Cell cycle-regulated RB expression and function in breast adenocarcinoma and immortalized mammary epithelial cells /
Loss of Rb or lowered Rb expression has been included as a prognostic indicator for breast cancer. Low or no Rb expression has been correlated with poorly differentiated, highly proliferative, highly metastatic breast adenocarcinomas. In previous immunohistochemical and immunoblot assays of breast a...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1999.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=730316781&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Loss of Rb or lowered Rb expression has been included as a prognostic indicator for breast cancer. Low or no Rb expression has been correlated with poorly differentiated, highly proliferative, highly metastatic breast adenocarcinomas. In previous immunohistochemical and immunoblot assays of breast adenocarcinomas and cell lines, Rb protein was identified using antibodies that did not distinguish between functional (hypophosphorylated) and nonfunctional (hyperphospholylated) Rb. In the present study we used three established cell lines as models for normal mammary epithelial cells (MECs) (MCFIOA), estrogen receptorpositive, non-metastatic, differentiated breast adenocarcinoma (MCF-7), and poorly differentiated, estrogen receptor negative, highly metastatic breast adenocarcinoma (MDA-MB-231). The cells were synchronized in G0/G1 by serum deprivation then stimulated to progress through the cell cycle by addition of serum. Total and hypophosphorylated Rb protein and mRNA, Rb localization and kinetics of phosphorylation, and Rb function were then determined and compared. Immunoblot Analysis revealed that total Rb was significantly higher in the tumor cells than in the immortalized MECS. Post-transcriptional mechanisms were thought to account for the differences since the amount of RB mRNA was the same across cell lines. Using quantitative direct immunofluorescence microscopy with an antibody recognizing hypophosphorylated (G99-549) Rb, we observed that Rb was phosphorylated more rapidly in response to serum stimulation in MCF-7 cells as compared to MCFIOA cells. In contrast, Rb dephosphorylation appeared to be deregulated in MDA-MB-231 cells. Overall the difference between peak and trough values for hypophosphorylated Rb was larger in breast adenocarcinoma cells as compared to immortalized MECs. Rb function was assessed by formation of Rb/E2F/ DNA complexes in gel shift assays. Function correlated with Rb phosphorylation, and was highest at times when amounts of hypophosphorylated Rb were high. Rb remained functional longer in immortalized MECs than in either tumor line despite the fact that the tremor cells had more total Rb protein. Our results suggest the importance of assessment of functional (hypophosphorylated) versus total Rb protein in clinical prognostication of breast adenocarcinoma. |
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| Item Description: | Vita. "Major Subject: Veterinary Physiology". |
| Physical Description: | xi, 87 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 62-85). |