Identification of CD20 Mutations in Malignant Lymphoma: Can They Be Predictors of Response to Rituximab?.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 288-288 ◽  
Author(s):  
Yasuhito Terui ◽  
Takuma Sakurai ◽  
Yuji Mishima ◽  
Yuko Mishima ◽  
Natsuhiko Sugimura ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Malignant Lymphoma ◽  
Laser Scanning ◽  
Transmembrane Domain ◽  
Point Mutations ◽  
K562 Cells ◽  
Cytoplasmic Domain ◽  
Isolated Cells ◽  
Mammalian Expression ◽  
Cd20 Expression

Abstract Purpose: Although rituximab is commonly used as induction and maintenance therapy for CD20+ malignant lymphoma, some patients become refractory to treatment and the mechanism of resistance is unclear. The aim of this study was to investigate the relationship between CD20 mutations and rituximab resistance. Methods: To investigate whether CD20 mutations affect the response to rituximab, fresh CD19+ lymphoma cells were isolated from the lymph nodes, peripheral blood or bone marrow of 48 patients with NHL using magnetic activated cell sorting (MACS). CD19+/CD20+ cells were subsequently sorted by flow cytometry. RNA was prepared from the isolated cells and RT-PCR was performed. The resulting PCR products were sequenced, subcloned into the mammalian expression vector pTARGET, transfected into K562 cells and CD20 expression was examined by flow cytometry and laser scanning confocal microscopy. Results: In all 48 patients, overall response rate (CR+CRu+PR) to rituximab was 93.8% (45/48), but two cases became PD after PR. DNA sequence analysis revealed that point mutations were mostly observed in two CD20 domains - the third transmembrane domain and the C-terminal cytoplasmic domain. One patient had point mutations in the transmembrane domain, three cases showed point mutations in the C-terminal cytoplasmic domain and six cases had non-specific CD20 mutations, which did not affect CD20 expression. Thirty-eight patients showed no mutations of CD20 gene. CD20 expression was very weak in patients with point mutations in the C-terminal cytoplasmic domain, whereas expression was increased in patients with point mutations in the transmembrane domain. Conclusions: This study suggests that point mutations in CD20 may cause rituximab resistance and identification of CD20 mutations upon diagnosis may help to predict a patient’s response to rituximab.

Point mutation of C-terminal region of CD20 molecule predicts rituximab-induced complement-dependent cytotoxicity and clinical response to rituximab in non-Hodgkin’s lymphoma

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 7563-7563 ◽  
Author(s):  
Y. Terui ◽  
Y. Mishima ◽  
Y. Mishima ◽  
M. Yokoyama ◽  
K. Hatake ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Laser Scanning ◽  
Transmembrane Domain ◽  
Point Mutations ◽  
K562 Cells ◽  
Cytoplasmic Domain ◽  
Laser Scanning Confocal Microscopy ◽  
Cytoplasmic Domains ◽  
Isolated Cells ◽  
Cd20 Expression

7563 Background: Although rituximab is commonly used as induction and maintenance therapy for malignant lymphoma, some patients become refractory to treatment and the mechanism of resistance is unclear. The aim of this study was to investigate the relationship between CD20 mutations and rituximab resistance. Methods: To investigate whether CD20 mutations affect response to rituximab, fresh CD19+ lymphoma cells were isolated from the lymph nodes, or bone marrow of 68 patients with NHL. The cells were subsequently sorted by flow cytometry. RNA was prepared from the isolated cells and RT-PCR was performed. The PCR products were sequenced, subcloned into an expression vector pTARGET, transfected into K562 cells. CD20 expression was examined by flow cytometry and laser scanning confocal microscopy. Results: In all 68 patients, overall response rate (CR+CRu+PR) to rituximab was 91.2% (62/68), but t four cases became PD after PR. DNA sequence analysis revealed that point mutations were mostly observed in three CD20 domains - extracellular/cytoplasmic domains, the third transmembrane domain and the C-terminal cytoplasmic domain. Two cases had point mutations in extracellular/cytoplasmic domains, one patient had point mutations in the transmembrane domain, four cases showed point mutations in the C-terminal cytoplasmic domain and six cases had non-specific CD20 mutations, which did not affect CD20 expression. 56 patients showed no mutations of CD20 gene. CD20 expression was very weak in patients with point mutations in the C-terminal cytoplasmic domain, whereas expression was increased in patients with point mutations in the transmembrane domain. Conclusions: Point mutations in CD20 may cause rituximab resistance and identification of CD20 mutations upon diagnosis may help to predict a patient’s response to rituximab. No significant financial relationships to disclose.

scholarly journals The two phenylalanines in the GFFKR motif of the integrin α6A subunit are essential for heterodimerization

1997 ◽  
Vol 328 (2) ◽  
pp. 529-537 ◽  
Author(s):  
A. Annemieke DE MELKER ◽  
Duco KRAMER ◽  
Ingrid KUIKMAN ◽  
Arnoud SONNENBERG
Keyword(s):  
Point Mutations ◽  
K562 Cells ◽  
Cytoplasmic Domain ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Α Subunit ◽  
Focal Contacts ◽  
Arginine Residues ◽  
A Site ◽  
Laminin 1

The membrane-proximal domain of the integrin α subunit contains a conserved motif of five amino acid residues, GFFKR. We deleted this motif from the human α6A subunit and found that in COS-7 cells this mutant cannot associate with the β1 subunit and is retained in the endoplasmic reticulum. Point mutations in the GFFKR motif of the glycine residue or the two highly charged amino acids, or deletion of the lysine and arginine residues, had no effect on the ability of α6 to interact with β1 and to be expressed at the cell surface. In contrast, by replacing either of the two phenylalanines with alanine, or by deletion of both of these residues, α6 was incapable of associating with β1. The α6 point mutants that associated with β1 were expressed in K562 cells and their responsiveness to integrin-activating factors was determined. None of these transfectants bound spontaneously to laminin-1, but binding could be induced by either PMA or the stimulating anti-β1 antibody TS2/16 to the same extent as that of the wild-type transfectant. The ability of these mutants to initiate focal-contact formation in CHO cells plated on laminin-1 substrates also appeared to be unaltered. Thus the behaviour of α6 mutants involving the glycine, lysine or arginine residues was indistinguishable from that of wild-type α6 both in inside-out and outside-in signalling. In contrast, deletion of the cytoplasmic domain of α6 C-terminal of the GFFKR motif resulted in a loss of responsiveness of α6β1 to PMA stimulation and formation of focal contacts on laminin-1. However, this mutant was targeted to focal contacts formed by other integrins, even when they had not bound ligand. Together, these results suggest that the two phenylalanine residues of the GFFKR motif provide a site for interaction of the α6A subunit with β1, whereas the cytoplasmic domain C-terminal of this motif is involved in the regulation of bidirectional signalling via α6Aβ1.

scholarly journals Syndecan Transmembrane Domain Specifically Regulates Downstream Signaling Events of the Transmembrane Receptor Cytoplasmic Domain

2021 ◽  
Vol 22 (15) ◽  
pp. 7918
Author(s):  
Jisun Hwang ◽  
Bohee Jang ◽  
Ayoung Kim ◽  
Yejin Lee ◽  
Joonha Lee ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Growth Factor Receptor ◽  
Cytoplasmic Domain ◽  
Nih3t3 Cells ◽  
C Elegans ◽  
Downstream Signaling ◽  
Downstream Effectors ◽  
Signaling Events

Despite the known importance of the transmembrane domain (TMD) of syndecan receptors in cell adhesion and signaling, the molecular basis for syndecan TMD function remains unknown. Using in vivo invertebrate models, we found that mammalian syndecan-2 rescued both the guidance defects in C. elegans hermaphrodite-specific neurons and the impaired development of the midline axons of Drosophila caused by the loss of endogenous syndecan. These compensatory effects, however, were reduced significantly when syndecan-2 dimerization-defective TMD mutants were introduced. To further investigate the role of the TMD, we generated a chimera, 2eTPC, comprising the TMD of syndecan-2 linked to the cytoplasmic domain of platelet-derived growth factor receptor (PDGFR). This chimera exhibited SDS-resistant dimer formation that was lost in the corresponding dimerization-defective syndecan-2 TMD mutant, 2eT(GL)PC. Moreover, 2eTPC specifically enhanced Tyr 579 and Tyr 857 phosphorylation in the PDGFR cytoplasmic domain, while the TMD mutant failed to support such phosphorylation. Finally, 2eTPC, but not 2eT(GL)PC, induced phosphorylation of Src and PI3 kinase (known downstream effectors of Tyr 579 phosphorylation) and promoted Src-mediated migration of NIH3T3 cells. Taken together, these data suggest that the TMD of a syndecan-2 specifically regulates receptor cytoplasmic domain function and subsequent downstream signaling events controlling cell behavior.

scholarly journals Kinetics of in vitro natural killer activity against K562 cells as detected by flow cytometry

Cytometry ◽  
1998 ◽  
Vol 32 (4) ◽  
pp. 280-285 ◽  
Author(s):  
Loris Zamai ◽  
Adriana R. Mariani ◽  
Giorgio Zauli ◽  
Luigi Rodella ◽  
Rita Rezzani ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Natural Killer ◽  
Natural Killer Activity ◽  
K562 Cells ◽  
Killer Activity ◽  
Kinetics Of

scholarly journals Gating Motions of KirBac1.1 Cytoplasmic Domain with Respect to Transmembrane Domain Revealed by FRET

2013 ◽  
Vol 104 (2) ◽  
pp. 129a
Author(s):  
Shizhen Wang ◽  
Sarah Heyman ◽  
Decha Enkvetchakul ◽  
Colin G. Nichols
Keyword(s):  
Transmembrane Domain ◽  
Cytoplasmic Domain

Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane

1999 ◽  
Vol 112 (11) ◽  
pp. 1721-1732 ◽  
Author(s):  
M.J. Francis ◽  
E.E. Jones ◽  
E.R. Levy ◽  
R.L. Martin ◽  
S. Ponnambalam ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Transmembrane Domain ◽  
Menkes Disease ◽  
Cytoplasmic Domain ◽  
Endocytic Pathway ◽  
Site Directed Mutagenesis ◽  
Trans Golgi Network ◽  
C Terminus ◽  
Golgi Network

The protein encoded by the Menkes disease gene (MNK) is localised to the Golgi apparatus and cycles between the trans-Golgi network and the plasma membrane in cultured cells on addition and removal of copper to the growth medium. This suggests that MNK protein contains active signals that are involved in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membrane, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisation motifs, using site-directed mutagenesis, protein expression, chemical treatment and immunofluorescence. We observed that a di-leucine motif (L1487L1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane. We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endocytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basis with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus.

Analysis of ploidy level of Artemisia annua L. based on flow cytometry and confocal laser scanning microscopy

2021 ◽  
Vol 50 (1) ◽  
pp. 29-35
Author(s):  
Shu-Gen Wei ◽  
Ling-Yun Wan ◽  
Ying Wei ◽  
Li-Li He ◽  
Jin-E Fu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Artemisia Annua ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Ploidy Levels ◽  
Artemisia Annua L

Eighty nine Artemisia samples treated with different concentrations of colchicine were used as breeding samples, with diploid Artemisia as the control. The ploidy levels of samples were determined by flow cytometry and confocal laser scanning microscopy (CLSM). An analysis of the flow cytometry results identified three suspected tetraploid plants and seven suspected triploid plants. The results of this study may be useful for breeding new Artemisia lines.

Mitomycin Induced Apoptosis in Human Leukemia K562 Cells

2012 ◽  
Vol 599 ◽  
pp. 71-75
Author(s):  
Shu Li Shao ◽  
Bin Zhao ◽  
Wei Wei Zhang ◽  
Wei Zhao ◽  
Guang Hui Wu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Gel Electrophoresis ◽  
Light Microscope ◽  
Previous Experiment ◽  
K562 Cells ◽  
S Phase ◽  
Fluorescence Microscope ◽  
Human Leukemia ◽  
Induced Apoptosis ◽  
Human Leukemia K562 Cells

Objective: The research aimed to study the effects of mitomycin on human leukemic K562 cells, and to explore the mechanism of mitomycin induced apoptosis．In order to provide previous experiment basis for mitomycin applying clinical treatments Methods: The multiplication and apoptosis status of K562 cells treated different time by different concentration mitomycin were observed by light microscope, fluorescence microscope, TEM, agrose gel electrophoresis of DNA and flow cytometry. Results: The results showed that mitomycin could induce K562 cells apoptosis, and the best concentration was 12.5μg/ml for 48 h. The optimal concentration of apoptosis induced by apoptosis rate is （28.8±1.04）% （P<0.01）. Mitomycin could affect the S phase among cellular multiplication, cell could be blocked by mitomycin and then apoptosis in this phase. Conclusions: Mitomycin can induce the apoptosis of human leukemic K562 cells. It is of great significance to guide clinical medication.

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