A rapid method for quantifying cytoplasmic versus nuclear localization in endogenous peripheral blood leukocytes by conventional flow cytometry

Brittain, George C.; Gulnik, Sergei V.

doi:10.1002/cyto.a.23103

Cited by 5 publications

(3 citation statements)

References 54 publications

(53 reference statements)

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“…SLL/CLL cells within lymph node biopsies have been reported to express p-p65(Ser529) at higher levels than normal B cells (46), but the stimuli which induce p65(Ser529) phosphorylation in B-CLL appear to be undefined. Further prompting our investigation of ODN-induced p65(Ser529) phosphorylation in B-CLL cells, was past evidence that stimulus-induced Ser529 phosphorylation parallels p65 nuclear translocation in T cells (29, 47) and that Ser529 phosphorylation influences nuclear NF-kB activity and promoter binding in other lineages (45, 48, 49).…”

Section: Resultsmentioning

confidence: 90%

Mechanistic Insights into CpG DNA and IL-15 Synergy in Promoting B Cell Chronic Lymphocytic Leukemia Clonal Expansion

Gupta

Yan

Barrientos

et al. 2018

The Journal of Immunology

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Malignant cell growth within patients with B cell chronic lymphocytic leukemia (B-CLL) is largely restricted to lymphoid tissues, particularly lymph nodes. The recent in vitro finding that TLR-9 ligand (oligodeoxynucleotide [ODN]) and IL-15 exhibit strong synergy in promoting B-CLL growth may be particularly relevant to growth in these sites. This study shows IL-15-producing cells are prevalent within B-CLL-infiltrated lymph nodes and, using purified B-CLL cells from blood, investigates the mechanism for ODN and IL-15 synergy in driving B-CLL growth. ODN boosts baseline levels of phospho-RelA(S529) in B-CLL and promotes NF-κB-driven increases in and mRNA, followed by elevated IL-15Rα and IL-2/IL-15Rβ (CD122) protein. IL-15→CD122 signaling during a critical interval, 20 to 36-48 h following initial ODN exposure, is required for optimal induction of the cycling process. Furthermore, experiments with neutralizing anti-IL-15 and anti-CD122 mAbs indicate that clonal expansion requires continued IL-15/CD122 signaling during cycling. The latter is consistent with evidence of heightened mRNA in the fraction of recently proliferated B-CLL cells within patient peripheral blood. Compromised ODN+IL-15 growth with limited cell density is consistent with a role for upregulated IL-15Rα in facilitating homotypic IL-15 signaling, although there may be other explanations. Together, the findings show that ODN and IL-15 elicit temporally distinct signals that function in a coordinated manner to drive B-CLL clonal expansion.

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Section: Resultsmentioning

confidence: 90%

Mechanistic Insights into CpG DNA and IL-15 Synergy in Promoting B Cell Chronic Lymphocytic Leukemia Clonal Expansion

Gupta

Yan

Barrientos

et al. 2018

The Journal of Immunology

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“…FACS analysis was used to detect nuclear CREB3L1, using a previously published protocol ( 32 ) ( Figure 3F ). CREB3L1 at baseline was expressed only in the cytoplasm in all four cell lines.…”

Section: Resultsmentioning

confidence: 99%

Chemotherapy Controls Metastasis Through Stimulatory Effects on GRP78 and Its Transcription Factor CREB3L1

et al. 2020

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To achieve a cure for metastatic breast cancer, further understanding of molecular drivers of the metastatic cascade is essential. Currently, chemotherapy regimens include doxorubicin and paclitaxel which act in part by inducing the unfolded protein response (UPR). The master regulator of the UPR, glucose regulated protein 78 (GRP78), localizes on the surface of tumor cells and is associated with metastatic disease. Cyclic AMP responsive element binding protein 3-like 1 (CREB3L1), a member of the UPR, is a breast cancer metastasis suppressor that acts on cyclic AMP to promote the expression of target genes including GRP78. The aim of the present study was to evaluate the effects of chemotherapy on CREB3L1 and cell-surface GRP78 expression and its association with the development of breast cancer metastasis. For this purpose, we use breast cancer cells migration in vitro assays and an in vivo metastatic mouse model. The results showed that chemotherapy activated CREB3L1 and enhanced cell-surface GRP78 expression specifically in triple-negative breast cancer cells (TNBC), reducing their migration and metastatic potential. CREB3L1 knockout (KO) in the triple negative MDAMB231 cell line using CRISPR/Cas9 technology led to inhibition of GRP78 expression and abrogation of the CREB3L1 metastatic suppression function. Inoculation of CREB3L1-KO MDAMB231 cells into a mouse metastatic model induced a massive metastatic profile which chemotherapy failed to prevent. These findings elucidate a potential pathway to the development of a novel treatment strategy for metastatic TNBC based on modulating CREB3L1 and cell-surface GRP78 expression by chemotherapy and GRP78-targeted drugs.

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“…Mononuclear cells were isolated and washed twice using 1 mL of IMDM medium (STEMCELL, Vancouver, Canada) and centrifuged at 400 g for 5 minutes. Aliquots of 10 5 cells were labelled with 25 μL of FITC‐labelled mAb against CD34 (Ebioscience, San Diego) and subsequently sorted by flow cytometry (MoFlo Astrios EQ; Beckman Coulter) 17 . Purified CD34+ cells were cultured in serum‐free stem cell medium (StemSpan SEFM II, STEMCELL) for 5 to 7 days and then analysed by flow cytometry to confirm purity.…”

Section: Methodsmentioning

confidence: 99%

Hydrops fetalis associated with anti‐CD36 antibodies in fetal and neonatal alloimmune thrombocytopenia: Possible underlying mechanism

Chen

et al. 2020

Transfusion Medicine

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Objectives: In the present study, we asked whether anti-CD36 antibodies impair the maturation of erythropoietic stem cells to mature red blood cells (RBCs), leading to anaemia and hydrops fetalis (HF). Background: Recent studies have shown the importance of anti-CD36 antibodies in the development of Fetal/Neonatal Alloimmune Thrombocytopenia (FNAIT). In comparison to other types of antibody-mediated FNAIT, anti-CD36 antibodies are frequently associated with anaemia and HF. As mature RBCs do not express CD36, the reason for this phenomenon is currently not fully understood. Material and methods: A case of FNAIT with signs of HF was characterised in this study. Maternal anti-CD36 antibodies were isolated by an absorption/elution approach. We cultured haematopoietic stem cells (HSCs) with purified anti-CD36 antibodies, and the formation of burst-forming unit-erythroid and colony-forming unit-erythroid (CFU-E/BFU-E) cells was analysed. Apoptosis of HSCs was also investigated. Results: Analysis of the mother showed type-1 CD36 deficiency. Anti-CD36 antibodies were found in maternal serum, as well as on fetal platelets, by ELISA, and the specificity of these antibodies was further substantiated by flow cytometry. In comparison to control IgG, incubation of HSCs with purified anti-CD36 antibodies led to a significant reduction in CFU-E/BFU-E cell formation, and this result was associated with an increased number of apoptotic CD34+ erythroid/myeloid precursor cells. Administration of intrauterine transfusion with washed RBCs was effective in improving fetal anaemia. Conclusions: Anti-CD36 antibodies may cause anaemia and trigger HF through apoptosis of CD34+ erythroid/myeloid precursor cells. However, the contribution of other cells must also be taken into account.

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A rapid method for quantifying cytoplasmic versus nuclear localization in endogenous peripheral blood leukocytes by conventional flow cytometry

Cited by 5 publications

References 54 publications

Mechanistic Insights into CpG DNA and IL-15 Synergy in Promoting B Cell Chronic Lymphocytic Leukemia Clonal Expansion

Mechanistic Insights into CpG DNA and IL-15 Synergy in Promoting B Cell Chronic Lymphocytic Leukemia Clonal Expansion

Chemotherapy Controls Metastasis Through Stimulatory Effects on GRP78 and Its Transcription Factor CREB3L1

Hydrops fetalis associated with anti‐CD36 antibodies in fetal and neonatal alloimmune thrombocytopenia: Possible underlying mechanism

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