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

Gene. 2003 Mar 13:306:67-78. doi: 10.1016/s0378-1119(03)00404-9.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Astrocytes / drug effects
  • Astrocytes / metabolism*
  • Astrocytes / virology
  • Blotting, Northern
  • Cells, Cultured
  • DNA, Complementary / drug effects
  • DNA, Complementary / genetics
  • DNA, Complementary / metabolism
  • Fetus
  • Gene Expression Profiling*
  • Gene Expression Regulation, Developmental
  • HIV-1 / growth & development*
  • Humans
  • Oligonucleotide Array Sequence Analysis / methods*
  • Tumor Necrosis Factor-alpha / pharmacology*

Substances

  • DNA, Complementary
  • Tumor Necrosis Factor-alpha