Sprouty 2 regulates DNA damage-induced apoptosis in Ras-transformed human fibroblasts

Abstract

We have reported that expression of Sprouty 2 (Spry2) is necessary for tumor formation by HRas(V12)-transformed fibroblasts. We now report on the role of Spry2 in the inhibition of UV(254 nm) radiation-induced apoptosis in HRas(V12)-transformed human fibroblasts. Silencing Spry2 in this context resulted in increased apoptosis, associated with decreased Akt activation and decreased phosphorylation of HDM2 at Ser-166, which has been shown to stabilize HDM2. As a consequence, when cells with silenced Spry2 were UV-irradiated, they exhibited diminished levels of HDM2 and elevated levels of p53. In agreement with these findings, overexpression of Spry2 in the parental non-transformed fibroblasts led to increased Akt activation and to the stabilization of HDM2. It also led to diminished expression of p53 and decreased apoptosis following UV irradiation. Silencing Spry2 in HRas-transformed cells decreased Rac1 activation, but independent expression of Spry2 in the non-transformed parental cells had no effect on Rac1, suggesting a specific involvement in the activation of Rac1 by Ras. Silencing Spry2 in HRas(V12)-transformed cells resulted in diminished interaction between HRas and Tiam1, a Rac1-specific nucleotide exchange factor. Expression of constitutively active Rac1 in cells with silenced Spry2 partly reversed the effect of Spry2 down-regulation. Furthermore, loss of Spry2 expression in HRas(V12)-transformed cells augmented the cytotoxicity of the DNA-damaging, chemotherapeutic agent cisplatin, a process that was also reversed by active Rac1. Together, these data show that Spry2 inhibits apoptosis in response to DNA damage by regulating Akt, HDM2, and p53, by a process mediated partly by Rac1.

FIGURE 1.

Role of Spry2 on the inhibition of UV-induced apoptosis by HRas. A, normal parental non-transformed human fibroblasts (MSU1.1) and their HRas oncogene-transformed derivatives (PH3MT) were irradiated with UV (30 J/m²), incubated at 37 °C in media containing 10% serum for 4 h, and then assayed for early apoptotic events with annexin-FITC as indicated under “Experimental Procedures.” Cells that stained positive for annexin only were analyzed. The percentage of apoptotic cells following UV treatment was normalized to the percentage of cells undergoing apoptosis in the absence of treatment and is expressed in the graph as -fold change. A representative of three experiments is shown. A, MSU1.1 and PH3MT cells were treated and analyzed as described. A Western blot showing the level of Spry2 and HRas proteins is also shown. B, MSU1.1 and PH3MT cells were analyzed as described in the presence or absence of AG1478 (6 μm) and wortmannin (50 nm), as indicated. A representative of three experiments is shown. C, HRas-transformed cell lines with endogenous (PH3MT-SC) or down-regulated (PH3MT-2A3) levels of Spry2 (inset) were exposed to UV radiation (90 J/m²), and the percentage of cells undergoing apoptosis in the presence (black bars) or absence (white bars) of such treatment is shown. A representative of seven experiments is shown. D, the parental non-transformed human fibroblasts expressing an empty vector (MSU1.1-VC (VC)) or high levels of Spry2 (MSU1.1-S62 (S62)) (inset) were treated and analyzed as described. A representative of three experiments is shown.

FIGURE 2.

Effect of Spry2 down-regulation on the regulation of p85, Akt, HDM2, and p53. A, the indicated cell strains were deprived of serum for 12 h and then treated with EGF (100 ng/ml) for the indicated times (in min). Whole cell lysates (WCL) were prepared and assayed by Western blotting to determine the levels of the indicated proteins; pp85, phospho-85; pAkt, phospho-Akt; pHDM2, phospho-HDM2. B, bar graphs showing the quantification of the bands in the Western blot in panel A. C, the cell lines were exposed to UV radiation (90 J/m²) and allowed to grow under normal conditions for the indicated time periods (in hours) following UV treatment. WCL were prepared and then analyzed by Western blotting with the indicated antibodies. D, bar graphs showing the quantification of the bands in panel C.

FIGURE 3.

Effect of Spry2 overexpression on the activation of Akt, HDM2, and p53. A and B, a vector control cell strain, MSU1.1-VC, and a cell strain overexpressing Spry2, MSU1.1-S62, were treated with EGF and analyzed as in Fig. 2, A and B. pp85, phospho-85; pAkt, phospho-Akt; pHDM2, phospho-HDM2. C and D, the indicated cell strains were exposed to UV radiation as above and analyzed as in Fig. 2, C and D.

FIGURE 4.

Effect of Spry2 on Rac1 activation in HRas-transformed cells. A, WCL from the HRas-transformed cell strain expressing a high endogenous level of Spry2 (PH3MT-SC (SC)) or a reduced level of Spry2 (PH3MT-2A3 (2A3)) and the parental non-transformed human fibroblast cell strain expressing a low endogenous level of Spry2 (empty vector) (MSU1.1-VC (VC)) or expressing a high level of Spry2 (MSU1.1-S62 (S62)) were pulled down (PD) with PAK-CRIB-conjugated beads. The amount of Rac1 bound to the beads, as well as the Rac1 present in the WCL was determined. The amount of active Cdc42 was determined only in the PH3MT cells. B, WCL from PH3MT-SC and PH3MT-2A3 cell strains were immunoblotted or immunoprecipitated (IP) with an antibody specific to HRas and then immunoblotted to detect Tiam1 and HRas with the indicated antibodies. C, HRas-transformed fibroblasts (PH3MT) were stably transfected with an empty vector or a vector encoding a Myc-tagged, dominant negative form of Rac1 (Rac1^N17). WCL from these stable clones were analyzed by Western blotting for Rac1 and β-actin expression. The same cell strains were treated and analyzed as in Fig. 1A. The average of two independent experiments is shown. D, HRas-transformed cells with down-regulated Spry2 (PH3MT-2A3) but stably expressing GFP-Rac1^V12 (2A3-R1) or GFP alone (2A3-VC) were analyzed by Western blotting for pAkt; Akt; p53; HDM2; GFP; Spry2; or β-actin. E, the PH3MT-2A3 VC and PH3MT-2A3 R1 cell strains were UV-irradiated and analyzed as in Fig. 1A. The average of three independent experiments is shown. F, the indicated cell strains (5,000 cells/dish) were grown in agarose in a culture medium containing 2.5% serum for 3 weeks, as described in Ref. . Representative pictures of the colonies that formed in agarose are shown. R1, PH3MT-2A3-R1.

FIGURE 5.

Effect of Spry2 in cisplatin cytotoxicity. A–C, the PH3MT, PH3MT-2A3, and MSU1.1 cell strains were treated with cisplatin at a concentration of 0, 2, 5, 10, 20, and 40 μm concentrate for 6 h, allowed to grow under normal conditions for 24–48 h, and assayed for cisplatin cytotoxicity as described under “Experimental Procedures.” The number of cells is proportional to the optical density at 490 nm (A_{490 nm}). The -fold decrease in A_{490 nm}, relative to the untreated samples, is shown. For each panel, a representative of three independent experiments (in each n = 4) is shown. Where error bars are not shown, they are within the data point. D, a public data set from a previous study measuring genetic changes associated with cisplatin resistance in lung cancer was mined to determine the association between Spry2 expression and increased cisplatin IC₅₀. Cell lines derived from patients with small cell (SCLC), squamous cell (SqCLC), or non-small cell (NSCLC) lung cancer were sorted according to their relative Spry2 expression. For each cell line, the expression of Spry2 and Akt is reported as a dichotomous variable, i.e. either high (H) or low (L), depending on whether the level of these genes in that cell line was greater or less than their median expression in the entire subset. Each circle represents a cell line, whereas the bar represents the average IC₅₀ for that group. A similar analysis was performed for Akt expression.