Title

Regulation of sialomucin complex expression in normal rat mammary epithelial cells and tumor cells

Date of Award

1999

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

Kermit L. Carraway, Committee Chair

Abstract

Sialomucin complex (SMC, Rat Muc4) is a heterodimeric glycoprotein complex consisting of a mucin subunit ASGP-1 (a&barbelow;scites s&barbelow;ialog&barbelow;lycop&barbelow;rotein-1) and a transmembrane subunit ASGP-2. SMC is highly expressed on the surface of ascites 13762 rat mammary adenocarcinoma cells, approximately 100 times the level in lactating mammary gland and 104 times that in virgin mammary gland. The SMC transcript is expressed at a level 9-fold higher than that in normal mammary epithelial cells. The 5' end and flanking region of the normal rat and 13762 tumor SMC genes were cloned and characterized, allowing the generation of a preliminary exon/intron map. Using PCR-based DNA walking, 600 bp and 2.5 kb of new sequence was obtained from tumor and normal genomic DNA, respectively. The two sequences are identical in their overlapping regions, suggesting that there are no mutations in this region that contribute to SMC overexpression in the tumor cells. Moreover, the 2.5 kb sequence has promoter activity in transfected HBL-100 cells. These data suggest that SMC is overexpressed in the tumor cells by other mechanisms than uncontrolled transcription caused by a promoter mutation.SMC is sharply increased at mid-pregnancy in a manner similar to beta-casein. Unlike beta-casein, SMC appears to be regulated post-transcriptionally. Its transcript is present in both virgin and pregnant mammary tissue, and SMC synthesis is induced rapidly in cultured primary mammary epithelial cells from either normal pregnant or virgin rats. SMC protein, but not transcript levels, are significantly reduced when mammary cells are cultured in Matrigel, a reconstituted basement membrane which stimulates casein expression. SMC precursor is synthesized in Matrigel at a 10-fold lower rate. Matrigel has no effect on either the level of SMC or its transcript in cultured 13762 mammary tumor cells. These results indicate that SMC is a novel product of normal mammary gland and milk, which is post-transcriptionally regulated by extracellular matrix in normal mammary gland, but not in 13762 mammary adenocarcinoma cells.SMC is also post-transcriptionally regulated by transforming growth factor-beta (TGFbeta). The repression of SMC expression by TGFbeta is rapid, is independent of TGFbeta-induced cell cycle arrest and does not require new protein synthesis. Unlike Matrigel, TGFbeta does not reduce SMC protein synthesis, as SMC precursor accumulation is equivalent in TGFbeta-treated and untreated cells. Instead, SMC precursor in TGFbeta treated cells is more persistent and does not become processed as rapidly into mature ASGP-1 and ASGP-2, indicating that TGFbeta disrupts processing of SMC precursor. These results indicate that SMC is also regulated by TGFbeta by a novel post-translational mechanism. Thus, SMC is regulated by multiple post-transcriptional mechanisms, which serve to repress potential deleterious effects of overexpression.

Keywords

Biology, Cell

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:9961279