Masters Theses

Huo Li

Date of Award

12-1996

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Wesley D. Wicks

Committee Members

John Koontz, Ranjan Ganguly

Abstract

The retinoblastoma gene product (pRb) plays an important role in constraining cellular proliferation and in regulating the cell cycle. pRb's regulation of the expression of transforming growth factor β2 (TGF-P2), an inhibitory growth factor gene, was suggested to be mediated by Activating Transcription Factor 2 (ATF-2)(Kim et al., 1992), Factor a (ATF-a) (Gong et al., 1995), and their DNA target element (ATF site: CGTCA) in the TGF-P2 promoter. The differential regulation of TGF-P2 promoter activity (first observed by Gong et al., 1995) by pRb has been explored in more depth and the functional interaction sites of ATF-2 and ATFa on pRb have been mapped and compared in this study. Optimal transient transfection conditions using lipofectamine as transfection inducer for the introduction of a foreign TGF-β2 CAT reporter construct into CHO cells have been selected. Under these conditions, the activation by co-transfected ATF-a, ATF-2 and the differential regulation mediated by pRb on the TGF-β2 promoter have been explored further in terms of varying input DNA concentrations and the time course for the expression of the regulated TGF-β2 CAT. Although the regulatory pattern was similar on days 1, 2 and 3, the greatest response was observed 48 hr after transient transfection. The functional interaction sites for both ATF-2 and ATF-a on pRb were probed by cotransfecting cells with a series of truncated pRb mutants and ATF-2 or ATF-a. Our results demonstrate that ATF-2 and ATF-a share the same interaction sites on pRb in vivo: primarily in the so called A/B pocket domains of pRb, since deletions of amino acid sequences in these domains caused dramatic decreases in the ability of pRb to affect the TGF-P2 promoter with or without the overexpression of ATF's. The interaction sites also appear to involve the C-terminal domain of pRb (referred to as the C pocket), although the degree of effect was less substantial than that seen with the A/B pocket mutants. The levels of expression of foreign wild type (wt) and mutant (mt) pRb's in the presence or absence of overexpression of ATF-2 and ATF-a have been monitored using an antibody against the HA tag located on the C-terminus of pRb. All mutant forms of pRb have been shown to be expressed at levels similar to wt pRb and overexpression of ATF-2 and ATF-a did not effect the levels of expression of wt and mt pRb's. We also studied the differential regulation of ATF-2's and ATF-a's transcriptional activations by pRb in an alternative cell line, the human uterine mesodermal tumor cell line SKUT-1 which lacks endogenous pRb. The introduction of foreign pRb does not cause cellular apoptosis in SKUT-1 cells. The same regulatory pattern as in CHO cells has been observed, although there was minor variation in the degree of stimulation or inhibition.

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