Functional Studies of Id1

In order to determine the effect of Id-1 expression on human PCA cell growth, we transfected an Id-1 expression vector into a PCA cell line, LNCaP, which exhibited undetectable levels of Id-1 in the absence of fetal calf serum (FCS).

Ectopic Id-1 Expression and its Effect on PCA Growth. Id-1 expression in LNCaP cells was high in the presence of 10% FCS; it decreased with lower FCS concentrations, and became undetectable after culturing in serum free media (SFM) for 48 h. To determine the effect of ectopic Id-1 expression, a retroviral vector containing the full-length human Id-1 cDNA (pBabe-Id-1) was transfected into LNCaP cells, and 10 stable clones were selected (18). In the absence of FCS, 7/10 clones expressed Id-1 at different levels in the absence of FCS (Figure 3). The remaining 3 clones remained Id-1 negative. The effect of Id-1 in PCA cell proliferation was determined in the transfectant clones. The results showed that the introduction of Id-1 increased LNCaP cell proliferation and Id-1 expression (Figure 4). These data indicate that ectopic expression of Id-1 gene activity enhances the growth of transfectant clones in the absence of FCS.

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Serum-dependent-Id-1 expression in LNCaP cells. Cells were cultured in RPMI 1640 medium containing different FCS concentrations for 24 to 48 h before Western blot analysis. Note that Id-1 expression declines as the level of FCS decreases. (B) Id-1 expression levels in stable transfectant clones (Id-l-Cl-10), vector control (pBabe), and parental LNCaP cells cultured in SFM for 48 h. Note that 7/10 clones express different levels of Id-1 protein, while Id-1 expression is undetectable in the controls. Expression of actin was used as an internal loading control.

Effect of Id-1 Expression on DNA Synthesis and Cell Cycle Distribution. Next, we determine whether Id-1-induced cell growth was due to its ability to initiate DNA synthesis in PCA cells in SFM (18). Cell cycle analyses showed an increased number of S phase cells in Id-1-expressing cells (Figure 5). In addition, the increase in S phase cells was correlated with the levels of Id-1 expression; i.e., higher Id-1 expression levels, higher S phase number, thus higher cell proliferation rate. This finding was confirmed by the analysis of DNA synthesis by BrdU incorporation (Figure 6). The DNA synthesis rate in clones with higher Id-1 levels was similar to those of control LNCaP-pBabe cultured in 10% FCS. These data indicate that the increased cell growth involves shortening of the cell cycle with a

Figure 4. Growth rate of LNCaP and Id-1 transfectants cells, 103 cells were cultured in 24-well plates in SFM. Every 24 h, cell number was determined using Trypan-blue. Values represent the mean ± SD of three experiments. Note that the cell growth rate is associated with increased Id-1 expression.

Time (Hours)

concurrent increase in DNA synthesis (18).

concurrent increase in DNA synthesis (18).

Figure 4. Growth rate of LNCaP and Id-1 transfectants cells, 103 cells were cultured in 24-well plates in SFM. Every 24 h, cell number was determined using Trypan-blue. Values represent the mean ± SD of three experiments. Note that the cell growth rate is associated with increased Id-1 expression.

Time (Hours)

Figure 5. Cell cycle distribution of Id-1 transfectants. Cells (5 x 105) cultured in SFN for 48 h, unless indicated. Flow cytometry was performed on an EPICS profile analyzer and analyzed using the ModFit LT2.0 software (Coulter) (18). Note the increased number of S phase cells in cells expressing Id-1.

Figure 5. Cell cycle distribution of Id-1 transfectants. Cells (5 x 105) cultured in SFN for 48 h, unless indicated. Flow cytometry was performed on an EPICS profile analyzer and analyzed using the ModFit LT2.0 software (Coulter) (18). Note the increased number of S phase cells in cells expressing Id-1.

Figure 6. BrdU incorporation. Cells cultured in SFM for 48 h before testing. At least 500 cells/experiment were counted. Values represent the mean ± SD ofthree separate experiments (18).

Effect of Id-1 Expression on Rb/pl6 Pathway. To investigate the mechanisms involved in Id-1 -induced LNCaP cell proliferation, we determined the expression levels of pl6INMa,CDK4, p21Wafl, p27Kipl, CDK2, and Rb in Id-1 expressing clones (18). The results showed that the expression of was very low or undetectable in all of the Id-1 expressing clones, while a 2.0-3.0-fold increase in p!6INK4a expression levels was observed in the controls and the Id-1

negative clones (Figure 7). These data demonstrate that Id-1 expression reduced the levels of pi 6INK4a protein expression in LNCaP cells. In addition, we found that the phosphorylated forms ofCDK4 and 2 were apparent in all ofthe Id-1 expressing clones, but not in the controls or Id-1 negative clones (I 'mure 7). No significant changes were found in the levels of expression of p21w 1 or p27Kipl in the Icl-l expressing clones. Further phosphorylated Rb was found in all of the clones; however there was no evidence of Rb phosphorylation in the controls or Id-1 negative clones (Figure 7). Our results indicate that Id- / induced inactivation ofthe pl6INK4a/Rb pathway may be responsible for the increased cell proliferation observed in PCA cells.

Figure 7. Western blotting analysis of pj6INK4a? CDK4

p21Wafl, p27K'p1, CDK2, and Rb expression in Id-1 transfectants and controls. Cells were cultured in SFM for 48 h before harvesting. Aliquots (50 |„ig protein) were immunoblotted with Santa Cruz Biotechnology antibodies Id-1 (C20, ppCDM ►

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1:500), p21Wafl (1:1000, N20), p27Kipl (1:1000), Transduction Laboratories CDK4 (1:250), Oncogene CDK2 (1:2000) and pRb (1:500, Ab-1). The relative amounts of each protein were quantified as ratios to actin (1:500, Amersham). Results represent three independent experiments.

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