Maintenance of the CD40-related immunodeficient response in hyper-IgM B cells immortalized with a LMP1-regulated mini-EBV

Lu, Kristina T.; Dryer, Rebecca L.; Song, Charles H.; Covey, Lori R.

doi:10.1189/jlb.0305159

Cited by 5 publications

(13 citation statements)

References 67 publications

(55 reference statements)

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“…Because LMP1 activation mimics signals through the CD40 pathway, LCL tet cells allow for independent comparisons of downstream functions in response to regulated LMP1 and CD40 signals. Characterization of these cells revealed a CD23 lo /CD38 hi surface phenotype of generated pt1-LCL tet cells that was distinctly different from the typical CD23 hi / CD38 lo activated phenotype of control LCL tet cells 18 and conventional EBV-immortalized LCLs. 19 We also observed that the pt1-LCL tet cells retained the CD23 lo /CD38 hi phenotype after extended periods of culture with LMP1 or CD40 signals.…”

Section: Introductionmentioning

confidence: 99%

“…One manifestation of the pt1 B-cell defect is uncharacteristically low CD23 expression in the presence of constitutive LMP1 signals 18 ( Figure 1A). To determine whether low CD23 surface expression is due to reduced transcription, pt1-LCL tet and control LCL tet cells were analyzed for CD23a/b expression by qPCR.…”

Section: Diminished Cd23 Expression Is Directly Related To Decreased mentioning

confidence: 99%

“…Mini-EBV transformation (LCL tet ) of primary B cells from pt1 and controls (D11 and C2 clones) was carried out with recombinant plasmid p1852 as previously described. 18 …”

Section: Cell Linesmentioning

confidence: 99%

“…(C) Further analysis of the CD23b-II probe was performed with control D11 and pt1-LCL tet nuclear extracts. The following antibodies were added in increasing amounts to the indicated lanes: p50 (lanes 3-6), p65 (lanes 7-10), p52 (lanes [11][12][13][14], purified rabbit IgG (lanes 15-16), and c-Rel (lanes [17][18][19][20]. (D) EMSA with a probe that spans the identified NF-B binding site in the CD23a promoter (nucleotides Ϫ109 to Ϫ76).…”

Section: Decreased C-rel Expression In Pt1-lcl Tet Cells Is Not a Funmentioning

confidence: 99%

“…By using these cells, we were able to assess c-Rel expression in cells that were newly immortalized (CD23 lo /CD38 hi ) and in culture for 1 week (pt1-1wk) and cells that had acquired a lymphoblastic (CD23 hi /CD38 lo ) phenotype upon extended growth in culture (pt1-4wk). 18 As shown in Figure 5, newly immortalized pt1-LCLs (lane 1) express significantly less c-Rel than other CD23 hi LCLs (lanes 3-4) or transformed B cell lines (lane 5). Also, as the pt1-LCLs were grown in culture and acquired a lymphoblastic phenotype, a population emerged in which CD23 was up-regulated, concurrent with an increase in c-Rel expression to levels significantly greater than newly immortalized LCLs ( Figure 5, lane 2).…”

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confidence: 99%

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c-Rel plays a key role in deficient activation of B cells from a non–X-linked hyper-IgM patient

Sinquett

Dryer

et al. 2006

Blood

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Our previous results demonstrated that B cells from a patient (pt1) with non-X-linked hyper-IgM syndrome (HIGM) possess an atypical CD23(lo) phenotype that is unaffected by CD40-mediated activation. To investigate the molecular mechanism underlying defective CD23 expression in pt1 B cells, we used lymphoblastoid cell lines that express LMP1 under the control of a tetracycline-inducible promoter (LCL(tet)). Our analysis revealed that the CD23(lo) phenotype in the pt1-LCL(tet) cells is a direct consequence of diminished CD23 transcription. We demonstrate a marked decrease in c-Rel-containing complexes that bind to the proximal CD23a/b promoters in pt1-LCL(tet) extracts, resulting from an overall lower expression of c-Rel in pt1-LCL(tet) cells. Analysis of c-Rel mRNA revealed relatively equal amounts in pt1-LCL(tet) and control LCL(tet) cells, indicating that diminished c-Rel protein expression is unrelated to decreased transcription. Finally, a critical role for c-Rel in CD23 regulation was demonstrated by effectively altering c-Rel expression that resulted in the direct modulation of CD23 surface expression. Collectively, these findings demonstrate that low levels of c-Rel are the underlying cause of aberrant CD23 expression in pt1 B cells and are likely to play a critical role in the pathophysiology of this form of HIGM.

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Section: Introductionmentioning

confidence: 99%

Section: Diminished Cd23 Expression Is Directly Related To Decreased mentioning

confidence: 99%

“…Mini-EBV transformation (LCL tet ) of primary B cells from pt1 and controls (D11 and C2 clones) was carried out with recombinant plasmid p1852 as previously described. 18 …”

Section: Cell Linesmentioning

confidence: 99%

Section: Decreased C-rel Expression In Pt1-lcl Tet Cells Is Not a Funmentioning

confidence: 99%

mentioning

confidence: 99%

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c-Rel plays a key role in deficient activation of B cells from a non–X-linked hyper-IgM patient

Sinquett

Dryer

et al. 2006

Blood

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Mutated in colorectal cancer (MCC) is a novel oncogene in B lymphocytes

Edwards¹,

Baron²,

Moore³

et al. 2014

J Hematol Oncol

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BackgroundIdentification of novel genetic risk factors is imperative for a better understanding of B lymphomagenesis and for the development of novel therapeutic strategies. TRAF3, a critical regulator of B cell survival, was recently recognized as a tumor suppressor gene in B lymphocytes. The present study aimed to identify novel oncogenes involved in malignant transformation of TRAF3-deficient B cells.MethodsWe used microarray analysis to identify genes differentially expressed in TRAF3−/− mouse splenic B lymphomas. We employed lentiviral vector-mediated knockdown or overexpression to manipulate gene expression in human multiple myeloma (MM) cell lines. We analyzed cell apoptosis and proliferation using flow cytometry, and performed biochemical studies to investigate signaling mechanisms. To delineate protein-protein interactions, we applied affinity purification followed by mass spectrometry-based sequencing.ResultsWe identified mutated in colorectal cancer (MCC) as a gene strikingly up-regulated in TRAF3-deficient mouse B lymphomas and human MM cell lines. Aberrant up-regulation of MCC also occurs in a variety of primary human B cell malignancies, including non-Hodgkin lymphoma (NHL) and MM. In contrast, MCC expression was not detected in normal or premalignant TRAF3−/− B cells even after treatment with B cell stimuli, suggesting that aberrant up-regulation of MCC is specifically associated with malignant transformation of B cells. In elucidating the functional roles of MCC in malignant B cells, we found that lentiviral shRNA vector-mediated knockdown of MCC induced apoptosis and inhibited proliferation in human MM cells. Experiments of knockdown and overexpression of MCC allowed us to identify several downstream targets of MCC in human MM cells, including phospho-ERK, c-Myc, p27, cyclin B1, Mcl-1, caspases 8 and 3. Furthermore, we identified 365 proteins (including 326 novel MCC-interactors) in the MCC interactome, among which PARP1 and PHB2 were two hubs of MCC signaling pathways in human MM cells.ConclusionsOur results indicate that in sharp contrast to its tumor suppressive role in colorectal cancer, MCC functions as an oncogene in B cells. Our findings suggest that MCC may serve as a diagnostic marker and therapeutic target in B cell malignancies, including NHL and MM.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-014-0056-6) contains supplementary material, which is available to authorized users.

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c-Rel Deficiency Increases Caspase-4 Expression and Leads to ER Stress and Necrosis in EBV-Transformed Cells

Sinquett

Covey

2011

PLoS ONE

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LMP1-mediated activation of nuclear factor of kappaB (NF-κB) is critical for the ligand independent proliferation and cell survival of in vitro EBV-transformed lymphoblastoid cell lines (LCLs). Previous experiments revealed that a majority of LMP1-dependent responses are regulated by NF-κB. However, the extent that individual NF-κB family members are required for these responses, in particular, c-Rel, whose expression is restricted to mature hematopoietic cells, remains unclear. Here we report that low c-Rel expression in LCLs derived from a patient with hyper-IgM syndrome (Pt1), resulted in defects in proliferation and cell survival. In contrast to studies that associated loss of NF-κB with increased apoptosis, Pt1 LCLs failed to initiate apoptosis and alternatively underwent autophagy and necrotic cell death. Whereas the proliferation defect appeared linked to a c-Rel-associated decrease in c-myc expression, identified pro-survival and pro-apoptotic targets were expressed at or near control levels consistent with the absence of apoptosis. Ultrastructural examination of Pt1 LCLs revealed a high level of cellular and ER stress that was further supported by gene expression profiling showing the upregulation of several genes involved in stress and inflammation. Apoptosis-independent cell death was accompanied by increased expression of the inflammatory marker, caspase-4. Using gene overexpression and siRNA knockdown we demonstrated that levels of c-Rel directly modulated expression of caspase-4 as well as other ER stress genes. Overall, these findings reveal the importance of c-Rel in maintaining LCL viability and that decreased expression results in ER stress and a default response leading to necrotic cell death.

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Maintenance of the CD40-related immunodeficient response in hyper-IgM B cells immortalized with a LMP1-regulated mini-EBV

Cited by 5 publications

References 67 publications

c-Rel plays a key role in deficient activation of B cells from a non–X-linked hyper-IgM patient

c-Rel plays a key role in deficient activation of B cells from a non–X-linked hyper-IgM patient

Mutated in colorectal cancer (MCC) is a novel oncogene in B lymphocytes

c-Rel Deficiency Increases Caspase-4 Expression and Leads to ER Stress and Necrosis in EBV-Transformed Cells

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