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Title: Investigation of apoptosis by conditional gene expression
Author: Malcomson, Roger D. G.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1997
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It was decided to direct the expression of several apoptosis-related genes (namely c-myc, p53, p21WAF/CIP, and Nedd2) in tissue culture in order to provide useful tools to answer questions about the role of such genes in the control of apoptosis. Owing to the limitations of existing conventional expression technology, where test genes are constitutively overexpressed from a strong viral promoter often in transient expression assays, use was made of conditional minigenes: 1) A temperature-sensitive (ts) murine p53 (p53val135) was employed to investigate the sensitivity of activated c-Ha-ras-transformed rat embryo fibroblasts (Clone 6) to DNA-damage induced by the genotoxic chemotherapeutic drugs etoposide and bleomycin; 2) An oestrogen-regulable c-myc-oestrogen receptor hormone binding domain fusion protein (myc-ER) was used in conjunction with p53val135 in order to investigate whether forced expression of phenotypically wild-type p53 was sufficient to trigger apoptosis by c-myc in Clone 6 and Rat-1 fibroblasts; and 3) Vectors were constructed that contain apoptosis genes under the control of semi-synthetic promoters based upon the inducible E. coli lac operator-repressor system or a promoter containing yeast Gal-4 binding sites inducible by a tamoxifen-sensitive VP16GaERtm chimaeric trans-activator protein. Results showed that: 1) Expression of ts p53 at the permissive temperature protected Clone 6 cells from cytotoxic drug-induced apoptosis, probably by enforcing a cell cycle arrest in G1. 2) Co-expression of myc-ER and ts p53 yielded no stable cell lines probably due to biologically significant basal activation of both p53val135 and myc-ER under the culture conditions used. This is consistent with a co-operative role for c-myc and p53 in apoptosis triggering. 3) Control of expression in Rat-1 cells of the ICE-like protease Nedd2 (the mouse homologue of human ICH-1) by the VP16GalERtm system was tight enough to allow development of stably-transfected cells, which upon induction with 4-hydroxytamoxifen, rapidly underwent apoptosis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available