Michael Strauss

Wig-1, a new p53-induced gene encoding a zinc finger protein

Wild type p53 expressed from a temperature-sensitive (ts p53) construct induces both G1 cell cycle arrest and apoptosis in the p53-negative J3D mouse T lymphoma line (Wang et al., 1995). Using differential display analysis, we have identified one new p53-induced gene, wig-1 (for wild type p53-induced gene 1), whose 7.6u2009kb and 2.2u2009kb transcripts are upregulated in ts p53-transfected J3D cells following induction of wild type p53 expression by temperature shift to 32°C. The wig-1 transcripts were also induced in irradiated NIH3T3 and p21−/− fibroblasts but not in irradiated p53−/− fibroblasts. Whole body gamma irradiation caused induction of both wig-1 transcripts in mouse brain, testis, kidney, spleen and lung. A basal wig-1 expression was detected in brain, testis and kidney. The WIG-1 protein contains three zinc finger motifs and a putative nuclear localization signal.

Nonphysiological overexpression of low-density lipoprotein receptors causes pathological intracellular lipid accumulation and the formation of cholesterol and cholesteryl ester crystals in vitro

Recent therapeutic strategies for the treatment of familial hypercholesterolemia have been based on liver-directed gene transfer of a functional low-density lipoprotein (LDL) receptor cDNA under control of viral or strong housekeeping promoters. Strong viral promoters including cytomegalovirus, Rous sarcoma virus, and simian virus 40 promoters are commonly employed to reach significant physiological effects. These promoters mediate constitutive and nonphysiological overexpression in every transduced cell, while the endogenous LDL receptor expression is controlled by a complex feedback mechanism based on intracellular cholesterol concentration. To investigate intracellular consequences of persistent LDL receptor overexpression we constructed a recombinant adenovirus encoding the human LDL receptor under control of the Rous sarcoma virus promoter. The metabolic and morphological effects of LDL receptor expression were characterized by uptake experiments with human hepatoma cells using fluorescent and radiolabeled LDL. We observed that large amounts of LDL accumulate within LDL receptor transduced cells, which eventually lead to massive intracellular lipid deposition. Kinetic experiments with LDL-supplemented medium resulted in numerous crystal shaped structures in the cytosol of transduced cells as visualized by digital interference contrast optic within 60xa0min after LDL supplementation. Thin layer chromatography analyses of cellular lipids suggested these crystalline structures to be dependent on intracellular cholesterol and cholesterol ester levels. Mock-infected cells showed neither cholesterol lipid accumulation nor crystal formation. In conclusion, our data demonstrate that nonphysiological overexpression of the LDL receptor can cause massive lipid accumulation, which cannot be compensated by the hepatoma cell metabolism. This phenomenon may result in negative selection of LDL receptor overexpressing cells in vitro and in vivo.