Studien zur Regulation der Clusterin-Expression in der MCF-7- und MDA-MB-231-Mamma-Karzinom-Zelllinie

The secretory form of the clusterin protein (sCLU) consists of a 38-kDa glycosylated alpha- and beta-chain, which arise from the cleavage of the 65-kDa sCLU precursor protein. sCLU is produced in inflamed epithelia and apoptotic cells and inhibits apoptotic cell death. In human breast carcinomas, sCLU overexpression is associated with tumor progression. To date, little is known about the hormonal regulation of clusterin expression in human breast cancer. Consequently, the effects of steroid hormone treatment on clusterin mRNA and protein expression was studied in two breast carcinoma cell lines. Moreover, it was analyzed, if clusterin expression is influenced by the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), which inhibits tumor growth. In addition, we assessed the effect of TSA on the induction of p21 expression, cellular senescence and cell survival, all processes known to be influenced by cytostatic agents and apoptosis inducing drugs. mRNA and protein expression were examined by real-time RT-PCR, Western Blot analyses and immunohistochemistry in MDA-MB-231 and MCF7 breast cancer cells a) after cultivation with 17beta-estradiol (E2) and E2 in combination with different progestins (medroxyprogesterone acetate, R5020 or megestrol acetate), b) after 48 hours of treatment with different doses of TSA (0.2, 0.5, 0.75, 1 and 2 µM/L) and c) after treatment with 0.5 µM/L TSA for 0 to 96 hours. After cultivation with and without steroids, both cell lines expressed the 65-kDa sCLU precursor protein. However, clusterin mRNA and protein expression was not regulated by E2 or E2 in combination with progestins in both cell lines. Consequently, in the MDA-MB-231 and MCF7 breast cancer cell line, clusterin mRNA and protein expression is not subject to hormonal regulation. Therefore, MDA-MB-231 and MCF7 cells differ from T47D breast cancer cells, which show extensive regulation of clusterin mRNA expression and sCLU processing by steroid hormones. TSA doses >= 0.2 µM/L induced cellular senescence in MCF7 and MDA-MB-231 cells, which is accompanied by inhibition of cell proliferation. Moreover, TSA concentrations >= 0.5 µM/L showed a distinct cytotoxic effect, which became apparent by the continuous cell loss during cultivation time. In this context, it was shown that TSA treatment (0.5 µM/L) permanently increased p21 expression in MDA-MB-231 cells, but only transiently (after 12 and 24 hours of treatment) in MCF7 cells. Additionally, differences in clusterin protein processing were detected in MCF7 and MDA-MB-231 cells. Whereas in TSA-treated MDA-MB-231 cells, only the 65-kDa sCLU precursor protein was detected, in TSA treated MCF7 cells mature sCLU (38-kDa) was found in addition to the 65-kDa sCLU precursor protein. Furthermore, there were differences in the dose response. In MCF7 cells, an increase of clusterin isoform expression was observed only after treatment with 0.2 and 0.5 µM/L TSA, whereas in MDA-MB-231 cells all applied TSA doses raised the expression of the 65 kDa clusterin precursor protein. Clusterin and p21 peak expression coincided in MCF7 cells, however, a causal correlation between clusterin and p21 expression was neither detected in MCF7 nor in MDA-MB-231 cells by clusterin-p21 immunodouble-labeling. This is in contrast to results obtained in colon cancer cell lines. In summary, the presented data show that the studied breast cancer cell lines show differences in clusterin protein processing after cultivation with the HDAC-inhibitor TSA. In following studies, it has to be elucidated, if these different responses are also found by clinically used HDAC-inhibitors and if the cell line specific sensitivity to HDAC-inhibitors correlates with clusterin isoform expression and processing.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2010

Author dc:creator

• Pinell, Nicole Maria

Contributors dc:contributor

• Krusche, Claudia Astrid

• info:eu-repo/classification/ddc/610
• Brustkrebs
• Genregulation
• Medizin
• Clusterin
• MCF-7-Zellen
• MDA-MB-231-Zellen
• clusterin expression
• MCF7 cells
• MDA-MB-231 cells

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-114012%22

OAI record oai:identifier

https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A51755