Seong-Min Ahn | PubMed Health

PubMed2004

Retroviral delivery of TIMP-2 inhibits H-ras-induced migration and invasion in MCF10A human breast epithelial cells.

The matrix metalloproteases (MMPs) play important roles in invasion, metastasis and angiogenesis in various cell types. Tissue inhibitor of metalloprotease (TIMP)-2, an endogenous inhibitor of MMP-2, has been shown to inhibit invasion and metastasis. We have previously shown that MMP-2 is responsible for the H-ras-induced invasive and migrative phenotypes in MCF10A human breast epithelial cells. Here, we investigated the effect of TIMP-2 overexpression on migration and invasion in H-ras MCF10A cells. Human TIMP-2 gene was effectively introduced into H-ras MCF10A cells by retrovirus-mediated gene delivery. TIMP-2 overexpression mediated by retrovirus significantly inhibited migration as well as invasion of H-ras MCF10A cells in a dose-dependent manner. We also show the antiangiogenic effect of TIMP-2 gene delivery. Taken together, our study shows that retrovirus-mediated delivery of TIMP-2 efficiently inhibits metastatic progression of ras-transformed human breast epithelial cells, suggesting a potential use of the TIMP-2 gene therapy for the treatment of breast cancer.

Cell Line↗

PubMed2002

In vitro bioassay for transforming growth factor-beta using XTT method.

Research in the cytokine field has grown exponentially in recent years, and the validity of such studies relies heavily on the appropriate measurement of levels of cytokines in various biological samples. Transforming growth factor (TGF)-beta, a hormonally active polypeptide found in normal and transformed tissue, is a potent regulator of cell growth and differentiation. The most widely used bioassay for TGF-beta is the inhibition of the proliferation of mink lung epithelial cells. Though detection of [3H]thymidine incorporation is more sensitive than the MTT assay, it presents some disadvantages due to the safety and disposal problems associated with radioisotopes. In this study, we attempted to ascertain the experimental conditions which could be used for measuring the in vitro biological activity of TGF-beta in a safer and more sensitive way compared with the currently available methods. We compared the commonly used method, the MTT assay, to the XTT assay using different parameters including cell number, incubation time and the wave length used for detecting the product. We examined the anti-proliferative activities of TGF-beta in three different cell lines: Mv-1-Lu mink lung epithelial cells, MCF10A human breast epithelial cells and H-ras-transformed MCF10A cells. Herein, we present an experimental protocol which provides the most sensitive method of quantifying the biological activity of TGF-beta, with a detection limit of as low as 10 pg/ml: Mv-1-Lu or H-ras MCF10 A cells (1 x 10(5)/well) were incubated with TGF-beta at 37 degrees C in a humidified CO2 incubator for 24 hr followed by XTT treatment and determination of absorbance at 450 or 490 nm. Our results may contribute to the establishment of an in vitro bioassay system, which could be used for the satisfactory quantitation of TGF-beta.

Animals↗