Expression of Cyclin D1, p27 and p21 in MCF-7 cells treated with 4-OH Tamoxifen in the presence of growth factors Linköping University

1. Expression of Cyclin D1, p27 and p21 in MCF-7 cells treated with 4-OH Tamoxifen in the presence of growthfactors Anna Merca Department Of Oncology Division of Biomedicine and Surgery Faculty of Health Sciences Linköping University

2. Aims of the Study General Aim: To determine the effect of 4-OH Tamoxifen (4-OH TAM) on cell cycle regulators in MCF-7 cells in the presence of growth factors such as heregulinβ1(HRGβ1) and/or estradiol (E2). Specific Aim: To determine the expression of Cyclin D1, p27 Kip1 and p21 Waf1/CIP1 in MCF-7 cells.

3. Estrogen

4. Tamoxifen (TAM)

5. SERM-ER promoter Gene Activation (charged SERM-ER complex) Gene Silent (neutral SERM-ER complex) Estrogenic Anti-estrogenic Surface Signaling EGFR HER2/neu TKs Phosphorylation Cascade CoR CoA CoA CoR AF-2b AF-2b AF-1 AF-1 SERM-ER promoter

6. Cyclin B cdc2 Cyclin D1 cdk4/6 P Cyclin A Cyclin A pRB Cyclin E cdk2 cdc2 E2F cdk2 pRB P (early G1) G0 G1 M p21 p27 G2 S (late G1 ) E2F The Cell Cycle

7. Material MCF-7 cells

8. Methods Cell Lysis SDS- PAGE + Western Blotting Vindeløv Method for Cell Cycle Analysis

10. 4-OH TAM 1μM - + - - + - - + - - + - 4-OH TAM 4μM - - + - - + - - + - - + E2 1nM - - - + + + - - - + + + HRGβ1 10ng/ml -- - - - - + + + + + + 24h 48h 72h Results Cyclin D1 expression in MCF-7 cells treated with 4-OH TAM, +/- E2, +/- HRGβ1

11. 4-OH TAM 1μM - + - - + - - + - - + - 4-OH TAM 4μM - - + - - + - - + - - + E2 1nM - - - + + + - - - + + + HRGβ1 10ng/ml -- - - - - + + + + + + 24h 48h 72h Results p21 Waf1 expression in MCF-7 cells treated with 4-OH TAM, +/- E2, +/- HRGβ1

12. 4-OH TAM 1μM - + - - + - - + - - + - 4-OH TAM 4μM - - + - - + - - + - - + E2 1nM - - - + + + - - - + + + HRGβ1 10ng/ml -- - - - - + + + + + + 24h 48h 72h Results p27 Kip expression in MCF-7 cells treated with 4-OH TAM, +/- E2, +/- HRGβ1

13. 4-OH TAM 1μM - + - - + - - + - - + - 4-OH TAM 4μM - - + - - + - - + - - + E2 1nM - - - + + + - - - + + + HRGβ1 10ng/ml -- - - - - + + + + + + 24h 48h 72h Results Beta Actin expression in MCF-7 Cells treated with 4-OH TAM, +/- E2, +/- HRGβ1

17. P Cyclin D1 pRB E2F Cyclin D1 Cdk4 DNA pRB P Cell Cycle Regulation E2 E2F MCF-7 cells

18. Cyclin B cdc2 Cyclin D1 cdk4/6 P Cyclin A Cyclin A pRB Cyclin E cdk2 cdc2 E2F cdk2 pRB P G0 (early G1) G1 M p21 p27 G2 S (late G1 ) E2F Cell CycleRegulation

19. Conclusions HRGβ1-stimulated expression of Cyclin D1 and p21 in MCF-7 cells wasunaffected by addition of E2 and/or 4-OH TAM. E2-induced increase in expression of Cyclin D1 wasobserved in all treatment periods (24, 48, and 72h), addition of 4-OH TAM attenuated CyclinD1 levels. E2-induced increase in expression of p27 wasobservedonly after 48 and 72h treatment periods. HRGβ1-stimulated expression of p27 was not affected by addition of 4-OH TAM at 48 and 72h. Addition of E2 to HRGβ1 attenuated the levels of p27 at 48 and 72h treatments. Addition of 4-OH TAM in increasingconcentration to all treatmentslessened the percentage of cells going to S-phase of the cell cycle.

20. Future Experiments To confirm the current results: repeat western blots on Cyclin D1, p27, and p21, repeat the Vindeløv Method for Cell Cycle Analysis, Additional Experiments: determine Cyclin E expression by western blotting assess apoptosis using M30 antibody