Caveolin, a 21-24 kDa integral membrane protein is a principal structural component of caveolae membranes in vivo. Caveolae are small, stationary depressions (rafts) on the plasma membrane consisting of cholesterol and proteins of which caveolin is the important one. They aid the vesicular transport and have also been found to play a role in signal transduction. Caveolin is also associated with certain cancers such as breast, prostate, ovarian and renal. In the present work, I studied the expression of the caveolin-1 mRNA in three breast cell lines; Hs 578Bst, Hs 578T and MDA-MB-231 using RT-PCR and sequence analysis. Caveolin has been thought to be a candidate tumor suppressor gene as it has been mapped to a tumor suppressor region on chromosome 7. However, the present study refutes the role of caveolin-1 as a tumor suppressor as its expression is increased in the tumor cell lines as compared to the normal breast cell line.
Caveolae are small, concave, omega-shaped indentations of the plasma membrane that have been implicated in vesicular transport processes and more recently, in signal transduction (Lisanti et al, 1994, Couet et al, 1997). Caveolae are found in most cell types but are extremely abundant in terminally differentiated cell types such as adipocytes (Scherer et al, 1994), simple squamous epithelia (Simionescu and Simionescu, 1983) smooth muscle cells (Forbes et al, 1979) and fibroblasts (Bretscher and Whytock, 1977). Caveolin, a 21-24 kDa integral membrane protein, is a principal structural component of caveolae membranes in vivo (Glenney, 1989, Rothberg et al, 1992 and Kurzchalia et al, 1992). Caveolin is a protein of interest due to these reasons.
Recent studies have shown that caveolin is only the first member of a growing gene family of caveolin proteins; caveolin has been re-termed as caveolin-1. Three different caveolin genes; Cav-1, Cav-2 and Cav-3 encoding four different subtypes of caveolin have been described (Couet et al, 1997). There are two different subtypes of caveolin-1 that differ in their respective translation initiation sites (Scherer et al, 1995). Caveolin-1 has been mapped to the tumor suppressor region of chromosome 7q31.1 wherein, more tumor suppressor genes are suspected to reside. The D7S522 marker is located ~67kb upstream from Cav-2 and Cav-2 is located ~19kb upstream from Cav-1. Hence, caveolin-1 is believed to be a candidate tumor suppressor. In vitro studies by Lee et al, 1998 also support the role of caveolin-1 as a tumor suppressor. However, studies by Hurlstone et al, 1999 refute the role of caveolin-1 as a breast tumor suppressor gene in vivo. The objective of the present study was to study the expression of caveolin-1 mRNA in various breast cell lines.
One normal breast cell line, Hs 578Bst and two breast carcinoma cell lines; Hs 578T from the same patient as Hs 578Bst and an unrelated breast carcinoma cell line MDA-MB-231 were studied for the expression of mRNA for caveolin-1 using the specific primers and molecular techniques such as RT-PCR and DNA Sequence Analysis. The two cell lines from the same patient were derived from epithelial cells while MDA-MB-231 was derived from a pleural effusion of an unrelated patient.
|Total RNA samples from the three cell lines. Lane 1contains total RNA from Hs 578Bst, lane 2 (Hs 578T) and lane 3 (MDA-MB-231). The two rRNA subunits, 28S and 18S can be seen clearly.|
|RT-PCR analysis: Hs578BsT (lanes 1 & 4), Hs578T (lanes 2 & 5), MDA-MB-231 (lanes 3 & 6), actin (lanes 1-3) and caveolin-1 (lanes 4-6). RT-PCR was performed as described in Materials and Methods and 10 mL of the products were run on a 0.8% agarose gel stained with ethidium bromide. Actin primers generated an expected ~700 bp (lanes 1-3) product while caveolin-1 primers generated an expected ~400 bp product (lanes 4-6).|
|Arrows indicate the position of the forward and reverse primers used in this experiment. The two primers span the intron between exon 1 and exon 2.|
|DNA sequence analysis of the RT-PCR products using Caveolin-1 specific primers shown in Materials and Method. The sequence read from the RT-PCR products (experimental) showed 100% homology to the published Caveolin-1 sequence (GenBank:NM_001753).|
|Hs 578Bst||Hs 578T||MDA-MB-231|
|Caveolin-1||100%||215% (~2 fold)||302% (~3 fold)|
Sincere thanks to Sabrina Volpi and Rocco Coli for their standing help and guidance throughout this project. Thanks to Dr. Berish Rubin for giving me this opportunity to undertake this independent piece of work and for ordering the cell lines and other material for the same. I am very grateful to Dr. Raj Kandpal and Nagrajan Ganachari for helping me grow the cell lines studied here and for maintaining them throughout this project. I’m also thankful to Dr. Michael Risley for useful discussion and Dr. Robert Ross, Sharon Thomas and Brooke Barton for help with Sigma Gel. Thanks to Manish Pandya for help with the computer. At last, thanks to my colleagues Milu Ahmed and Kerem Pilavci for useful discussions during the course of the work.
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|This document was last modified 01/31/2006.|
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