Retardation of cell growth by avian reovirus p17 through the activation of p53 pathway

Abstract

The second open reading frame of avian reovirus S1 gene segment encodes a 17 kDa non-structural protein, named p17. The biological role of p17 is fully unknown so far. Using trypan blue dye exclusion and MTT assay, we demonstrated that the ectopic expression of p17 results in the reduction of viable cell number and cell proliferation rate of Vero, BHK, 293, and HeLa cells. Measurement of LDH activity and DNA fragmentation analysis revealed that p17 expression did not cause cell death or apoptosis. These data indicated that the p17 possessed the growth retardation function. Semi-quantitative RT-PCR and Western blotting revealed that p17-expressing cells induced the expression of CDK inhibitor p21cip1/waf1 in a time- and dose-dependent manner, but the transcripts of CDK inhibitor p15INK4b, p16INK4a, or p27kip were not altered. In the presence of p17, the p53 protein level and p53-driven reporter activity were elevated significantly. Dominant negative p53 alleviated the p21 accumulation, p53 activation, and growth inhibition effect induced by p17. Taken together, these studies revealed a possible intrinsic function of p17 in growth regulation through the activation of p53 and p21cip1/waf1.

Fig. 1

Detection of ARV p17 expression. (A) RT-PCR analysis with p17 (upper panel) or actin (lower panel) specific primers are displayed. Five micrograms of p17-pcDNA3.1(−) was transfected into Vero cells. After incubation of various time periods, total RNA was harvested. The p17 and actin transcripts are indicated. (B) p17 expression within the nucleus. Vero cells transfected with p17-pcDNA3.1(−). After incubation for 48 h, immunofluorescence staining was performed by using rabbit polyclonal antibody against p17 and anti-rabbit-FITC. Addition of 0.3 μg/ml PI as counterstaining to indicate nucleus localization. Phase contrast image shows the morphology of cells (a). Arrows indicate cells expressing p17 (b). Flowing PI staining, arrows indicate nucleus localization (c). The three images were same field. The magnification was 400×.

Fig. 2

Retardation of cell growth by ARV p17. (A) The growth curve and doubling time of Vero cells transfected with vector pcDNA3.1(−) or with p17-pcDNA3.1(−). The data represent the average of triplicate plates with standard deviation. The cultured condition was shown. The x-axis represents the time period incubation post-transfection. The viable cells were counted with a hemocytometer in the presence of trypan blue and are represented in the y-axis. (B) p17 expression retards cell proliferation by using the MTT assay. Vero cells transfected with pcDNA3.1(−) or p17-pcDNA3.1(−) or sigma C-pcDNA3.1(−). The x-axis indicates cultured time period and condition. The y-axis represents the row OD value. For a 24 and 48 h observation, p17-expressing cells reduced the cell proliferation. More remarkable effect was observed when cells were cultured in lower serum medium (upper). Cell growth rate of cells transfected with control vector or sigma C-pcDNA3.1(−) was comparable (lower).

Fig. 3

p17 expression did not cause cell death or apoptosis. (A) Vero cells transfected with various amounts of p17-pcDNA3.1(−) and cultured in 5% serum or 0.1% serum for 48 h. Cell death was quantified by LDH release into the medium, which was performed in duplicate on treated plates. Three independent experiments were conducted to calculate cell death and standard deviation. Cells lysed in 2% Triton X-100 represent the normalized release of maximal LDH. (B) DNA fragmentation analysis revealed that 17 expression did not cause DNA laddering. Ectopic expression of ARV apoptosis-inducing protein sigma C and infection with ARV S1133 infection was included here as positive control.

Fig. 4

Induction of p21^cip1/waf1 by p17. Vero cells transfected with pcDNA3.1(−) or p17-pcDNA3.1(−). Incubation for various time periods as indicated. RNA or total protein was harvested. (A) Semi-quantitative RT-PCR using specific primer pairs to measure the transcripts of CDK inhibitors. Only the p21^cip1/waf1 transcript was increased in a time-dependent manner. (B) p21^cip1/waf1 protein level was elevated in response to p17 expression but not in control cells.

Fig. 5

Activation of p53-dependent pathway in a time- and dose-dependent manner by p17. (A) Western blot analysis using anti-p53 (upper panel) or anti-actin (lower panel) antibody of Vero cells transfected with p17-pcDNA3.1(−). (B) Cells cotransfected with 1 μg p53-luc reporter plasmids and various amounts of p17-pcDNA3.1(−). After 48 h incubation, luciferase activity was measured and represented as activation folds relative to cells without p17 expression.

Fig. 6

Dominant negative p53 expression releas of the p17-mediated effects. (A) Dominant negative p53 expression blocked the p17-induced p53-drived reporter activity. Luciferase assay of cells transfected with pcDNA3.1(−) or p17-pcDNA3.1(−) and p53-driven luciferase reporter upon dominant negative p53 expression. The amounts of the reporter and effector plasmids (μg) transfected were as indicated. pRKbetaGAL expressed β-galactosidase was included here as transfection efficiency normalization standard. The total amount of DNA mixture of each reaction was identical. Three independent experiments were conducted to calculate the standard deviation. (B) MTT assay revealed that dominant negative p53 expression rescued the growth retardation upon p17 expression. β-Galactosidase activity was measured to normalize transfection efficiency. The data represented here are the average of three independent experiments. (C) Western blot analysis demonstrated that the dominant negative p53 expression reduced the p21^cip1/waf1 expression induced by p17 (lanes 3, 4, 5, and 6).