Allele-dependent transcriptional regulation of the human integrin α2 gene

Genetically controlled variation in α2β1 expression by human blood platelets was previously described. Sixty-two haplotype sequences corresponding to the proximal 5′ regulatory region (−1096 to +1) of the α2 gene were compared, and a dimorphic sequence −52C>T was identified that is located precisely between 2 tandem Sp1/Sp3 binding elements previously shown to be absolutely required for transcriptional activity of this gene in epithelial cell lines and the erythroleukemic cell line K562. The gene frequency of −52T in a random Caucasian population is approximately 0.35, and the expression of −52T correlates directly with reduced densities of platelet α2β1. In mobility shift analyses, the −52T substitution attenuates complex formation with both Sp1 and Sp3. When transfected into the erythroleukemia cell line Dami, promoter-luciferase constructs bearing the −52T sequence exhibit a 5-fold decrease in activity relative to the −52C construct. In transfected CHRF-288-11 megakaryocytic cells, the corresponding activity decreases by 10-fold. The −52T sequence appears to be in linkage disequilibrium with the previously defined allele A3 (807C; HPA-5b), known to be associated with diminished expression of platelet α2β1. In summary, a natural dimorphism has been identified within the proximal 5′ regulatory region of the human integrin α2 gene that is responsible for decreased expression levels of the integrin α2β1 on blood platelets through a mechanism that is probably mediated by the nuclear regulatory proteins Sp1 and Sp3.

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Establishment and Characterization of a New Human Ph+ Erythroleukemic Cell Line, VES.

Blood ◽

10.1182/blood.v106.11.4558.4558 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4558-4558

Author(s):

Mirna Golemovic ◽

Lana Rnjak ◽

Klara Dubravcic ◽

Sanja Davidovic-Mrsic ◽

Ivana Franic ◽

...

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Multiparameter Flow Cytometry ◽

Fish Analysis ◽

Suitable Model ◽

Short Period ◽

Erythroleukemia Cell ◽

Erythroleukemic Cell ◽

Erythroleukemic Cell Line

Abstract Continuous human malignant hematopoietic cell lines are invaluable tool for hematological research. Here we report on a new erythroleukemic cell line termed VES that was established from the bone marrow mononuclear cells (BMNC) of a 22-year-old woman with Ph+ CML during her second post-transplant period. Actually, after unsuccessful allo-transplantation, the patient received auto-transplant that also failed to engraft. At the time of initiation of BMNC into culture for the analysis of stromal cell capacity, her bone marrow was tested Ph-negative by both FISH and PCR. Instead of establishment of an adherent stromal layer in vitro, blast-like cells appeared in the supernatant on day 27. These blast cells expressed highly amplified bcr/abl locus as shown by FISH, whereas the patient’s bone marrow was still Ph-negative at the time. Following a very short period, the cultured cells started to express stable features of erythroleukemia cell line. Optimal growth was obtained by suspending the cells at concentration of 0.3x106 cells/ml in IMDM containing 10% FBS, 1% penicillin-streptomycin and 1% L-glutamine solution. After 3 days of culture, cell concentration varied between 1.3 and 1.8x106 cells/ml. VES cells were tested negative for the presence of EBV virus. For analysis of clonogenicity, VES cells at concentration of 103/ml were grown in a serum-free methylcellulose medium without cytokines (H4236; StemCell Tech., Canada). Following 7-day culture, 103 VES cells produced 251 ±42 and after 14 days 431 ±30 GF-independent colonies. Furthermore, on day 14 of the cell culture it was possible to observe reddish color of the colonies indicating the presence of hemoglobin. Cytological examination of cytospin preparations indicated homogenous population of leukemia blasts (>80%) the majority of them (>90%) being glycophorinA positive and MPO negative. Imunophenotyping and multiparameter flow cytometry revealed proerythroblastic lineage-associated profile: GlyA/CD235a+, CD11b+, CD15+, CD29+, CD33+, and CD117+. Conventional cytogenetics revealed complex karyotype with multiple numerical/structural abnormalities (MAKA), while metaphase FISH revealed the following aberrations: t(9;22), 5q31, 7q31, +8, +6. Since FISH analysis detected highly amplified bcr/abl locus, we tested VES cells’ sensitivity to imatinib (Gleevec). Treatment with imatinib for 3 days (MTS assay) inhibited proliferation of VES cells with IC50 value of 0.2mM. Following 14 days of culture in methylcellulose medium with addition of 0.2mM imatinib, the clonogenic potential of VES cells was reduced by 55% in relation to untreated control. We demonstrated that imatinib induced apoptosis in VES cells in time- and dose-dependant manner, assessed with AnnexinV and PI staining, while there were no significant changes observed in the cell cycle except for a mild increase in cells in G0/G1 phase. Although further analyses are required, we believe that VES cells represent a new Ph+ erythroleukemia cell line and a suitable model for studying Ph+ malignant hematopoiesis in vitro.

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On the Mechanism by which Cell Contact Induces the Release Reaction of Blood Platelets; the Effect of Cationic Polymers

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649347 ◽

1972 ◽

Vol 27 (01) ◽

pp. 121-133 ◽

Cited By ~ 14

Author(s):

P Massini ◽

E. F Lüscher

Keyword(s):

Human Blood ◽

Calcium Ions ◽

Blood Platelets ◽

Cell Contact ◽

Second Phase ◽

Cationic Polymers ◽

Release Reaction ◽

Per Se ◽

Human Blood Platelets

SummaryHuman blood platelets are aggregated by the basic polymers polylysine and DEAE- dextran. Under certain conditions a second phase of aggregation, concomitant with the release reaction, is elicited. The presence of ADP, calcium ions and a plasmatic cofactor within the primary aggregates are necessary for the induction of the release reaction. These experiments demonstrate that cell contact per se does not lead to a release reaction ; in order to become effective it must take place in the presence of ADP.

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Chromatographic Isolation of the LDH-Isoenzymes of Human Blood Platelets and an Investigation of Their Enzyme Kinetics

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651273 ◽

1968 ◽

Vol 20 (03/04) ◽

pp. 301-313 ◽

Cited By ~ 2

Author(s):

W Schneider ◽

K Schumacher ◽

B Thiede ◽

R Gross

Keyword(s):

Human Blood ◽

Blood Platelets ◽

Deae Cellulose ◽

Order Reaction ◽

Kinetic Properties ◽

Ldh Isoenzymes ◽

Kinetic Investigations ◽

Substrate Concentrations ◽

Chromatographic Isolation ◽

Human Blood Platelets

SummaryThe LDH-isoenzymes of human blood platelets show a distinct predominance of the isoenzymes 2 and 3 upon chromatography on DEAE-cellulose. Small amounts of LDH-1 are also present, while only traces of LDH-4 and -5 can be detected.Enzyme kinetic investigations of the principal isoenzymes LDH-1, -2 and -3 clearly show that the differences in inhibition constants with pyruvate as substrate which are demonstrable at 25° largely disappear at 37°. On the other hand, the differences among the isoenzymes in their affinity for pyruvate and lactate as substrate as well as in with respect to the optimal substrate concentrations of pyruvate are more marked at 37° than at 25°. Also, the type of inhibition found with lactate as substrate is increasingly the expression of a higher order reaction in going from LDH-1 to LDH-3. A dependence of the LDH distribution pattern upon the metabolism of the cell is discussed. A comparison of our results with thrombocytes with those of other workers with erythrocytes and leucocytes makes it unlikely that the LDH pattern is directly dependent upon the existence of an oxidative metabolism. Rather, the redox potential of the cell could be of importance for the nature of the pattern of isoenzymes and for their differing kinetic properties.

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Interaction of Vasopressin with Human Blood Platelets: Dependency on Mg2+

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661662 ◽

1986 ◽

Vol 56 (03) ◽

pp. 260-262 ◽

Cited By ~ 6

Author(s):

Isabella Roos ◽

Fabrizia Ferracin ◽

Alfred Pletscher

Keyword(s):

Phosphatidic Acid ◽

Human Blood ◽

Arginine Vasopressin ◽

Specific Binding ◽

Blood Platelets ◽

Bivalent Cations ◽

Platelet Membranes ◽

Maximal Rise ◽

Human Blood Platelets ◽

Stimulation Of

SummaryArginine-vasopressin (AVP) in the presence of Mg2+ but not in the absence of bivalent cations led to accumulation of [32P]-phosphatidic acid ([32P]-PA) in human blood platelets. Mg2+ also enhanced the specific binding of [3H]-AVP to intact platelets. The concentrations of the cation which enabled AVP to cause half maximal rise of [32P]-PA and those inducing half maximal [3H]-AVP-binding were of the same order. It is concluded that the stimulation of phosphatidyl inositide breakdown by AVP in presence of Mg2+ is at least partially due to a Mg2+-induced enhancement of specific AVP-binding to the platelet membranes.

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