pRb-Independent growth arrest and transcriptional regulation of E2F target genes

Abstract

The retinoblastoma tumor suppressor (pRb) has traditionally been studied as a negative regulator of cell cycle progression through its interactions with the E2F family of transcription factors. Utilizing prostate epithelial cell lines established from Rb+/+ and Rb-/- prostate tissues, we previously demonstrated that Rb-/- epithelial cells were not transformed and retained the ability to differentiate in vivo despite the lack of pRb. To further study the effects of pRb loss in an epithelial cell population, we utilized oligonucleotide microarrays to identify any pRb-dependent transcriptional regulation during serum depletion-induced growth arrest. These studies identified 120 unique transcripts regulated by growth arrest in Rb+/+ cells. In these wild-type cells, the majority (80%) of altered transcripts were downregulated, including 40 previously identified E2F target genes. Although the transcriptional repression of E2F target genes is characteristic of pRb pocket protein family activity, further analysis revealed that, compared to Rb+/+ cells, Rb-/- cells exhibited a nearly identical response for all transcripts including those of E2F target genes. These findings demonstrate that pRb is not strictly required for the vast majority of transcriptional alterations associated with growth arrest.

Figure 1

Growth kinetics and cell cycle analysis of wtPrE and Rb^-/-PrE. Rb^+/+ and Rb^-/- cell lines were cultured in the presence or absence of 5% FBS supplement and analyzed by (A) trypan blue staining to determine viable cell number at 0, 24, and 48 hours; (B) DAPI staining to determine cell cycle populations at 48 hours; and (C) Western blot analysis for pRb, p107, and p130 at 0, 24, and 48 hours.

Figure 2

Comparison of gene expression profiles of growth-arrested Rb^+/+ and Rb^-/- cell lines. Gene expression alterations in response to serum withdrawal in Rb^+/+ cell lines were plotted along the x-axis, and gene expression alteration in response to serum withdrawal in Rb^-/- cell lines was plotted along the y-axis. The dashed line represents a 1:1 correlation between wild-type cell line fold change and Rb^-/-PrE cell line fold change. Shaded areas represent the limits of the twofold change between the two cell lines. Each filled circle represents the combined wild-type and Rb^-/-PrE cell line data for an individual gene transcript.

Figure 3

Semiquantitative RT-PCR confirmation of G₁/S phase genes repressed following growth arrest. wtPrE and Rb^-/-PrE cells were cultured in the presence or absence of serum for 48 hours. Equal cDNA generated from total RNA was utilized in RT-PCR reactions with primers specific to Rrm2, c-Myb, Mcmd, Fen1, Prim1, or Tyms. The HPRT transcript is a housekeeping gene that serves as a loading control. Only the reactions that best represented the linear range of amplification are depicted.

Figure 4

Semiquantitative RT-PCR confirmation of G₂/M phase genes repressed following growth arrest. wtPrE and Rb^-/-PrE cells were cultured in the presence or absence of serum for 48 hours. Equal cDNA generated from total RNA was utilized in RT-PCR reactions with primers specific to Plk1, Ki67, Ccnb1rs1, Cycb2, Cdc25c, or Cdc2a. The actin transcript serves as a loading control.

Figure 5

Semiquantitative RT-PCR confirmation of genes identified by microarray analysis as being differentially regulated between wtPrE and Rb^-/-PrE during growth arrest. wtPrE and Rb^-/-PrE cells were cultured in the presence or absence of serum for 48 hours. Equal cDNA generated from total RNA was utilized in RT-PCR reactions with primers specific to proliferin or Ifi203. The actin transcript serves as a loading control.