Influence of ZAP-70 Expression On Migration of Chronic Lymphocytic Leukemia (CLL) and Lymphoma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2345-2345
Author(s):  
Eva Calpe ◽  
Carles Codony ◽  
Maria Joao Baptista ◽  
Eva Fernandez ◽  
Marta Crespo ◽  
...  
Keyword(s):  
Cell Migration ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Cell Line ◽  
Adhesion Molecules ◽  
Cell Lines ◽  
Calcium Flux ◽  
Clinical Prognosis ◽  
Ramos Cell ◽  
The Impact

Abstract Abstract 2345 Poster Board II-322 ZAP-70 (ξ-associated protein) is a tyrosine kinase (PTK) of the Syk/ZAP family normally expressed in T and NK cells. Increased ZAP-70 expression in CLL correlates with unmutated IgVH genes, a short time of progression, and a short survival. Mechanisms by which increased ZAP-70 protein expression can influence clinical outcome are not fully understood. After B-cell receptor (BCR) stimulation, activated ZAP-70 can cooperate with Syk, the tyrosine kinase of the BCR signal pathway normally expressed in B-cells, thus inducing activation of the CLL cells. Stimulation of the BCR leads to the triggering of Syk protein kinase to the cytoplasmatic tails of the receptor initiating subsequent activation of critical effector enzymes such as PI3K and PLCγ2. The PI3K/Akt pathway is related to survival and protection from BCR-induced apoptosi. Against this background, we analyzed the functional consequences of abnormal expression of ZAP-70 protein in B-lymphoma cell lines that normally does not express ZAP-70, particularly analyzing the impact of increased ZAP-70 expression oncell metabolism and cell migration. Mec-1 (CLL), Raji and Ramos (Burkitt) cell lines were stably transfected with the ZAP-70 expression vector pEGFP-ZAP-70 as well as the control pEGFP. BCR stimulation was induced in the stable Ramos cell line by IgM at several time points, and phosphorylation of downstream proteins was analyzed by Western Blot. Proliferation, cell cycle, calcium flux, adhesion molecules and cell migration were analyzed in all stable cell lines after ZAP-70 phosphorylation. After IgM stimulation, in Ramos cell line phosphorylation of ZAP-70 was observed at residue Y319 and, simultaneously, phospho-Syk expression was reduced. In addition, Erk protein was strongly activated in ZAP-70 transfected cell line compared to the control cell line, this activation lasting more than 24 hours. Notably, calcium flux detected by flow cytometry was higher in ZAP-70 transfected Ramos. Migration experiments showed that after IgM stimulation, transfected ZAP-70 Ramos migrated significantly more rapidly than cells transfected with the vector alone. Of note, after IgM stimulation, ZAP-70 transfected Ramos cells expressed a higher number of adhesion molecules (CXCR7 and others) on surface membrane than non-transfected cells. This effect on migration stimulation was also observed with the Raji cell line transfected with ZAP-70. In conclusion, addition of ZAP-70 expression to a heterologous B-cell system enhances BCR downstream signalling, calcium mobilization, and migration. Ongoing studies are determining the influence on the gene expression profile dependent of ZAP-70 expression. Altogheter, these data suggest that additional ZAP-70 expression in CLL and other lymphoproliferative disorders enhances tumoral activity, this explaining in part the poor clinical prognosis of patients with increased expression of ZAP-70. Disclosures: No relevant conflicts of interest to declare.

The Protein Tyrosine Phosphatase STS-1 Interacts with Bcr-Abl and Impairs the Response of Philadelphia-Positive Acute Lymphoblastic Leukemia (ALL) to Tyrosine Kinase Inhibitors

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3236-3236
Author(s):  
Marcus Liebermann ◽  
Daniela Hoeller ◽  
Susanne Badura ◽  
Tamara Tesanovic ◽  
Hubert Serve ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Signalling Pathways ◽  
Abl Kinase ◽  
Clinical Resistance

Abstract Abstract 3236 Bcr-Abl is a leukemogenic fusion gene that by itself is sufficient for cellular transformation (Daley et al.) and is the hallmark of chronic myeloid leukemia and Philadelphia chromosome positive (Ph+) ALL. The Bcr-Abl fusion protein is a constitutively active tyrosine kinase (TK) which disrupts multiple cellular signalling pathways controlling apoptosis, cell cycle, proliferation and DNA repair. In Ph+ ALL, a subtype of ALL with a particularly poor prognosis, targeted inhibition of Bcr-Abl activity by Abl kinase inhibitors such as imatinib has improved treatment outcome but has not abrogated the frequent development of clinical resistance. In addition to mutations in the Bcr-Abl tyrosine kinase domain (TKD), it has become apparent that other resistance mechanisms contribute to disease progression. The activity of proteins involved in the above-mentioned signalling pathways and possibly resistance to TK inhibitors (TKI) is controlled at least partially by posttranslational modifications such as phosphorylation, which is regulated by the balance between kinases and protein tyrosine phosphatases (PTP). We previously showed that PTP1B is a negative regulator of Bcr-Abl-mediated transformation and modulates sensitivity to the TKI imatinib (Koyama et al). We hypothesized that other phosphatases for which Bcr-Abl is a substrate may also contribute to resistance, one candidate being Suppressor of T-cell receptor Signalling 1 (STS-1), which negatively regulates the endocytosis of receptor TK involved in a variety of hematologic malignancies. It was the aim of this study to determine whether: i) Bcr-Abl is a substrate of STS-1 ii) STS-1 is able to dephosphorylate Bcr-Abl iii) expression of STS-1 reduces the proliferation of Bcr-Abl expressing cells by inhibiting Bcr-Abl kinase activity iv) the level of STS-1 expression modulates the sensitivity of Bcr-Abl positive cells to TKI In order to answer these questions, we used 293T cells, a human primary embryonal kidney cell line, and the IL3-dependent murine pro B cell line Ba/F3. Both cell lines were modified with constructs encoding both forms of Bcr-Abl (p185/p210) and Sts-1. For experiments with endogenous Bcr-Abl (p185) and Sts-1 we used Sup B15 cells, a human B cell precursor leukemia, and its TKI-resistant subline (Sup B15 RT), which was generated in our lab and is highly resistant not only to imatinib but also to 2nd generation TKIs (Nilotinib & Dasatinib), with no evidence of TKD mutations or transcriptional up-regulation of Bcr-Abl. In all above described cell lines the interaction between Bcr-Abl and Sts-1 could be shown in an overexpressed system (293T & Ba/F3) and on an endogenous level (Sup B15 & Sup B15 RT) by using co-IPs followed by SDS-PAGE and Western blotting. The functional relevance was examined by testing the ability of Sts-1 to dephosphorylate Bcr-Abl. Complete dephosphorylation of Bcr-Abl was shown for p185bcr-abl and p210bcr-abl in 293T cells. To verify that the functional activity was also present in hematopoietic cells, we analyzed the ability of Sts-1 to dephosphorylate Bcr-Abl in Ba/F3 and Sup B15 cells. Dephosphorylation was observed in both cell lines but was less pronounced than in 293T cells. We therefore more closely examined the most important tyrosine (Tyr) residues of Bcr-Abl and identified Tyr245 and Tyr412 as the major targets of Sts-1. Phosphorylation of Tyr245 and Tyr412 was decreased by ∼60% in Ba/F3 cells and ∼39% in Sup B15 cells. These two residues are known to be important for regulating cell proliferation, survival and cell motility. In a competitive proliferation assay in the absence of IL3, the proliferation rate of BA/F3 cells infected with Bcr-Abl and Sts – 1 was reduced compared to a Bcr-Abl infected control population. When treated with imatinib the Sts-1 expressing cells showed an approximately 5-fold reduced proliferation rate compared to cells lacking Sts-1, or to imatinib-resistant cells harbouring the Bcr-Abl “gatekeeper mutation” T315I. The expression level of Sts-1 was found to be approximately 3-fold lower in the Sup B15 RT compared to the WT cell line. Regulation appeared to occur at the transcriptional level as shown by quantitive RT-PCR. These results show that Bcr-Abl is a substrate of Sts-1, that this phosphatase modulates Bcr-Abl kinase activity and may abrogate the response to TKI. This suggests that phosphatases may contribute to the development of clinical resistance of Ph+ leukemias to TKIs. Disclosures: Ottmann: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

Novel α4-integrin ligands on an endothelial cell line

2000 ◽  
Vol 78 (2) ◽  
pp. 99-113 ◽  
Author(s):  
Kim-Sue RS Tudor ◽  
Tracy L Deem ◽  
Joan M Cook-Mills
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
B Cell ◽  
Cell Line ◽  
Adhesion Molecules ◽  
Cell Lines ◽  
Cellular Interactions ◽  
Lymphocyte Adhesion ◽  
Cell Membrane Proteins ◽  
Integrin Ligands

The unique combination of adhesion molecules expressed on endothelial cells is thought to mediate the specificity of leukocyte-endothelial cell interactions. In this study, murine endothelial cell lines were used as a model to identify novel adhesion molecules that participate in these cellular interactions. Lymphocyte adhesion to the continuous endothelial cell lines mHEVa and mHEVc required alpha 4-integrin. Interestingly, lymphocyte alpha 4-integrin bound to VCAM-1 as well as an unknown ligand on the mHEVa cell line. We have demonstrated that this VCAM-1-independent adhesion to the mHEVa cells was not mediated by other known alpha 4-integrin ligands (fibronectin, alpha 4-integrin itself, or MAdCAM-1). Two novel alpha 4-integrin ligands (p50 and p10) were isolated from the mHEVa cell line but not the mHEVc cell line by B cell alpha 4-integrin-specific ligand binding of radiolabeled mHEV cell membrane proteins. These results provide the first direct evidence that novel ligands for alpha 4-integrin exist on membranes from endothelial cells. Key words: B cell, T cell, endothelial cells, integrin, vascular cell adhesion molecule-1, VCAM-1.

scholarly journals Transformation-associated alterations in interactions between pre-B cells and fibronectin

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2311-2320 ◽  
Author(s):  
FM Lemoine ◽  
S Dedhar ◽  
GM Lima ◽  
CJ Eaves
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cell Attachment ◽  
Attachment Site ◽  
Receptor Antibodies

Abstract Marrow stromal elements produce as yet uncharacterized soluble growth factors that can stimulate the proliferation of murine pre-B cells, although close contact between these two cell types appears to ensure a better pre-B cell response. We have now shown that freshly isolated normal pre-B cells (ie, the B220+, surface mu- fraction of adult mouse bone marrow) adhere to fibronectin (FN) via an RGD cell-attachment site, as shown in a serum-free adherence assay, and they lose this functional ability on differentiation in vivo into B cells (ie, the B220+, surface mu+ fraction). Similarly, cells from an immortalized but stromal cell-dependent and nontumorigenic murine pre-B cell line originally derived from a Whitlock-Witte culture were also found to adhere to fibronectin (FN) via an RGD cell-attachment site. Moreover, in the presence of anti-FN receptor antibodies, the ability of this immortalized pre-B cell line to proliferate when co-cultured with a supportive stromal cell line (M2–10B4 cells) was markedly reduced (down to 30% of control). This suggests that pre-B cell attachment to FN on stromal cells may be an important component of the mechanism by which stromal cells stimulate normal pre-B cell proliferation and one that is no longer operative to control their more differentiated progeny. Two differently transformed pre-B cell lines, both of which are autocrine, stromal-independent, tumorigenic in vivo, and partially or completely differentiation-arrested at a very early stage of pre-B cell development, did not bind to FN. In addition, anti-FN receptor antibodies were much less effective in diminishing the ability of these tumorigenic pre-B cells to respond to M2–10B4 cell stimulation, which could still be demonstrated when the tumorigenic pre-B cells were co- cultured with M2–10B4 cells at a sufficiently low cell density. Analysis of cell surface molecules immunoprecipitated from both the nontumorigenic and tumorigenic pre-B cell lines by an anti-FN receptor antibody showed an increase in very late antigen (VLA) alpha chain(s) in both tumorigenic pre-B cell lines and a decrease in the beta 1 chain in one. Interestingly, all of the pre-B cell lines expressed similar amounts of messenger RNA for the beta 1 chain of the FN receptor. These results suggest that alteration of FN receptor expression on pre-B cells may represent a mechanism contributing to the outgrowth of leukemic pre-B cells with an autocrine phenotype and capable of stromal cell-independent, autonomous growth.

scholarly journals Rate of replication of the murine immunoglobulin heavy-chain locus: evidence that the region is part of a single replicon.

1987 ◽  
Vol 7 (1) ◽  
pp. 450-457 ◽  
Author(s):  
E H Brown ◽  
M A Iqbal ◽  
S Stuart ◽  
K S Hatton ◽  
J Valinsky ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Gene Cluster ◽  
Cell Lines ◽  
Heavy Chain ◽  
Dna Content ◽  
Immunoglobulin Heavy Chain ◽  
S Phase ◽  
C Value ◽  
Murine Erythroleukemia

We measured the temporal order of replication of EcoRI segments from the murine immunoglobulin heavy-chain constant region (IgCH) gene cluster, including the joining (J) and diversity (D) loci and encompassing approximately 300 kilobases. The relative concentrations of EcoRI segments in bromouracil-labeled DNA that replicated during selected intervals of the S phase in Friend virus-transformed murine erythroleukemia (MEL) cells were measured. From these results, we calculated the nuclear DNA content (C value; the haploid DNA content of a cell in the G1 phase of the cell cycle) at the time each segment replicated during the S phase. We observed that IgCH genes replicate in the following order: alpha, epsilon, gamma 2a, gamma 2b, gamma 1, gamma 3, delta, and mu, followed by the J and D segments. The C value at which each segment replicates increased as a linear function of its distance from C alpha. The average rate of DNA replication in the IgCH gene cluster was determined from these data to be 1.7 to 1.9 kilobases/min, similar to the rate measured for mammalian replicons by autoradiography and electron microscopy (for a review, see H. J. Edenberg and J. A. Huberman, Annu. Rev. Genet. 9:245-284, 1975, and R. G. Martin, Adv. Cancer Res. 34:1-55, 1981). Similar results were obtained with other murine non-B cell lines, including a fibroblast cell line (L60T) and a hepatoma cell line (Hepa 1.6). In contrast, we observed that IgCh segments in a B-cell plasmacytoma (MPC11) and two Abelson murine leukemia virus-transformed pre-B cell lines (22D6 and 300-19O) replicated as early as (300-19P) or earlier than (MPC11 and 22D6) C alpha in MEL cells. Unlike MEL cells, however, all of the IgCH segments in a given B cell line replicated at very similar times during the S phase, so that a temporal directionality in the replication of the IgCH gene cluster was not apparent from these data. These results provide evidence that in murine non-B cells the IgCH, J, and D loci are part of a single replicon.

scholarly journals A Novel Approach for Treatment of T-Cell Malignancies: Targeting T-Cell Receptor Vβ Families

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 28-29
Author(s):  
Jie Wang ◽  
Katarzyna Urbanska ◽  
Prannda Sharma ◽  
Mathilde Poussin ◽  
Reza Nejati ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Receptor ◽  
Brentuximab Vedotin ◽  
T Cell Lymphomas ◽  
T Cell Malignancies

Background: Peripheral T-cell lymphomas (PTCL) encompass a highly heterogeneous group of T-cell malignancies and are generally associated with a poor prognosis. Combination chemotherapy results in consistently poorer outcomes for T-cell lymphomas compared with B-cell lymphomas.1 There is an urgent clinical need to develop novel approaches to treatment of PTCL. While CD19- and CD20-directed immunotherapies have been successful in the treatment of B-cell malignancies, T-cell malignancies lack suitable immunotherapeutic targets. Brentuximab Vedotin, a CD30 antibody-drug conjugate, is not applicable to PTCL subtypes which do not express CD30.2 Broadly targeting pan-T cell markers is predicted to result in extensive T-cell depletion and clinically significant immune deficiency; therefore, a more tumor-specific antigen that primarily targets the malignant T-cell clone is needed. We reasoned that since malignant T cells are clonal and express the same T-cell receptor (TCR) in a given patient, and since the TCR β chain in human α/β TCRs can be grouped into 24 functional Vβ families targetable by monoclonal antibodies, immunotherapeutic targeting of TCR Vβ families would be an attractive strategy for the treatment of T-cell malignancies. Methods: We developed a flexible approach for targeting TCR Vβ families by engineering T cells to express a CD64 chimeric immune receptor (CD64-CIR), comprising a CD3ζ T cell signaling endodomain, CD28 costimulatory domain, and the high-affinity Fc gamma receptor I, CD64. T cells expressing CD64-CIR are predicted to be directed to tumor cells by Vβ-specific monoclonal antibodies that target tumor cell TCR, leading to T cell activation and induction of tumor cell death by T cell-mediated cytotoxicity. Results: This concept was first evaluated in vitro using cell lines. SupT1 T-cell lymphoblasts, which do not express a native functioning TCR, were stably transduced to express a Vβ12+ MART-1 specific TCR, resulting in a Vβ12 TCR expressing target T cell line.3 Vβ family specific cytolysis was confirmed by chromium release assays using co-culture of CD64 CIR transduced T cells with the engineered SupT1-Vβ12 cell line in the presence of Vβ12 monoclonal antibody. Percent specific lysis was calculated as (experimental - spontaneous lysis / maximal - spontaneous lysis) x 100. Controls using no antibody, Vβ8 antibody, and untransduced T cells did not show significant cytolysis (figure A). Next, the Jurkat T cell leukemic cell line, which expresses a native Vβ8 TCR, was used as targets in co-culture. Again, Vβ family target specific cytolysis was achieved in the presence of CD64 CIR T cells and Vβ8, but not Vβ12 control antibody. Having demonstrated Vβ family specific cytolysis in vitro using target T cell lines, we next evaluated TCR Vβ family targeting in vivo. Immunodeficient mice were injected with SupT1-Vβ12 or Jurkat T cells with the appropriate targeting Vβ antibody, and either CD64 CIR T cells or control untransduced T cells. The cell lines were transfected with firefly luciferase and tumor growth was measured by bioluminescence. The CD64 CIR T cells, but not untransduced T cells, in conjunction with the appropriate Vβ antibody, successfully controlled tumor growth (figure B). Our results provide proof-of-concept that TCR Vβ family specific T cell-mediated cytolysis is feasible, and informs the development of novel immunotherapies that target TCR Vβ families in T-cell malignancies. Unlike approaches that target pan-T cell antigens, this approach is not expected to cause substantial immune deficiency and could lead to a significant advance in the treatment of T-cell malignancies including PTCL. References 1. Coiffier B, Brousse N, Peuchmaur M, et al. Peripheral T-cell lymphomas have a worse prognosis than B-cell lymphomas: a prospective study of 361 immunophenotyped patients treated with the LNH-84 regimen. The GELA (Groupe d'Etude des Lymphomes Agressives). Ann Oncol Off J Eur Soc Med Oncol. 1990;1(1):45-50. 2. Horwitz SM, Advani RH, Bartlett NL, et al. Objective responses in relapsed T-cell lymphomas with single agent brentuximab vedotin. Blood. 2014;123(20):3095-3100. 3. Hughes MS, Yu YYL, Dudley ME, et al. Transfer of a TCR Gene Derived from a Patient with a Marked Antitumor Response Conveys Highly Active T-Cell Effector Functions. Hum Gene Ther. 2005;16(4):457-472. Figure Disclosures Schuster: Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding; AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria.

scholarly journals A Novel Analysis Method for Evaluating the Interplay of Oxygen and Ionizing Radiation at the Gene Level

2021 ◽  
Vol 12 ◽  
Author(s):  
Jeannette Jansen ◽  
Patricia Vieten ◽  
Francesca Pagliari ◽  
Rachel Hanley ◽  
Maria Grazia Marafioti ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Radiation Treatment ◽  
Hierarchical Cluster ◽  
Specific Gene ◽  
Analysis Method ◽  
Whole Genome Analysis ◽  
Gene Level ◽  
The Impact

Whilst the impact of hypoxia and ionizing radiations on gene expression is well-understood, the interplay of these two effects is not. To better investigate this aspect at the gene level human bladder, brain, lung and prostate cancer cell lines were irradiated with photons (6 Gy, 6 MV LINAC) in hypoxic and normoxic conditions and prepared for the whole genome analysis at 72 h post-irradiation. The analysis was performed on the obtained 20,000 genes per cell line using PCA and hierarchical cluster algorithms to extract the most dominant genes altered by radiation and hypoxia. With the help of the introduced novel radiation-in-hypoxia and oxygen-impact profiles, it was possible to overcome cell line specific gene regulation patterns. Based on that, 37 genes were found to be consistently regulated over all studied cell lines. All DNA-repair related genes were down-regulated after irradiation, independently of the oxygen state. Cell cycle-dependent genes showed up-regulation consistent with an observed change in cell population in the S and G2/M phases of the cell cycle after irradiation. Genes behaving oppositely in their regulation behavior when changing the oxygen concentration and being irradiated, were immunoresponse and inflammation related genes. The novel analysis method, and by consequence, the results presented here have shown how it is important to consider the two effects together (oxygen and radiation) when analyzing gene response upon cancer radiation treatment. This approach might help to unrevel new gene patterns responsible for cancer radioresistance in patients.

Epigenetic silencing of the immunoglobulin heavy-chain gene in classical Hodgkin lymphoma-derived cell lines contributes to the loss of immunoglobulin expression

Blood ◽  
2004 ◽  
Vol 104 (10) ◽  
pp. 3326-3334 ◽  
Author(s):  
Alexey Ushmorov ◽  
Olga Ritz ◽  
Michael Hummel ◽  
Frank Leithäuser ◽  
Peter Möller ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Epigenetic Silencing ◽  
Chain Gene ◽  
Classical Hodgkin Lymphoma ◽  
Regulatory Regions ◽  
The Impact

Abstract Immunoglobulin production is impaired in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) in spite of functional clonal rearrangements. The presence of “crippling” mutations in coding and regulatory regions, as well as down-regulation of B-cell-specific transcription factors, has been suggested as a potential reason for the lack of immunoglobulin (Ig) chain gene transcription. We have investigated the impact of epigenetic silencing in suppressing Ig heavy (H)-chain expression. Chromatin immunoprecipitation (ChIP) was used to analyze transcription factor binding to octamer motifs present in the IgH regulatory regions. Transcription factors were bound to these motifs in control cell lines, however, they were absent in the cHL-derived cell lines KMH2, L1236, and L428. Ectopic expression of octamer-binding transcription factor (Oct2) and/or B-cell Oct binding protein/Oct-binding factor (BOB.1/OBF.1) did not result in any measurable binding to these sites. Increased histone 3 Lysine 9 (H3-K9) methylation was observed in the promoter region of the IgH locus in L428 and L1236 cells. This is a typical feature of heterochromatic, transcriptionally silent regions. Treatment of cHL-derived cell lines with the DNA demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) partially reactivated IgH transcription and affected chromatin modifications. Our results suggest an important role of epigenetic silencing in the inhibition of IgH transcription in HRS cells. (Blood. 2004;104:3326-3334)

scholarly journals PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line

2016 ◽  
Vol 113 (38) ◽  
pp. 10619-10624 ◽  
Author(s):  
Vipul Kumar ◽  
Frederick W. Alt ◽  
Richard L. Frock
Keyword(s):  
Ionizing Radiation ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Strand Break ◽  
Recombination Reaction ◽  
Dna Breaks ◽  
Dsb Repair ◽  
B Lineage ◽  
B Lines

Classical nonhomologous end joining (C-NHEJ) is a major mammalian DNA double-strand break (DSB) repair pathway. Core C-NHEJ factors, such as XRCC4, are required for joining DSB intermediates of the G1 phase-specific V(D)J recombination reaction in progenitor lymphocytes. Core factors also contribute to joining DSBs in cycling mature B-lineage cells, including DSBs generated during antibody class switch recombination (CSR) and DSBs generated by ionizing radiation. The XRCC4-like-factor (XLF) C-NHEJ protein is dispensable for V(D)J recombination in normal cells, but because of functional redundancy, it is absolutely required for this process in cells deficient for the ataxia telangiectasia-mutated (ATM) DSB response factor. The recently identified paralogue of XRCC4 and XLF (PAXX) factor has homology to these two proteins and variably contributes to ionizing radiation-induced DSB repair in human and chicken cells. We now report that PAXX is dispensable for joining V(D)J recombination DSBs in G1-arrested mouse pro-B–cell lines, dispensable for joining CSR-associated DSBs in a cycling mouse B-cell line, and dispensable for normal ionizing radiation resistance in both G1-arrested and cycling pro-B lines. However, we find that combined deficiency for PAXX and XLF in G1-arrested pro-B lines abrogates DSB joining during V(D)J recombination and sensitizes the cells to ionizing radiation exposure. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested pro–B-cell lines. Finally, we also find that PAXX deficiency has no impact on V(D)J recombination DSB joining in ATM-deficient pro-B lines. We discuss implications of these findings with respect to potential PAXX and XLF functions in C-NHEJ.

Sign in / Sign up

Export Citation Format

Share Document