Coilin levels modulate cell cycle progression and γH2AX levels in etoposide treated U2OS cells

Abstract

Coilin is considered the Cajal body (CB) marker protein. In this report, we investigated the role of coilin in the DNA damage response and found that coilin reduction correlated with significantly increased levels of soluble γH2AX in etoposide treated U2OS cells. Additionally, coilin levels influenced the proliferation rate and cell cycle distribution of cells exposed to etoposide. Moreover, coilin overexpression inhibited nucleolar localization of endogenous coilin in etoposide treated U2OS cells. Collectively, these data provide additional evidence for coilin and CBs in the DNA damage response.

Figure 1. Coilin reduction induces soluble γH2AX levels upon etoposide treatment

(A) U2OS cells were transfected with coilin siRNA or control siRNA for 24 hours, then untreated or treated with etoposide for an additional 16 hours. Cell lysates were western blotted and probed with anti-coilin (top panel), anti-γH2AX (middle panel) and anti-β tubulin antibodies (lower panel). β tubulin serves as a loading control. (B) Quantitative analysis of γH2AX relative to β-tubulin levels. The ratio of γH2AX relative to β-tubulin from treated control and coilin siRNA transfected cells were normalized to the ratio obtained from treated cells not transfected with siRNA. The increased relative γH2AX level in coilin siRNA transfected cells was statistically significant (*, p = 0.02, n = 3) compared to control siRNA γH2AX levels.

Figure 2. Coilin impacts cell proliferation rates in etoposide treated U2OS cells

(A) Proliferation assay on U2OS cells transfected with control siRNA (control KD) or coilin siRNA (coilin KD). Data were normalized to that obtained for the untreated control KD condition (* = p < 0.05 versus control KD, # = p < 0.05 versus control KD+, n = at least 12 for each condition). (B) Proliferation assay on U2OS cells transfected with GFP or GFP-coilin and treated with etoposide (+) or untreated, normalized to the untreated GFP (GFP-coilin) transfected condition. Treated cells expressing GFP-coilin were significantly reduced in their proliferation compared to untreated or treated cells expressing GFP alone (* p = < 0.05 versus GFP, # = p < 0.05 versus GFP+, n = at least 12 for each condition).

Figure 3. Transiently transfected coilin increases the percent of cells in S and G2/M after etoposide treatment

(A) U2OS cells expressing GFP or GFP-coilin were untreated or subjected to etoposide treatment (+) 24 h after transfection, followed by FACS analysis. Cell cycle distribution was conducted only on cells expressing GFP or GFP-coilin. (B) The bar graph represents the mean percentage of each cell cycle phase ± standard error about the mean from three independently conducted experiments. * = p < 0.05 versus G1, S and G2/M phases of treated GFP expressing cells.

Figure 4. Coilin levels influence nucleolar accumulation in etoposide treated U2OS cells

(A) Distribution of coilin (red) in untreated and etoposide treated U2OS cells. DAPI staining was used to detect the nucleus (blue). In untreated cells (upper panel), an arrow indicates a cell with nucleoplasmic coilin but lacking CB, arrowheads denote coilin in CBs, and double arrowheads show nucleolar coilin accumulation. In etoposide treated cells, the percent of cells with nucleolar coilin accumulation (double arrowhead) is increased compared to that found in untreated cells. Scale bars = 10 μm. (B) Untreated and etoposide treated U2OS cells were co-stained with antibodies to detect coilin (green) and fibrillarin (red). Double arrowheads mark nucleolar accumulations, and arrowheads indicate CBs. (C) Over expression of GFP-coilin suppresses the nucleolar accumulation of endogenous coilin in etoposide treated U2OS cells. Cells were transfected with GFP or GFP-coilin and 24h post transfection cells were treated with etoposide or left untreated, and then stained to detect coilin (red) or GFP/GFP-coilin (green). DAPI (blue) was used to detect the nucleus. Arrowheads denote CBs and double arrowheads indicate nucleolar coilin. For untreated cells transfected with the empty GFP vector, arrows indicate two cells, one of which is expressing GFP, which have nucleoplasmic coilin but lack CBs. For untreated cells transfected with GFP-coilin vector, arrows also indicate two cells, one of which is expressing GFP-coilin. Treated cells expressing low and medium levels of GFP-coilin are shown. Scale bars = 10μm.