Rad51 inhibition is an effective means of targeting DNA repair in glioma models and CD133+ tumor-derived cells

Abstract

High grade gliomas (HGGs) are characterized by resistance to radiotherapy and chemotherapy. Targeting Rad51-dependent homologous recombination repair may be an effective target for chemo- and radiosensitization. In this study we assessed the role of Rad51-dependent repair on sensitivity to radiation and temozolomide (TMZ) as single agents or in combination. Repair protein levels in established glioma cell lines, early passage glioblastoma multiforme (GBM) cell lines, and normal human astrocytes (NHAs) were measured using western blot. Viability and clonogenic survival assays were used to measure the effects of Rad51 knockdown with radiation (XR) and TMZ. Immunocytochemistry was used to evaluate kinetics of Rad51 and γ-H2AX repair foci. Immunohistochemistry was used to assess Rad51 protein levels in glioma specimens. Repair proteins including Rad51 are upregulated in HGG cells compared with NHA. Established glioma cell lines show a dose-dependent increase in Rad51 foci formation after XR and TMZ. Rad51 levels are inversely correlated with radiosensitivity, and downregulation markedly increases the cytotoxicity of TMZ. Rad51 knockdown also promotes more residual γ-H2AX foci 24 h after combined treatment. Newly established GBM cell lines also have high Rad51 levels and are extremely sensitive to Rad51 knockdown. Clinical samples from recently resected gliomas of varying grades demonstrate that Rad51 levels do not correlate with tumor grade. Rad51-dependent repair makes a significant contribution to DNA repair in glioma cells and contributes to resistance to both XR and TMZ. Agents targeting Rad51-dependent repair would be effective adjuvants in standard combination regimens.

Fig. 1.

(A) Western blot analysis of BRCA2, KU70, Chk2, and Rad51 protein levels in 2 high-grade glioma cell lines (T98G, U373) and normal human astrocytes (NHA). (B) Rad51 levels in 4 glioma cell lines (A7, T98G, U373, U87) and NHA, with and without benzyl guanine (Bg) and TMZ treatment. Asynchronous cells were exposed to Bg + TMZ and harvested for western blot on day 0 (c, control) and 3 days post treatment (D3 = day 3 control, D3Bg = day 3 with Bg, D3BgTMZ = day 3 with Bg and TMZ). Actin levels are shown as a loading control. On day 3, flow cytometry data demonstrate a marked TMZ-induced G2 phase arrest in the glioma cells compared to untreated controls (C).

Fig. 2.

Rad51 foci numbers induced by radiation, temozolomide, and combination treatment in 4 high-grade glioma cell lines (A7, T98G, U373, U87) and normal human astrocytes (NHAs). Asynchronously growing cells were irradiated (0, 1, or 2 Gy) and fixed for fluorescent immunocytochemistry 4 h post XR or treated with TMZ then irradiated on day 3 and fixed 4 h later. Data are expressed as proportion of cells with ≥5 foci (A) and mean foci/cell in T98G (B) and U87 cells (C) post 0 Gy, 2 Gy ± TMZ. Error bars are standard errors. Typical appearances of Rad51 foci in T98G cells after 0, 1, and 2 Gy are shown in the right panel.

Fig. 3.

The effect of Rad51 knockdown using short interfering RNA on clonogenic survival of T98G glioma cells following XR (A). Asynchronous T98G cells were transfected with Rad51 siRNA on day 0 (dashed line) or scrambled control (solid line) then irradiated. Data points represent means of surviving (SF) from 3 different flasks. Error bars are standard errors. Stable transfection using shRNA constructs producing different levels of knockdown were also established and used for single dose irradiation experiments. Differential Rad51 expression levels were confirmed in 3 different cell lines on western blot (B). Survival data are summarized in 3 (C) as plating efficiency (upper panel) and surviving fraction (lower panel). Data points represent means from 2 different experiments, 3 flasks per data point in each experiment.

Fig. 4.

Cytotoxicity of TMZ ± BG on T98G (high MGMT, circles) and U373 (low MGMT, squares) glioma cells (A). Asynchronous cells were exposed to TMZ ± BG for 3 h then replated in fresh medium and assessed for colony formation 10–14 days later. The effect of Rad51 knockdown on cytotoxicity of TMZ was assessed using siRNA (B), showing significantly enhanced toxicity with combined treatment in T98G cells. Knockdown was associated with abolition of Rad51 foci assessed by immunocytochemistry in T98G cells (C, upper panel) and reduced protein levels on western blot (C, lower panel).

Fig. 5.

The effect of TMZ ± BG on clonogenic survival after XR. Asynchronous cells were exposed to TMZ 100 µM then irradiated with single doses in the range 0.1–5 Gy on day 3. Data points represent means from 3 flasks at each data point and are shown as surviving fractions (SF), normalized for the effect of TMZ on unirradiated cells. T98G (high MGMT cells with BG) upper panel (A), U373 (low MGMT cells) lower panel (B). Solid line, XR only, dashed line XR + TMZ. (C) The effect of Rad51 knockdown using short interfering RNA on clonogenic survival of T98G glioma cells following XR, or XR + TMZ. Solid triangle symbols, solid line = XR alone, open triangle symbols dashed line = XR + Rad51siRNA, open circular symbols = XR + TMZ, open square symbols = XR + TMZ + Rad51siRNA expressed as plating efficiency (PE). Relative survival of combined treatment with TMZ + Rad51siRNA (square symbols dashed line) compared to XR alone (triangular symbols solid line) is shown separately (D).

Fig. 6.

γ-H2AX foci at 24 h post treatment with XR, TMZ, Rad51 siRNA, and combined treatment of T98G cells. Cells were treated with XR alone + control siRNA (white bar), XR + Rad51siRNA (close hatched bar), XR + TMZ + control siRNA (grey bar) or XR + TMZ + Rad51siRNA or untreated (black bar). Cells were fixed for immunocytochemistry 24 h later and fluorescent foci were counted manually. γ-H2AX data points represent means from 3 different samples, error bars are SEM.

Fig. 7.

Immunohistochemistry using fluorescent antibodies to detect Cd133 and nestin protein in 3 newly established glioma cell lines, GBM 2, 4, and 7 (A). (B) Western blot of repair proteins Rad51 and MGMT in GBM 4 and 7 cells compared to T98G and U87.

Fig. 8.

Cytotoxicity of temozolomide in the 2 newly established glioma cell lines GBM 4 (A) and 7 (B). Cells were exposed to graded doses of TMZ for 3 h on day 0 then irradiated with XR doses 0, 2, or 5 Gy on day 8 then assessed for viability on day 5 post XR using the SRB assay. Data points represent means from 6 experimental points at each dose combination in a single experiment.

Fig. 9.

(A) Western blot showing Rad51 knockdown in GBM4 and GBM 7 cells. (B) and (C) Survival data in GBM4 and GBM 7 cells following a 3 h exposure to TMZ (0, 10, 30, 50 µM) on day 1 followed by XR (0, 2, or 5 Gy) on day 6, assessed by SRB assay on day 3 post XR. Data points represent means from 6 experimental points within one experiment. Error bars are standard deviations.

Fig. 10.

Glioma specimens stained for Rad51 protein compared to a positive control (testis). Panel (A) testis, showing nuclear Rad51 staining in the seminferous tubules; (B) a grade III astrocytoma, showing nuclear staining; and (C) a grade IV tumor with no Rad51 staining.