Investigation of apoptosis by conditional gene expression

Apoptosis is a form of cell death that occurs in individual cells in normal and diseased tissues. It occurs as a consequence of activation of a program of molecular events that results in the dismantlement and clearance of cells, often without induction of an inflammatory response. Many of the molecular components that regulate and execute apoptosis have been identified. Some of these regulators are, or are related to, genes that are altered in oplasia, such as c- myc,p53 and Bcl -2. The effector molecules belong to a class of cysteine proteases, known the ICE -like proteases (e.g. Nedd2). Whilst the known components can have identifiable activity in apoptosis id (or) cell cycle control, it has been suggested that they interact to control apoptosis. Further, it is not known nv the regulatory molecules interact upon downstream effectors to control apoptosis induction. Until recently, has not been possible to examine the role of apoptosis genes in combination other than by combinatorial mouse lockouts - a very time- consuming and expensive exercise. It was therefore decided to direct the expression of , veral apoptosis -related genes (namely c -myc, p53, p21 WAF /CIP, and Nedd2) in tissue culture in order to .ovide 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 inigenes: 1) A temperature- sensitive (ts) murine p53 (p53va1135) was employed to investigate the sensitivity of :tivated c- Ha -ras- transformed rat embryo fibroblasts (Clone 6) to DNA -damage induced by the genotoxic iemotherapeutic drugs etoposide and bleomycin; 2) An oestrogen -regulable c- myc -oestrogen receptor hormone nding domain fusion protein (myc -ER) was used in conjunction with p53va1135 in order to investigate whether ,rced expression of phenotypically wild -type p53 was sufficient to trigger apoptosis by c -myc in Clone 6 and at -1 fibroblasts; and 3) Vectors were constructed that contain apoptosis genes under the control of semi - rnthetic promoters based upon the inducible E. coli lac operator- repressor system or a promoter containing east Gal-4 binding sites inducible by a tamoxifen -sensitive VP16GaElen chimaeric trans- activator protein.Results showed that: 1) Expression of ts p53 at the permissive temperature protected Clone 6 cells from rtotoxic drug- induced apoptosis, probably by enforcing a cell cycle arrest in G1. 2) Co- expression of myc -ER id ts p53 yielded no stable cell lines probably due to biologically significant basal activation of both p53val 135 id 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 3mologue of human ICH- I) by the VP I6GalER`m system was tight enough to allow development of stably - ansfected cells, which upon induction with 4- hydroxytamoxifen, rapidly underwent apoptosis. In contrast, ansfections with a LacI- repressible Nedd2 expression vector could not produce repressed stable expression vets that were low enough to be compatible with colony survival following selection in tissue culture.In this work, conditional expression technology was applied to the problem of control of apoptosis and shows tat gene expression experiments that increase the susceptibility of cells to apoptosis can be carried out in a :gulated fashion. Using this approach, a cytoprotective role of wild type p53 -mediated growth arrest was discovered that was abrogated by c -myc. In addition, cell lines were developed that are suitable for the biochemical characterisation of the action of Nedd2 protease