Gene expression networks underlying retinoic acid-induced differentiation of human retinoblastoma cells

Invest Ophthalmol Vis Sci. 2003 Mar;44(3):996-1007. doi: 10.1167/iovs.02-0434.

Abstract

Purpose: To understand the genetic regulatory pathways underlying the retinoic acid (RA) induction of cone arrestin, gene array technology and other molecular tools were used to profile global gene expression changes in human retinoblastoma cells.

Methods: Weri-Rb-1 retinoblastoma cells were cultured in the absence or presence of RA for various periods. DNA microarray analysis profiled gene expression followed by real-time PCR and Northern and immunoblot analyses to confirm the change in expression of selected retinal genes and their gene products. Additional methodology included flow cytometry analysis, immunocytochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay.

Results: DNA microarray analysis of approximately 6800 genes revealed RA-induced upregulation of cone-specific genes and downregulation of rod-specific genes in Weri-Rb-1 cells. Other significantly upregulated mRNAs included chicken ovalbumin upstream promoter-transcription factor (COUP-TF1), retinoid X receptor (RXR)-gamma, thyroid hormone receptor (TR)-beta2, and guanylyl cyclase-activating protein (GCAP)-1. Real-time PCR and/or Northern blot analysis confirmed the expression changes of a subset of genes including the upregulation of a pineal- and retina-specific transcription factor, CRX. RA treatment also led to G(0)/G(1) cell cycle arrest and increased both the intensity of human cone arrestin (hCAR)-immunoreactivity and the number of apoptotic cells. The cell-cycle-arrest stage correlated with the observed microarray results in which the RA treatment downregulated critical genes such as cyclins (cyclin E, cyclin D3) and cyclin-dependent kinases (CDK5, CDK10).

Conclusions: These data suggest that RA induces a subpopulation of retinoblastoma cells to differentiate toward a cone cell lineage while selectively leading other cells into apoptosis.

Publication types

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

MeSH terms

  • Antineoplastic Agents / pharmacology*
  • Blotting, Northern
  • Cell Cycle
  • Cell Differentiation / drug effects
  • DNA, Neoplasm / metabolism*
  • Flow Cytometry
  • Fluorescent Antibody Technique, Indirect
  • Gene Expression Profiling
  • Gene Expression Regulation, Neoplastic*
  • Humans
  • Neoplasm Proteins / metabolism*
  • Oligonucleotide Array Sequence Analysis
  • RNA, Messenger / metabolism
  • Retinal Neoplasms / genetics*
  • Retinal Neoplasms / pathology
  • Retinoblastoma / genetics*
  • Retinoblastoma / pathology
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transcription, Genetic
  • Tretinoin / pharmacology*
  • Tumor Cells, Cultured
  • Up-Regulation

Substances

  • Antineoplastic Agents
  • DNA, Neoplasm
  • Neoplasm Proteins
  • RNA, Messenger
  • Tretinoin