Indications for cell stress in response to adenoviral and baculoviral gene transfer observed by proteome profiling of human cancer cells.
Gene transfer to cultured cells is an important tool for functional studies in many areas of biomedical research and vector systems derived from adenoviruses and baculoviruses are frequently used for this purpose. In order to characterize how viral gene transfer vectors affect the functional state of transduced cells, we applied 2-D PAGE allowing quantitative determination of protein amounts and synthesis rates of metabolically labeled cells and shotgun proteomics. Using HepG2 human hepatoma cells we show that both vector types can achieve efficient expression of green fluorescent protein, which accounted for about 0.1% of total cellular protein synthesis 72 h after transduction. No evidence in contrast was found for expression of proteins from the viral backbones. With respect to the host cell response, both vectors induced a general increase in protein synthesis of about 50%, which was independent of green fluorescent protein expression. 2-D PAGE autoradiographs identified a 3.6-fold increase of gamma-actin synthesis in adenovirus transduced cells. In addition shotgun proteomics of cytoplasmic and nuclear extract fractions identified a slight induction of several proteins related to inflammatory activation, cell survival and chromatin function by both virus types. These data demonstrate that commonly used gene transfer vectors induce a response reminiscent of stress activation in host cells, which needs to be taken into account when performing functional assays with transduced cells.
Gerner
Christopher
C
Department of Medicine I, Division: Institute of Cancer Research, Medical University of Vienna, Vienna, Austria.
Haudek-Prinz
Verena J
VJ
Lackner
Andreas
A
Losert
Annemarie
A
Peter-Vörösmarty
Barbara
B
Lorenz
Olga
O
Grusch
Michael
M
D000199:Actins / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics*; D017209:Apoptosis / Q000502:physiology; D001345:Autoradiography; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism* / Q000821:virology*; D003593:Cytoplasm / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D007249:Inflammation / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013312:Stress, Physiological; D053719:Tandem Mass Spectrometry; D014161:Transduction, Genetic; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics
D000199:Actins; D011994:Recombinant Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins