S-phase reduction in T47D human breast cancer epithelial cells induced by an S100P antisense-retroviral construct.
S100P is expressed in several malignant neoplasms. It was previously demonstrated that S100P is involved in the very early stages of breast carcinogenesis. In the present study we used a retrovirus-mediated transfer of antisense-S100P in order to check whether the decrease in expression of this protein could lead to alterations in the cell cycle of epithelial cells of human breast cancer. The T47D breast carcinoma cell line, a human breast epithelial cell that expresses high levels of S100P, was a tool used in this study to investigate the alteration in cell cycle induced by a retrovirus-mediated transfer of antisense-S100P. First we used the real-time PCR technique to quantify the gene expression. The results showed a reduction of 63% of expression within the T47D-S100P-A/S infected population compared with control T47D-LXSN clones. To determine the impact of the S100P antisense technique on protein expression in T47D cells, we performed immunofluorescence staining and analyzed the resulting images using a confocal microscope. The images showed much less pronounced antibody marking of the S100P protein in the T47D-S100P-A/S compared with control cells. To evaluate whether the antisense approach caused any alteration in the cell cycle, we concluded the study with flow cytometric analysis of the cell distribution. Our findings indicated that, in our model, S100P-antisense cells showed a 23% reduction of cells at the S-phase. Using transduction techniques with an S100P antisense-retroviral construct we were able to demonstrate a significant reduction in S-phase of the T47D cell cycle. To the best of our knowledge, this is the first time that an antisense approach has been used against S100P mRNA in breast cancer epithelial cells. The results showed here seem to further classify S100P as a protein that might be involved in the cell cycle imbalance observed during breast carcinogenesis.
Beissel
Bettina
B
Centro de Química e Meio Ambiente, IPEN/CNEN-SP, 04039-032 São Paulo, SP, Brazil.
Silva
Ismael D C G
ID
Pesquero
João B
JB
Russo
Jose
J
Schor
Nestor
N
Bellini
Maria Helena
MH
D001943:Breast Neoplasms / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology*; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D018613:Microscopy, Confocal; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D016372:RNA, Antisense; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002135:Calcium-Binding Proteins; D009363:Neoplasm Proteins; D016372:RNA, Antisense; C103003:S100P protein, human