Identifizierung während der Apoptose transkriptionell hochregulierter Gene mittels einer viralen Genfalle und Charakterisierung eines dieser Gene: Phosphatidylinositol 4-Phosphat 5-Kinase Ibeta

Summary Apoptosis is of basic importance for tissue homoeostasis of every organism. Dysregulation of programmed cell death can lead either to degenerative deseases or neoplasias. The fundamental molecular mechanisms are of major importance for the understanding of the pathogenesis of disorders and are essential for further therapy attempts. The discovery of new apoptotic genes is of decisive importance in particular for the development of new pharmacological agents in the treatment of cancer and neurodegenerative diseases. The aim of this thesis was to find novel genes whose expression is induced during apoptosis, and to characterize one of these genes functionally. The IL-3-dependent cell line FDC P1 was used as a study system. The existence of a constitutively expressed apoptosis cascade has led to the notion that survival factors repress this machinery and, if such factors are unavailable, cells die by default. IL-3 dependent cell lines do not stop proliferation if IL-3 is deprived, on the contrary they undergo apoptosis. In order to identify genes that are transcriptionally regulated in cells undergoing apoptosis by survival factor deprivation, a gene trap approach was used. For the identification of genes that are expressed only transiently during a biological process a strategy which makes use of the site-specific Cre/loxP recombination system was developed. By the combination of gene trap mutagenesis with site-specific recombination it is possible to select for integrations into transiently expressed genes. Using this strategy the gene Phosphatidylinositol 4-Phosphat 5-Kinase I (PIP5K) could be isolated from the FDC P1 cell line. To demonstrate the transcriptional induction of the identified genes Northern Blot experiments were carried out. These experiments showed the increase of the transcript only in a closely limited time window that afterwards decrease again to the output level. In proliferation experiments it could be shown that overexpression of PIP5K has an influence on the cell cycle. Such cells besides of an increased proliferation (approx. triple higher cell numbers) also showed a reduced spontaneous apoptosis ratio (approx. 70% less apoptotic cells). In soft agar experiments also an increased stress resistance of the cells could be observed (approx. eightfold under normal IL-3 conditions). For further experiments stable PIP5K-expressing HeLa cell populations were isolated. In a systematic biochemical analysis of cell cycle modulators, PIP5K regulated cell cycle proteins were identified. The increased expression of PIP5K led to an induction of PCNA, hsMAD2 and DNA polymerase , and, furthermore, to a repression of p21/Cip1/Waf1. Finally, the role of PIP5K was examined in HeLa cells through alternative stimuli induced apoptosis. It was shown that apoptosis accelerates in cells which where treated with cytostatic drugs or radiation. PIP5K can both increase and decrease apoptosis, depending upon the stimulus. While PIP5K showed an antiapoptotic effect during IL-3 withdrawal, after cytostatic treatment it acted proapoptotic. Finally, these results reflect again a subtle coordinated regulation of cell cycle and apoptosis.