Reexpression of hSNF5 in malignant rhabdoid tumor cell lines causes cell cycle arrest through a p21(CIP1/WAF1)-dependent mechanism

Abstract

Loss of hSNF5 function is usually observed in malignant rhabdoid tumor (MRT), a highly aggressive pediatric neoplasm. Previous studies have shown that reexpression of hSNF5 in MRT cell lines causes G1 cell cycle arrest with p16(INK4A), p21(CIP1/WAF1), and cyclin D1 playing key roles in MRT cell growth control. However, we have shown that reexpression of hSNF5 induced cell cycle arrest in the absence of p16(INK4A) expression. These results indicate that the mechanism of hSNF5-induced cell cycle arrest is context dependent. Here, we investigated the relationship between p21(CIP1/WAF1) and hSNF5 in the regulation of growth using several MRT cell lines. We found that G1 cell cycle arrest occurred concomitant with an increase in p21(CIP1/WAF1) mRNA and protein levels and preceded p16(INK4A) mRNA and protein upregulation. Chromatin immunoprecipitation data confirmed that hSNF5 appeared at both p21(CIP1/WAF1) and p16(INK4A) promoters after reexpression. We further showed that p21(CIP1/WAF1) induction showed both p53-dependent and p53-independent mechanisms. We also showed that reduction of p21(CIP1/WAF1) expression by RNAi significantly inhibited hSNF5-induced G(1) arrest. Our results show that both p21(CIP1/WAF1) and p16(INK4A) are targets for hSNF5 and that p21(CIP1/WAF1) upregulation during hSNF5-induced G(1) arrest precedes p16(INK4A) upregulation. These findings indicate that SNF5 mediates a temporally controlled program of cyclin-dependent kinase inhibition to restrict aberrant proliferation in MRT cells.

Figure 1. G₁ cell cycle arrest induced by reexpression of hSNF5

(A) Cells were harvested at the indicated times after infection with Ad-hSNF5 and Ad-GFP. Total cell protein (30 μg) were separated on a 4-20% SDS-polyacrylamide gel and probed with either anti-SNF5 or anti-ß-actin. un; uninfected control. (B) Twenty-four hours after infection with Ad-hSNF5 or Ad-GFP, cells were harvested and analyzed by flow cytometry. Left, representative profiles; Right, Values are the mean of three independent experiments; bars, ± SD. *, P < 0.05 relative to the number of S-phase of un-infection control. (C) Cells were harvested at the indicated times after infection with Ad-hSNF5 and Ad-GFP. Total cell protein (30 μg) were separated on a 4-20% SDS-polyacrylamide gel and probed with appropriate antibodies. un; uninfected control.

Figure 2. hSNF5-induced p21^CIP1/WAF1 expression

(A) RNA was extracted at the indicated times after infection with Ad-hSNF5 and Ad-GFP. The mRNA levels were measured for each gene by QT-PCR and normalized for β-actin expression. Values are the mean of three independent experiments; bars, ± SD. *, P < 0.05 relative to the Ad-GFP and un-infected control. un; uninfected control. (B) At 24 hours after infection with Ad-hSNF5 and Ad-GFP, protein was extracted for ChIPs assays. ChIPs assays were performed using antibodies directed against hSNF5, BRG-1, p53 and H3K4me3 on −2283 kb and −1391 kb of p21^CIP1/WAF1 promoter. Values are the mean of triplicates; bars, ± SD. *, P < 0.05 relative to the Ad-GFP control. **, P < 0.01 relative to the Ad-GFP control. #, P > 0.05 relative to the Ad-GFP control.

Figure 3. p21^CIP1/WAF1 expression after reexpression of hSNF5 in p53 stable knock down MRT cells

(A) p53 knock-down cells (A204 p53KD and TTC642 p53 KD) and the control cells (A204 pLKO.1 and TTC642 pLKO.1) were harvested and RNA and protein was extracted. The mRNA levels were measured by QT-PCR for each gene and normalized for β-actin expression. Values are the mean of three independent experiments; bars, ± SD. Total protein (30 μg) was separated on a 4-20% SDS-polyacrylamide gel and probed with appropriate antibodies. (B) Every 24 hours after infection with Ad-hSNF5 and Ad-GFP, cells were harvested and RNA was extracted. The mRNA levels were measured for each gene by QT-PCR and normalized for β-actin expression. Values are the mean of three independent experiments; bars, ± SD. *, P < 0.05 relative to the Ad-hSNF5 infected parent cells. #, P > 0.05 the Ad-hSNF5 infected parent cells.

Figure 4. hSNF5-induced p16^INK4A expression

(A) Cells were infected with Ad-hSNF5 and Ad-GFP. RNA was extracted at the indicated times after infection. The mRNA levels were measured by QT-PCR analysis for each gene and normalized for β-actin expression. Values are the mean of three independent experiments; bars, ± SD. *, P < 0.05 relative to the Ad-GFP and un-infected control. un; uninfected control. (B, C) At 24 and 48 hours after infection with Ad-hSNF5 and Ad-GFP, cells were harvested and protein was extracted for ChIPs assays. ChIPs assays were performed using antibodies directed against hSNF5(B), BMI-1(B), BRG-1(B) and H3K4me3(C) on −450 kb site of p16^INK4A promoter. Values are the mean of triplicates; bars, ± SD. *, P < 0.05 relative to the Ad-GFP control. **, P < 0.01 relative to the Ad-GFP control. #, P > 0.05 relative to the Ad-GFP control. (D) Total protein (30 μg) were separated on a 4-20% SDS-polyacrylamide gel and probed with BMI-1 antibody.

Figure 5. Inhibition of G₁ cell cycle arrest by reexpression of hSNF5 in p21^CIP1/WAF1 stable knock down MRT cells

(A) p21^CIP1/WAF1 knock-down cells (A204 p21KD and TTC642 p21 KD) and the control cells (A204 pLKO.1 and TTC642 pLKO.1) were harvested and RNA and protein was extracted. The mRNA levels were measured by QT-PCR for each gene and normalized for β-actin expression. Values are the mean of three independent experiments; bars, ± SD. Total protein (30 μg) was separated on a 4-20% SDS-polyacrylamide gel and probed with p21^CIP1/WAF1 antibodies. (B) Every 24 hours after infection with Ad-hSNF5 and Ad-GFP, cells were harvested and analyzed by flow cytometry. Values are the mean of three independent experiments; bars, ± SD. *, P < 0.05 relative to each Ad-GFP control.

Figure 6. Inhibition of pRb dephosphorylation after hSNF5 reexpression in p21^CIP1/WAF1 knock-down MRT cell lines

(A, B) p21^CIP1/WAF1 knock-down cells (A204 p21KD and TTC642 p21 KD) and the control cells (A204 pLKO.1 and TTC642 pLKO.1) were harvested and protein was extracted. Total cell protein (30 μg) were separated on a 4-20% SDS-polyacrylamide gel and probed with appropriate antibodies.