Analysis of p53 mutations in a large series of lymphoid hematologic malignancies of childhood

p53 mutations are found in a wide variety of cancers, including hematologic malignancies. These alterations apparently contribute to development of the malignant phenotype. We analyzed a large series of lymphoid (330 cases) and a smaller series of myeloid (29 cases) malignancies of childhood for p53 mutations by single-strand conformational polymorphism (SSCP) following polymerase chain reaction. Samples with abnormal SSCP were reamplified and analyzed by direct sequencing method. p53 mutations were detected within the known mutational hotspots (exons 5 to 8) in 8 of 330 lymphoid malignancies, and in none of 29 myeloid malignancies, showing that the frequency of p53 mutations in childhood lymphoid malignancies was very low (8 of 330 cases [2%]). Four of these patients had very aggressive, fatal acute lymphocytic leukemia (ALL). None of 13 infants and none of 48 patients with T-lineage leukemia had detectable p53 mutations in their ALL cells. Exceptionally, p53 mutations were comparatively frequent in a small sample of B-cell non-Hodgkin's lymphomas (2 of 8 cases). Mutations were detected in samples from two patients with ALL at relapse; these were not detected in samples at initial diagnosis from the same patients, suggesting that p53 mutations may be associated with progression to a more malignant phenotype. Seven of eight alterations of p53 were missense mutations, and seven of eight samples may be heterozygous for the mutant p53, indicating that p53 protein may act in a dominant negative fashion.     

Effects of intermittent pressure loading on the development of ventricular hypertrophy in the cat.

Although the effects of persistent pressure loading on the development of ventricular hypertrophy have been studied extensively, the effects of intermittent pressure loading have not been examined. To study the effects of intermittent pressure loading we subjected the right ventricle of cats to intermittent pulmonary artery constriction over a 2-week period. Two intermittent pressure loading schedules were employed. The first consisted of a right ventricular systolic pressure of 60 mm Hg for 3.5 days and normal right ventricular pressure for 3.5 days; and the second consisted of a right ventricular systolic pressure of 60 mm Hg for 2.3 days and normal right ventricular pressure for 4.7 days. The intermittent pressure-loaded cats were compared with normal unoperated controls, sham-operated controls, and cats with persistent right ventricular pressure load for either 1-week or 1- to 2-month duration. The data indicate that intermittent pressure loading caused significant right ventricular hypertrophy. Since significant residual ventricular hypertrophy was present in both intermittent pressure loading groups, regression of ventricular hypertrophy involves a slower process than the progression of hypertrophy.     

Effects of angiographic contrast media on sino-atrial nodal function.

Injection of meglumine diatrizoate (Renografin-76) into the selectively perfused sinus node artery of the dog produces bradycardia which is unaltered by autonomic blockade or by changes in sinus node artery pressure. Contrast agents and other hyperosmolar substances prolong the R-R interval in proportion to their osmolarity. Selective injection of contrast media into other cannulated segments of the coronary tree produces no change in heart rate. Transfemoral arteriography, however, produces bradycardia with both right and left coronary injections. Both direct and reflex sinus node depression occur with coronary arteriography in the dog. Direct effects are mediated by hyperosmolarity.     

Cytogenetic studies in patients with secondary leukemia/dysmyelopoietic syndrome after different treatment modalities

Cytogenetic studies of 68 patients who developed secondary leukemia (SL)/dysmyelopoietic syndrome (DMS) after extensive chemotherapy and/or radiation therapy as well as patients who developed SL/DMS without such treatment showed that those patients who received radiation alone or with chemotherapy had more extensive numerical and structural abnormalities than those who received only chemotherapy. In terms of the specific chromosomal abnormalities, there are no differences between the various treatment groups. Hypodiploidy is the most common form of aneuploidy in these patients, with the most common numerical abnormality being the loss of chromosome 7. The most common structural abnormalities involved chromosomes 3 and 5. When compared with patients with de novo leukemia and DMS, the chromosomal abnormalities in these patients are more complex and extensive. Serial studies revealed that cytogenetic abnormalities do not precede the development of hematologic changes by significant time periods.     

A granulocyte reactive monoclonal antibody, 1G10, identifies the Gal beta 1-4 (Fuc alpha 1-3)GlcNAc (X determinant) expressed in HL-60 cells on both glycolipid and glycoprotein molecules

1G10, a monoclonal IgM antibody that identifies a differentiation antigen on human granulocytes and a subpopulation of monocytes, was found to react specifically with glycosphingolipids bearing the Gal beta 1-4(Fuc alpha 1-3)GlcNAc hapten (X determinant). This carbohydrate determinant was found on both glycolipid and glycoprotein molecules isolated from HL-60 cells (a promyelocytic leukemia cell line). Thus, this highly conserved carbohydrate-defined determinant previously described on mouse embryonic and mouse and human carcinoma cells is also expressed as a tissue-specific differentiation antigen on normal human granulocytes.     

Selection of an HEL-derived cell line expressing high levels of platelet factor 4

A platelet factor 4 (PF4)-expressing cell line, HELNeo, was derived from the human erythroleukemia cell line, HEL. This was achieved by stable transfection of HEL cells with a construct containing the rat PF4 promoter driving the gene coding for resistance to neomycin, followed by selection of neomycin-resistant clones. HELNeo cells were all nonadhering and about 5% of the cells had polyploid nuclei (> or = 8N), as compared with 1% in HEL cells. Immunohistochemistry showed that about 90% of the HELNeo cells contained PF4, whereas only approximately 5% of the HEL cells contained PF4. No significant parallel enrichment was observed for other megakaryocytic markers, such as the glycoprotein complex IIb/IIIa, von Willebrand factor, and platelet activation- dependent granule to external membrane glycoprotein (PADGEM), which were present to a similar extent in both HEL and HELNeo lines. The increased expression of PF4 in HELNeo cells was confirmed by transient expression assays and was associated with a fivefold increase in trans- acting factors binding to the PF4 promoter. These cells should be a rich source for purifying trans-acting factors binding to the PF4 gene. Moreover, our study shows how a lineage-specific promoter may be used to generate lineage-specific cell lines from a multilineage hematopoietic cell line.     

Human platelet fibrinogen: purification and hemostatic properties

Conditions were developed in which 80% to 90% of platelet fibrinogen could be routinely purified in nondegraded form from the fluid phase of platelet suspensions stimulated with the calcium ionophore, A23187, in the presence of calcium, leupeptin, and prostaglandin E1. Fibrinogen was separated from other released proteins by chromatography on diethylaminoethanol (DEAE)-cellulose using a continuous pH and ionic strength gradient. Purified platelet fibrinogen, greater than 98% homogeneous by immunoelectrophoresis and sodium-dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), consisted of intact A alpha, B beta and gamma A chains, but not gamma' chains, and was 95% to 96% clottable. Platelet fibrinogen was shown to compete for the binding of radiolabeled plasma fibrinogen to ADP-activated platelets in a manner identical to that of unlabeled plasma fibrinogen itself. Also, at equivalent protein concentrations, platelet and plasma fibrinogens supported platelet aggregation to an equivalent extent. Based upon these results, we conclude that there is no significant difference between platelet and plasma fibrinogen with respect to their size, their clottability, their affinity for the activated platelet fibrinogen receptor, or their capacity to support subsequent platelet aggregation.     

Monocyte antigen CD14 is a phospholipid anchored membrane protein

A cDNA clone encoding the human monocyte antigen CD14 was isolated by transient expression in COS cells of a cDNA library prepared from phorbol diester-treated HL60 cells. RNA blot analysis showed abundant expression of a single mRNA species in mature monocytes and an increased expression of the mRNA following induction of differentiation in leukemic cell lines. The DNA blot hybridization pattern was consistent with a single-copy gene. The predicted amino acid sequence lacks the characteristic transmembrane domain and stop transfer motif of conventionally anchored membrane proteins. COS cells transfected with the CD14 cDNA released virtually all CD14 protein in soluble form following treatment with glycosyl phosphatidylinositol-specific phospholipase C, and CD14 immunoreactivity was absent from the affected monocytes of a patient with paroxysmal nocturnal hemoglobinuria (PNH). The data show that, to the limit of experimental sensitivity, all monocyte CD14 is joined to the plasma membrane by a phosphatidylinositol phospholipid.