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Masters Thesis 2003

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

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Masters Thesis 2003

  1. 1. Expression of Cyclin D1, p27 and p21 in<br />MCF-7 cells treated with 4-OH <br />Tamoxifen in the presence<br />of growthfactors<br />Anna Merca<br />Department Of Oncology<br />Division of Biomedicine and Surgery<br />Faculty of Health Sciences<br />Linköping University<br />
  2. 2. Aims of the Study<br />General Aim: <br />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). <br />Specific Aim:<br />To determine the expression of Cyclin D1, p27 Kip1 and p21 Waf1/CIP1 in MCF-7 cells.<br />
  3. 3. Estrogen<br />
  4. 4. Tamoxifen (TAM)<br />
  5. 5. SERM-ER<br />promoter<br />Gene Activation <br />(charged SERM-ER complex)<br />Gene Silent <br />(neutral SERM-ER complex)<br />Estrogenic<br />Anti-estrogenic<br />Surface Signaling<br />EGFR<br />HER2/neu<br />TKs<br />Phosphorylation Cascade<br />CoR<br />CoA<br />CoA<br />CoR<br />AF-2b<br />AF-2b<br />AF-1<br />AF-1<br />SERM-ER<br />promoter<br />
  6. 6. Cyclin B<br />cdc2<br />Cyclin D1<br />cdk4/6<br />P<br />Cyclin A<br />Cyclin A<br />pRB<br />Cyclin E<br />cdk2<br />cdc2<br />E2F<br />cdk2<br />pRB<br />P<br />(early G1)<br />G0<br />G1<br />M<br />p21<br />p27<br />G2<br />S<br />(late G1 )<br />E2F<br />The Cell Cycle<br />
  7. 7. Material<br />MCF-7 cells<br />
  8. 8. Methods<br />Cell Lysis <br />SDS- PAGE + Western Blotting <br />Vindeløv Method for Cell Cycle Analysis<br />
  9. 9. Control 4-OH TAM<br /> 1M 4 M<br />- Estradiol <br />+Estradiol (1nM)<br /><ul><li>Heregulin β1 </li></ul>(50ng/ml)<br />- Estradiol <br />+Estradiol (1nM)<br />+ Heregulin β1 <br />(50ng/ml)<br />
  10. 10. 4-OH TAM 1μM - + - - + - - + - - + - <br />4-OH TAM 4μM - - + - - + - - + - - + <br />E2 1nM - - - + + + - - - + + + <br />HRGβ1 10ng/ml -- - - - - + + + + + +<br />24h<br />48h<br />72h<br />Results<br />Cyclin D1 expression in MCF-7 cells treated with <br />4-OH TAM, +/- E2, +/- HRGβ1<br />
  11. 11. 4-OH TAM 1μM - + - - + - - + - - + - <br />4-OH TAM 4μM - - + - - + - - + - - + <br />E2 1nM - - - + + + - - - + + + <br />HRGβ1 10ng/ml -- - - - - + + + + + +<br />24h<br />48h<br />72h<br />Results<br />p21 Waf1 expression in MCF-7 cells treated with <br />4-OH TAM, +/- E2, +/- HRGβ1<br />
  12. 12. 4-OH TAM 1μM - + - - + - - + - - + - <br />4-OH TAM 4μM - - + - - + - - + - - + <br />E2 1nM - - - + + + - - - + + + <br />HRGβ1 10ng/ml -- - - - - + + + + + +<br />24h<br />48h<br />72h<br />Results<br />p27 Kip expression in MCF-7 cells treated with <br />4-OH TAM, +/- E2, +/- HRGβ1<br />
  13. 13. 4-OH TAM 1μM - + - - + - - + - - + - <br />4-OH TAM 4μM - - + - - + - - + - - + <br />E2 1nM - - - + + + - - - + + + <br />HRGβ1 10ng/ml -- - - - - + + + + + +<br />24h<br />48h<br />72h<br />Results<br />Beta Actin expression in MCF-7 Cells treated with 4-OH TAM, +/- E2, +/- HRGβ1<br />
  14. 14.
  15. 15.
  16. 16.
  17. 17. P<br /> Cyclin D1<br />pRB<br />E2F<br />Cyclin D1<br />Cdk4<br />DNA<br />pRB<br />P<br />Cell Cycle Regulation<br />E2<br />E2F<br />MCF-7 cells<br />
  18. 18. Cyclin B<br />cdc2<br />Cyclin D1<br />cdk4/6<br />P<br />Cyclin A<br />Cyclin A<br />pRB<br />Cyclin E<br />cdk2<br />cdc2<br />E2F<br />cdk2<br />pRB<br />P<br />G0<br />(early G1)<br />G1<br />M<br />p21<br />p27<br />G2<br />S<br />(late G1 )<br />E2F<br />Cell CycleRegulation<br />
  19. 19. Conclusions<br />HRGβ1-stimulated expression of Cyclin D1 and p21 in MCF-7 cells wasunaffected by addition of E2 and/or 4-OH TAM.<br />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.<br />E2-induced increase in expression of p27 wasobservedonly after 48 and 72h treatment periods.<br />HRGβ1-stimulated expression of p27 was not affected by addition of 4-OH TAM at 48 and 72h. <br />Addition of E2 to HRGβ1 attenuated the levels of p27 at 48 and 72h treatments.<br />Addition of 4-OH TAM in increasingconcentration to all treatmentslessened the percentage of cells going to S-phase of the cell cycle.<br />
  20. 20. Future Experiments<br />To confirm the current results:<br />repeat western blots on Cyclin D1, p27, and p21,<br />repeat the Vindeløv Method for Cell Cycle Analysis,<br />Additional Experiments:<br />determine Cyclin E expression by western blotting<br />assess apoptosis using M30 antibody<br />

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