![TCL1A overexpression interferes with a proficient DDR and confers aneuploidy. (A) Immunoblot analysis of phosphorylated (p)ATMS1981 in freshly isolated CLL cells treated with 10 μM etoposide for the indicated time points. Samples with low TCL1A expression (by quantitative reverse transcription polymerase chain reaction [qRT-PCR]) show higher etoposide-induced phosphorylation and cleavage of ATM. (B) qRT-PCR analysis of TCL1A expression of patient samples from panel A. (C) Splenocytes from leukemic (Leuk.) (10 months) Eμ-TCL1A mice showed a lower expression of p53 than age-matched WT mice (top). TUNEL analysis of splenocytes from WT and Eμ-TCL1A mice shows more DNA breaks in TCL1A-transgenic cells (bottom). Images at original magnification ×60 on an Axio Scope.A1 fluorescent microscope. (D) JVM3 (N = 5, representative photographs next to bar chart) and DoHH2 (N = 4) cells, both ± transgenic TCL1A, as well as MEC1 cells (N = 5; ± shRNA-mediated TCL1A knockdown) were synchronized in G2 via treatment with 9 μM RO-3306 for 20 hours. At 30 minutes and 1 hour after release, the percentage of aberrant spindles was determined by immunohistochemistry; α-tubulin (α-Tub) (green) and Hoechst (blue). The TCL1A-transgenic JVM3 and DoHH2 cells showed a more pronounced accumulation of multipolar spindles as compared with controls, which was reproduced in opposite direction in MEC1 cells after shRNA-mediated TCL1A knockdown. Range of 300 to 700 individual cells per condition; Student t test. Immunofluorescence images were captured at original magnification ×60 using an IX83 fluorescent microscope (Olympus, Japan). (E) Flow-cytometric quantification of Hoechst staining in DoHH2 ± TCL1A (top, N = 3 per genotype) and JVM3 ± TCL1A (bottom, N = 5 per genotype). In both cell lines, increased DNA ploidy was found in the TCL1A-positive condition as compared with the respective control line. (F) G-banding–based karyotype analysis of DoHH2 ± TCL1A B cells. Comparison of representative karyograms of DoHH2-TCL1A (N = 42) to DoHH2 parental cells (N = 21) with a trend toward an increased genetic complexity (chromosome gains, losses, deletions, and translocations) in the TCL1A-transfected cells. Percent of cells with genomic aberrations: DoHH2, 14.3%; DoHH2TCL1A, 23.8%; P = .516, Fisher exact test, quantification not illustrated. (G) Spectral karyotyping analysis of Eμ-TCL1A leukemic B cells shows karyotypes with trisomies of chromosomes 15 and 19 and translocations of chromosome 5. (H) Splenocytes from preleukemic (Pre-leuk.) (N = 3, WBC <30 × 109 cells/L) or leukemic (N = 5, WBC >50 × 109 cells/L) Eμ-TCL1A mice showed an aberrant number of chromosomes compared with WT (N = 3) mice; Student t test.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/141/12/10.1182_blood.2022015494/8/blood_bld-2022-015494-gr5.jpeg?Expires=1735832670&Signature=0fJszvbjeHQBjTn~Ctrepd3EU689QPsmE-KGvVCSdCN3LFdaRdmzG7uXBKMO3M6lG7pVa4mB3ORWOFoODRnNXrpRO~fbf6bwILSIiXR9W4zEHf7QqgReZCxP6gzJK-Jwq1UB60~EXo7SxbJ8tC7WVBbojJh1mdPKFRS0lC-vy0maBZrWmj6KJGcGth1jvi2rFOkmyZpMRZojPCMLyCNNS-ZxlyM66kUqqRXGFNpoJjJFHq3--DyPtIiNZdX8G9TJe11N0BjodGD4q1usnWtgUv7um0TauVEyPtrGhFTkVh5IGD0CFRuEigPFRSAAPHqQRn8X9jtCpVJSkNR7sOzIzA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
TCL1A overexpression interferes with a proficient DDR and confers aneuploidy. (A) Immunoblot analysis of phosphorylated (p)ATMS1981 in freshly isolated CLL cells treated with 10 μM etoposide for the indicated time points. Samples with low TCL1A expression (by quantitative reverse transcription polymerase chain reaction [qRT-PCR]) show higher etoposide-induced phosphorylation and cleavage of ATM. (B) qRT-PCR analysis of TCL1A expression of patient samples from panel A. (C) Splenocytes from leukemic (Leuk.) (10 months) Eμ-TCL1A mice showed a lower expression of p53 than age-matched WT mice (top). TUNEL analysis of splenocytes from WT and Eμ-TCL1A mice shows more DNA breaks in TCL1A-transgenic cells (bottom). Images at original magnification ×60 on an Axio Scope.A1 fluorescent microscope. (D) JVM3 (N = 5, representative photographs next to bar chart) and DoHH2 (N = 4) cells, both ± transgenic TCL1A, as well as MEC1 cells (N = 5; ± shRNA-mediated TCL1A knockdown) were synchronized in G2 via treatment with 9 μM RO-3306 for 20 hours. At 30 minutes and 1 hour after release, the percentage of aberrant spindles was determined by immunohistochemistry; α-tubulin (α-Tub) (green) and Hoechst (blue). The TCL1A-transgenic JVM3 and DoHH2 cells showed a more pronounced accumulation of multipolar spindles as compared with controls, which was reproduced in opposite direction in MEC1 cells after shRNA-mediated TCL1A knockdown. Range of 300 to 700 individual cells per condition; Student t test. Immunofluorescence images were captured at original magnification ×60 using an IX83 fluorescent microscope (Olympus, Japan). (E) Flow-cytometric quantification of Hoechst staining in DoHH2 ± TCL1A (top, N = 3 per genotype) and JVM3 ± TCL1A (bottom, N = 5 per genotype). In both cell lines, increased DNA ploidy was found in the TCL1A-positive condition as compared with the respective control line. (F) G-banding–based karyotype analysis of DoHH2 ± TCL1A B cells. Comparison of representative karyograms of DoHH2-TCL1A (N = 42) to DoHH2 parental cells (N = 21) with a trend toward an increased genetic complexity (chromosome gains, losses, deletions, and translocations) in the TCL1A-transfected cells. Percent of cells with genomic aberrations: DoHH2, 14.3%; DoHH2TCL1A, 23.8%; P = .516, Fisher exact test, quantification not illustrated. (G) Spectral karyotyping analysis of Eμ-TCL1A leukemic B cells shows karyotypes with trisomies of chromosomes 15 and 19 and translocations of chromosome 5. (H) Splenocytes from preleukemic (Pre-leuk.) (N = 3, WBC <30 × 109 cells/L) or leukemic (N = 5, WBC >50 × 109 cells/L) Eμ-TCL1A mice showed an aberrant number of chromosomes compared with WT (N = 3) mice; Student t test.
