Customized rapid subtraction hybridization (RaSH) gene microarrays identify overlapping expression changes in human fetal astrocytes resulting from human immunodeficiency virus-1 infection or tumor necrosis factor-alpha treatment.
Genes displaying altered expression as a function of human immunodeficiency virus (HIV)-1 infection of cultured primary human fetal astrocytes (PHFA) were previously identified using a rapid subtraction hybridization (RaSH) method. This scheme identified both known and novel genes displaying elevated expression, astrocyte elevated genes (AEG), and decreased expression, astrocyte suppressed genes (ASG), in PHFA as a consequence of infection with HIV-1 or treatment with HIV-1 envelope glycoprotein (gp120). RaSH also identified both known and novel genes displaying enhanced (HR) or reduced (HS) expression in HIV-1 resistant versus HIV-1 susceptible human T-cell clones. In the present study, a customized microarray approach employing these RaSH-derived genes was used to distinguish overlapping gene expression changes occurring in PHFA as a function of treatment with HIV-1 and the neurotoxic agent tumor necrosis factor (TNF)-alpha. RaSH cDNAs were spotted (microarrayed) on nylon membranes and probed with temporally isolated reverse transcribed cDNAs from HIV-1-infected and TNF-alpha-treated PHFA. This strategy identified genes displaying parallel changes after TNF-alpha treatment as observed following HIV-1 infection. Confirmation of genuine differential expression was achieved by Northern blotting. These studies document that TNF-alpha can induce a set of corresponding changes in specific AEGs and ASGs as does HIV-1 infection in PHFA. Furthermore, this customized microarray approach with RaSH-derived clones represents an efficient and sensitive methodology for elucidating molecular changes in PHFA occurring as a consequence of treatment with pharmacological agents affecting astrocyte physiology.
Su
Zao-zhong
ZZ
Department of Pathology, Herbert Irving Comprehensive Cancer Center, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA.
Chen
Yinming
Y
Kang
Dong-chul
DC
Chao
Wei
W
Simm
Malgorzata
M
Volsky
David J
DJ
Fisher
Paul B
PB
D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism* / Q000821:virology; D015152:Blotting, Northern; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D005333:Fetus; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D015497:HIV-1 / Q000254:growth & development*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D018076:DNA, Complementary; D014409:Tumor Necrosis Factor-alpha