Ataxia telangiectasia mutated-dependent regulation of topoisomerase II alpha expression and sensitivity to topoisomerase II inhibitor

Abstract

Topoisomerase II alpha (TOP2A) has a crucial role in proper chromosome condensation and segregation. Here we report the interaction of TOP2A with ataxia telangiectasia mutated (ATM) and its phosphorylation in an ATM-dependent manner after DNA damage. In vitro kinase assay and site-directed mutagenesis studies revealed that serine 1512 is the target of phosphorylation through ATM. Serine 1512 to Alanine mutation of TOP2A showed increased stability of the protein, retaining TOP2A activity at least with regard to cell survival activity. Ataxia telangiectasia-derived cell lines showed high levels of TOP2A that were associated with hypersensitivity to the TOP2 inhibitor etoposide. These findings suggest that ATM-dependent TOP2A modification is required for proper regulation of TOP2 stability and subsequently of the sensitivity to TOP2 inhibitor. In a lymphoblastoid cell line derived from a patient who developed MLL rearrangement, positive infant leukemia, defective ATM expression, and increased TOP2A expression were shown. It was intriguing that hypersensitivity to TOP2 inhibitor and susceptibility to MLL gene rearrangement were shown by low-dose etoposide exposure in this cell line. Thus, our findings have clinically important implications for the pathogenesis of infantile acute leukemia as well as treatment-associated secondary leukemia following exposure to TOP2 inhibitors.

Figure 1

(a) Ataxia telangiectasia mutated (ATM) immunoprecipitant contains a 170‐kDa protein. ATM was immunoprecipitated from unirradiated (−) and irradiated (IR) (+) wild‐type lymphoblastoid cell lines (WT) and ATR65RM cells (AT). Immunoprecipitants were visualized by Coomassie Brilliant Blue staining. The band at approximately 170 kDa (arrow) was subjected to peptide sequencing. SM, size marker. (b) Separation of ATM and topoisomerase II alpha (TOP2A) by sucrose density gradient (1, lightest fraction [Fr.]; 9, heaviest fraction). (c) Left panel, WT lymphoblastoid cell line (WT) and AT65RM (AT) cell lysates were immunoprecipitated (IP) with anti‐ATM or anti‐TOP2A antibody, and immunoprecipitants were immunoblotted (IB) with anti‐ATM or anti‐TOP2A antibody, respectively. Whole‐cell extract (WCE) was included as a size control. Right panel, cell lysates from 293T cells were immunoprecipitated with anti‐ATM or anti‐TOP2A antibody, with control IgG.

Figure 2

(a) Incorporation of [³² P] orthophosphate (³² P) into topoisomerase II alpha (TOP2A). Autoradiography of TOP2A immunoprecipitant from irradiated (IR) (+) or unirradiated (−) cells. (b) Schematic diagram of GST‐TOP2A fragment. The phosphorylated fragments are shown in black. (c) In vitro phosphorylation of TOP2A fragments by ataxia telangiectasia mutated (ATM) immunoprecipitants. Fragment numbers correspond to (b). Immunoprecipitated ATM (upper panel), ³² P incorporation into GST‐TOP2A (middle panel), and GST‐TOP2A fragment loading by Coomassie Brilliant Blue (CBB) staining (bottom panel) is shown. (d) Ser1512 or Ser1524 substituted GST‐TOP2A were subjected to in vitro kinase assay. (e) In vitro kinase assay of GST‐TOP2A with or without wortmannin, and with 10 μM ATM inhibitor (ATMi; KU55933), 10 μM DNA‐PK inhibitor (DNA‐Pki; NU7026), 10 μM ATM and Rad3‐related inhibitor (ATRi; VE‐821), and 10 μM Chk2 inhibitor (Chk2i; BML‐277). Dimethylsulfoxide was used as control. (f) In vitro phosphorylation of GST‐TOP2A by FLAG‐mock (mock), FLAG‐ATM wild‐type (WT), and FLAG‐ATM kinase‐dead (KD) immunoprecipitants. (g) In cellulo TOP2A Ser1512 phosphorylation after 10‐Gy irradiation was analyzed using phospho‐specific antibody.

Figure 3

(a) Western blot analysis of wild‐type (WT) cells and ataxia telangiectasia (AT) cells. Right panel, cell cycle distribution analyzed by flow cytometry. (b) Flow cytometric analysis of nuclear topoisomerase II content. Ten thousand cells of the G ₂/M population were subjected to analysis. Topoisomerase II alpha (TOP2A) expression (FITC labeling) and cell cycle distribution (propidium iodide [PI] staining) are shown. Cell cycle histograms corresponding to each dot blot are shown (middle panel). Far right, overlaid histograms of TOP2A expression in WT (fine line) and AT (bold line) cells. (c) Half‐life of TOP2A was evaluated in GM0637 and GM05849C cells. Cells were treated for cycloheximine (1 μg/mL) for the indicated time period, and harvested. The TOP2A expression was determined using Western blotting. Right panel, quantitative analysis of TOP2A expression. (d) Half‐life of TOP2A WT and Ser1512Ala mutant transfected in 293T cell, evaluated in the same way as (c).

Figure 4

Left panel, survival responses of GM05849C/mock (diamonds), GM05849C/ATM (triangles), and GM0637 cells (squares) to etoposide (VP16). Right panel, survival responses of GM05849C (diamonds) and GM0637 cells (squares) to ICRF‐193. Data from two independent experiments are shown.

Figure 5

(a) Western blot analysis of wild‐type lymphoblastoid cells (WT) and cells carrying ataxia telangiectasia mutated (ATM) heterogenous single nucleotide polymorphism with reduced ATM expression (L2). Extracts from LCLs probed with antibodies to topoisomerase II alpha (TOP2A), and α‐tubulin. Right panel, histograms of each cell cycle distribution. (b) Apoptosis percentages after etoposide treatment. The WT, L2, and ataxia telangiectasia (AT) cells were treated with 10 μM etoposide for 16 h and subjected flow cytometric analysis. Apoptosis percentages were measured by counting subdiploid fractions after staining with propidium iodide. The results were obtained after three independent experiments. (c) MLL rearrangements were analyzed by long distance inverted PCR. Arrow, rearranged bands; asterisk, germline bands; SM, size marker.