Page 107 - Haematologica3

Doxorubicin can stabilize the complex of topoisomerase II and broken DNA strands, thereby preventing the broken DNA double helix from being resealed and causing stalled DNA replication. Furthermore, the formation of doxoru- bicin-DNA adducts could activate DNA damage responses independent of topoisomerase II.8 When cells experience DNA damage, the cell cycle can be arrested in the G1, S or G2 phase for DNA repair.9 If the DNA damage is beyond recovery or the level of double-stranded breaks exceeds the repair capacity, cells never enter mitosis but die or undergo senescence.9 It does, however, remain poorly understood how doxorubicin treatment regulates cell cycle arrest and cell death in B-cell lymphomas.
Cell cycle checkpoints are critical to control the progres- sion of the cell cycle of DNA-damaged cells. The active complex of CDK1 and cyclinB1 controls entry into the mitotic (M) phase, and the expression of CDK1 is constitu- tive. Tyr15 phosphorylation mediated by Wee1 and Myt1 would inactivate CDK1, thus inhibiting mitotic entry. CyclinB1 expression increases at late S phase and reaches the peak at late G2 phase. CyclinB1 down-regulation would arrest cells at G2 phase, thus reducing mitotic entry.10,11 Further study proved that cyclinB1 is rate limiting but not essential for mitotic entry and progression.12 Abrogation of the G2/M checkpoint, for instance, by reducing the phosphorylation level of CDK1, enhances premature mitotic entry upon DNA damage, leading to increased cell death via mitotic catastrophe.9,13 Previous studies have shown that combined treatment with geno- toxic drugs and Wee1 inhibitor efficiently controls leukemia progression.14-16 It remains unclear whether Wee1 inhibitor enhances the M phase entry of cell cycle-arrested B-cell lymphomas and, if so, whether G1, S or G2 phase- arrested lymphomas are sensitive to Wee1 inhibitor.
In the current study, we employed primary mouse B cells, and various mouse and human B-cell lymphoma lines to test how B cells respond to Ara-C or doxorubicin treat- ment and to elucidate the relationships among DNA dam- age, cell cycle arrest and the cell death pathway. Our data suggest that cyclinB1/A2 upregulation is an intrinsically programmed DNA damage response. We show that differ- ent types of B cells exhibit differential cell cycle arrest upon Ara-C or doxorubicin treatment. Overall, our studies may reveal new mechanistic insights into DNA damage responses and cell cycle regulation, identify biomarkers to predict chemosensitivity and facilitate the development of novel therapies for B-cell lymphomas and beyond.
Methods
Cell culture and SOMAscan assay
CH12 lymphoma cells were cultured as described previously.17 G1XP lymphomas were generated, established and cultured as described previously.18 Ramos, OCI-LY1, OCI-LY3, OCI-LY7 and DHL-16 were gifts from Dr. Wing C. Chan (University of Nebraska, NE, USA) and were cultured in 10% fetal bovine serum lymphocyte medium. Lymphoma cells were cultured at 0.5×106/mL, and treated with Ara-C (Cat. SY004943, Accela, San Diego, CA, USA), MK1775 (Cat. 2373, Biovision, Milpitas, CA, USA) or doxorubicin (Cat. 159101, MP Biomedicals) at indicated concentrations for 6 h or 24 h. Splenic B cells were isolated from wildtype (wt) naïve mice using a negative selection kit (Stem Cell Technologies, Canada), cultured with anti-CD40 and interleukin- 4 as described previously,19 and collected 4 days after culture for
Ara-C treatment or for western blot and flow cytometry analysis. Details of the SOMAscan assay and computational analysis are provided in the Online Supplementary Materials and Methods.
Western blot, flow cytometry, and knockdown of cyclins
Primary antibodies used in the western blots are listed in Online Supplementary Table S3. Secondary horseradish peroxidase-conju- gated anti-mouse, anti-goat and anti-rabbit antibodies were from Jackson ImmunoResearch (West Grove, PA, USA) and developed by ECLTM western blotting detection reagents (GE Healthcare, Little Chalfont, UK) according to the instructions provided with the kit. Details of the flow cytometry analysis including cell cycle analysis and cyclin knockdown are given in the Online Supplementary Materials and Methods.
Fluorescence microscopy
Cells were collected and placed on poly-L-lysine-treated cover slips for 30 min, fixed by 4% paraformaldehyde for 1 h at room temperature and permeabilized by 0.1% Triton X-100 for 30 min. After blocking with 2% bovine serum albumin for 30 min, cells were covered by Vecta shield mounting medium (Cat.H-1200, Vector Laboratories, Burlingame, CA, USA) and slides. Images were acquired with an Eclipse TE2000 (Nikon).
haematologica | 2018; 103(3)
G2-arrested B lymphoma is prone to Wee1 inhibition
In vivo treatment of the transplant G1XP lymphoma model
G1XP lymphomas were generated by crossing Cγ1Cre knock- in, Xrcc4, and Trp53 conditional knockout mice, as described pre- viously.18 Animal work was approved by the Institutional Animal Care and Use Committee of University of Colorado Anschutz Medical Campus (Aurora, CO, USA). Details of the treatment are provided in the Online Supplementary Materials and Methods.
Results
Ara-C treatment induced apoptosis via caspase3 activation and DNA fragmentation
Ara-C is analogous to deoxycytidine and is thought to be an S phase-specific agent.20,21 How Ara-C causes the death of B cells remains incompletely understood. We treated mouse B-cell lymphoma, CH12 cells,22 with Ara-C, and found that Ara-C increased the percentage of annex- in-V-positive CH12 cells and caused DNA fragmentation (Online Supplementary Figure S1A-C). Since the occurrence of DNA fragmentation is related to caspase3-induced apoptosis, we examined whether Ara-C treatment affect- ed caspase3 activation. Our data showed that cleaved cas- pase3 was increased in a dose-dependent manner upon Ara-C treatment (Online Supplementary Figure S1D,E). Recent studies found that chemotherapeutic agents can cause necroptosis, a regulated form of necrosis or inflam- matory cell death.23 We, therefore, examined the expres- sion of RIP3 (RIPK3) and CaMKII, essential components of the necroptosis pathway, upon Ara-C treatment.24-26 We found that Ara-C did not increase RIP3 or CaMKII in var- ious B-cell lymphomas (Online Supplementary Figure S1F). We, therefore, conclude that Ara-C causes apoptosis by activating caspase3 in B-cell lymphomas and does not induce necroptosis.
Ara-C treatment upregulated cyclinB1 and cyclinA2 in various types of B cells
To elucidate how primary B cells or B-cell lymphomas respond to DNA damage, we employed cutting-edge
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