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
Linkping 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 cellsin the presence of growth factors such as heregulin1(HRG1) 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
Vindelv Method for Cell Cycle Analysis
9. Control 4-OH TAM
1M4 M
- Estradiol
+Estradiol (1nM)

Heregulin 1

(50ng/ml)
- Estradiol
+Estradiol (1nM)
+ Heregulin 1
(50ng/ml)
10. 4-OH TAM1M-+ - -+ -- + - -+ -
4-OH TAM4M- -+ - - + - -+- - +
E2 1nM- - - ++ + - --+ ++
HRG110ng/ml -- - - -- ++++++
24h
48h
72h
Results
Cyclin D1 expression in MCF-7 cells treated with
4-OH TAM, +/- E2, +/- HRG1
11. 4-OH TAM1M-+ - -+ -- + - -+ -
4-OH TAM4M- -+ - - + - -+- - +
E2 1nM - - - ++ + - --+ ++
HRG110ng/ml-- - - -- ++++++
24h
48h
72h
Results
p21 Waf1 expression in MCF-7 cells treated with
4-OH TAM, +/- E2, +/- HRG1
12. 4-OH TAM1M-+ - -+ -- + - -+ -
4-OH TAM4M- -+ - - + - -+- - +
E21nM- - - ++ + - --+ ++
HRG1 10ng/ml-- - - -- ++++++
24h
48h
72h
Results
p27 Kip expression in MCF-7 cells treated with
4-OH TAM, +/- E2, +/- HRG1
13. 4-OH TAM1M-+ - -+ -- + - -+ -
4-OH TAM4M- -+ - - + - -+- - +
E2 1nM- - - ++ + - --+ ++
HRG1 10ng/ml-- - - -- ++++++
24h
48h
72h
Results
Beta Actin expression in MCF-7 Cells treated with 4-OH TAM, +/- E2, +/- HRG1
14. 15. 16. 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
HRG1-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.
HRG1-stimulated expression of p27 was not affected by addition of 4-OH TAM at 48 and 72h.
Addition of E2 to HRG1 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 Vindelv Method for Cell Cycle Analysis,
Additional Experiments:
determine Cyclin E expression by western blotting
assess apoptosis using M30 antibody