Malignant breast carcinomas have the ability to invade normal tissues and spread to other sites giving rise to metastasis. Cancer invasion and metastasis requires the degradation of the basement membrane and the extra cellular matrix. The majority of the destruction of the matrix components during metastasis is known to be carried out by stimulated release of Matrix Metalloproteinases. In a normal mammary gland, constitutive expression of MMPs is low, except during times of development and pregnancy. MMP-13 also known as Collagenase-3 is a member of collagenase subfamily, was initially identified in breast cancer. It was later found to be over expressed in other malignant tumors, suggesting that MMP-13 is associated with aggressive tumors. Therefore, expression of MMP-13 can be used as a marker for tumor progression. Furthermore, understanding the regulation of MMP-13 expression can lead to the identification of potential targets to develop therapeutic agents for treatment of tumor invasion and metastasis.
Figure 1-RT-PCR analysis of MMP-13 transcript levels in MCF 10A, MCF 7, MDA-MB 231 and HTB 122 cell lines. (a) RT-PCR products generated from RNA isolated from each of the cell lines, amplified for 50 cycles by primers recognizing nucleotides 959-977 and 1264-1283 of the MMP-13 mRNA sequence from NCBI (NM_002427) (b) RT-PCR products generated from RNA isolated from each of the cell lines, amplified for 36 cycles by primers recognizing nucleotides 959-977 and 1264-1283 of the MMP-13 mRNA sequence from NCBI (NM_002427) (C). RT-PCR products generated from RNA isolated from each of the cell lines, amplified for 16 cycles by primers recognizing nucleotides 637-657 and 1061-1081of the beta-Actin mRNA sequence from NCBI (AK 223055).
Figure 2-The ClustalW alignment of the partial sequence of the 324 bp RT-PCR product amplified using MMP-13 primers in MDA-MB 231 cells with a segment of Homo sapiens MMP-13 mRNA sequence from NCBI (NM_002427).
Figure 3-Western blot analysis of MMP-13 protein in MCF 10 A, MCF 7, MDA-MB 231 and HTB 122 cell lines. The top panel shows proteolyzed MMP-13 (48 kDa) detected in MCF 10 A, MCF 7, MDA-MB 231 and HTB 122 cell lines. The bottom panel shows the expression of GAPDH (35 kDa) in MCF 10 A, MCF 7, MDA-MB 231 and HTB 122 cell lines.
The goal of this project was to investigate the dependence of the expression of MMP-13 on the invasiveness, in different breast cancer cell lines with different invasive properties. This study involved, investigation of expression of MMP-13 in a normal human mammary cell line (MCF 10A), a non invasive breast cancer cell line (MCF 7) and two highly invasive breast cancer cell lines (MDA-MB 231 and HTB 122).
The RT-PCR analysis of transcript levels showed high levels of expression in MCF-7 and MDA-MB 231 compared to MCF 10A, whereas HTB-122 did not show expression under identical incubation conditions (Fig 1b). Based on these results it is difficult to make a conclusion relating the invasiveness of the cell line and the expression of MMP-13.
ClustalW alignment with mRNA of MMP-13 in Homo sapiens confirmed that the 324 bp RT-PCR product is from MMP-13 (fig 2).
Western blot analysis of the proteolyzed MMP-13 in cell extracts of the MCF 10A, MCF 7 and MDA-MB 231 cell lines showed higher expression levels than the levels detected in HTB 122 (Fig 3). The observed differences in the transcripts and the MMP-13 enzyme levels could be due to different post transcriptional regulation mechanisms in the different cell lines.
Further studies should be done to understand signaling pathways and regulatory mechanisms (transcriptional and post transcriptional) involved in expression of MMP-13 to see how these affect expression of MMP-13 in cell lines with different phenotypes.
Please refer to the attached full paper for more details.
I would like to thank Dr. Berish Rubin for providing the opportunity to do this project and for his guidance throughout the course of this study. I would also like to thank Mr. Jinsong Qiu and Mr. Brian Fox for their help and assistance with the various molecular methods and techniques employed throughout this project. I also thank Dr. R. Kandpal for his advice on this project.
|This document was last modified 01/31/2006.|
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