mRNA and protein expression by northern and western analysis in control and TGFβ1-treated (10 ng/ml) MvlLu cells. As a control, the cell cycle distribution of each sample was determined by fluorescence-activated cell sorter (FACS) analysis. To corroborate our western data we performed immunostaining of control and TGFβ1-treated cells using a standard protocol. By northern and western analysis we found that TGFβ1 treatment of MvlLu cells resulted in a marked inhibition of topo IIα mRNA and protein expression. However, by immunostaining it appeared qualitatively that TGFβ1 had decreased topo IIα expression in all cells, suggesting that the decreased expression occurred independent of cell cycle phase. To test the hypothesis that cell cycle arrest is necessary for inhibition of topo IIα by TGFβ1 we simultaneously determined topo IIα protein expression and cell cycle position for each of 2-5 X 105 cells per sample by quantitative immunostaining using FACS analysis. We found that TGFβ1 inhibits topo IIα expression 42.6 ±9.8% in G1, 37.8 ±15.3% in S, and 79.2 ±4.0% in G2/M. Thus, the subpopulation of cells that continued cycling in the presence of TGFβ1 had markedly decreased topo IIα expression during all phases of the cell cycle. We conclude that TGFβ1 inhibits topo IIα expression in MvlLu cells. The inhibition of topo IIα protein expression by TGFβ1 is independent of cell cycle arrest. These results indicate that there are novel mechanisms that enable TGFβ1 to inhibit topo IIα expression without causing cell cycle arrest, and that TGFβ1 may protect cells from cell cycle-acting chemotherapy independent of its ability to arrest growth in the G1 phase.