Figure 1
Expression of PER 2 in human breast epithelial and breast cancer cell lines. Total cellular protein was isolated from two immortalized human breast epithelial cell lines (HME-tert and MCF-10A), two ERα-positive human breast tumor cell lines (MCF-7 and T47D lines) and one ERα-negative human breast tumor cell lines (MDA-MB-231 line). One hundred micrograms of total cellular protein from each cell line was separated by 10% polyacrylamide gel electrophoresis and subjected to Western blot analysis using an antibody directed against the human PER 2 protein.
Figure 2
Effect of PER 2 and CRY 2 on the growth of MCF-7 cells. Cell proliferation assays were conducted on parental, vector-transfected, PER 2 overexpressing MCF-7 cells, CRY 2 overexpressing MCF-7 cells, and cells over expressing both PER 2 and CRY 2. Cells were counted on a hemacytometer using the trypan blue stain every day for four days. N = 3 independent experiments in triplicate. *a = p < 0.05 vs. pCS2+pCDNA3.1, *b = p < 0.05 vs. Per2, *c = p < 0.05 vs. pCS2
Figure 3
PER 2 expression reduces MCF-7 cell growth in soft agar. Soft agar clonogenic assays were performed with MCF-7 cells. MCF-7 cell were transfected with pTracer™-CMV2 or pTracer™-CMV2-hPer 2 and sorted. Two thousand five hundred cells per well were seeded in 12-well plates (3.8 cm2) in culture medium containing 0.35% low-melting agarose over a 0.7% agarose base layer and incubated for 12 days. Colonies larger than 100 μm in diameter were counted.
Table 1
Effect of PER 2 and CRY 2 on the cell cycle of MCF-7 cells
| G1 | S | G2/M | |
| GFP | 7414 | 15.57 | 10.29 |
| pCS2 | 65.36 | 18.03 | 16.61 |
| PER2 | 82.24 | 7.16 | 10.60 |
| pCS2 + pcDNA3.1 | 62.61 | 17.49 | 19.90 |
| PER2 + CRY2 | 89.19 | 0.58 | 10.22 |
Cell cycle analyses were conducted on parental, vector-transfected, PER 2 overexpressing MCF-7 cells, CRY 2 overexpressing MCF-7 cells, and cells over expressing both PER 2 and CRY 2. After three days of expression, propidium iodide(PI) staining and samples were analyzed by flow cytometry.
Figure 4
PER 2 expression induces apoptosis in MCF-7 breast cancer cells. (A) MCF-7 cells were transiently transfected with empty vector, vector expressing PER 2, or both vectors expressing PER 2 or CRY 2. After 72 hours, total cell extracts were prepared and subjected to Western blot analysis using an antibody directed against the 85 kDa cleaved fragment of PARP [poly (ADP-ribose) polymerase] (representative of three independent studies). (B) Densitometric analysis of cleaved PARP protein (average value of three independent studies). a. PER 2 or PER 2 + CRY 2 vs vector control, p < 0.001, b PER 2 + CRY 2 vs PER 2, p < 0.01
Figure 5
PER 2 expression increases P53 but decreases cyclin D1 expression in MCF-7 cells. (A) MCF-7 cells were transiently transfected with and empty vector (pcDNA 3.1) [U] or vector expressing PER 2 [T]. After 48 hours, total cell extracts were prepared and subjected to Western blot analysis using an antibodies directed against the P53 and PER 2 proteins. (B) Densitometric analysis of P53 protein expression. * p < 0.05. (C) Western blot analysis using an antibodies directed against the cyclin D1(cD1) and PER 2 proteins. (D) Densitometric analysis of cD1 protein expression. * p < 0.05.