ALBERT

Description: Cell cycle kinetics of malignant lymphoma were investigated using in vivo labeling with iododeoxyuridine (IdUrd) and subsequent flow cytometry (FCM) of IdUrd/DNA and Ki-67/DNA. This approach provides an extensive cell kinetic profile from only one single tumor biopsy, including data upon the percentage of S-phase cells, the IdUrd labeling index (LI), Ki-67-derived growth fraction, duration of the S-phase, duration of the G1-phase, potential doubling time, cell production rate, and total cell cycle time. Tissue samples from 33 patients were studied: non-Hodgkin's lymphoma (NHL; n = 22), Hodgkin's disease (HD; n = 7), and reactive hyperplasia (n = 4). In NHL, the percentage of S- phase cells, LI, growth fraction, duration of the S-phase, and cell production rate were significantly correlated with the histologic malignancy grade according to the Working Formulation (P 〈 or = .02). Data found in HD were not essentially different from those in low-grade NHL and reactive hyperplasia. Remarkably, the duration of the S-phase, the duration of the G1-phase, and the total cell cycle time appeared to be rather independent of histologic malignancy grade within the NHL category. A significant correlation was observed between the IdUrd LI and the percentage of S-phase cells, the growth fraction, the potential doubling time, and the cell production rate (P 〈 .001), but not with the duration of the separate cell cycle phases (P 〉 .05). Our data show (1) that it is feasible to obtain detailed information on the in vivo growth characteristics of malignant lymphoma; and (2) that the transition time through the different cell cycle phases widely varies, even within distinct histologic subgroups.

Description: Human myeloid progenitor cells temporarily express HLA class II molecules during the differentiation pathway to granulocytes and macrophages. The significance of major histocompatibility complex (MHC) class II molecules at this stage of development is unknown. As a first stop of inquiry into their function, we have characterized the profile of major self-peptides bound to the HLA-DR molecules expressed by KG-1 cells, a line that shares many of the phenotypic characteristics of colony-forming unit-granulocyte-macrophage progenitors. Searches of protein data bases showed that all matching peptides bound to the HLA- DR molecules of KG-1 cells corresponded to intracellular, rather than exogenous or transmembrane, precursor proteins. Because the absence of a conventional self-peptide repertoire could be related to altered trafficking of class II molecules, the biosynthesis of HLA-DR and the invariant chain proteins was determined. The MHC class II associated invariant chain protein is synthesized normally in KG-1 cells, but processed fragments of invariant chain, class II-associated invariant chain peptides (CLIPs), occupy the antigen-binding groove of KG-1 class II molecules at a much lower frequency compared with that of mature antigen-presenting cells. Low CLIP occupancy of HLA-DR is a characteristic shared by KG-1 cells, normal CD34+ progenitor cells, and HLA-DR+ breast carcinoma cells. The unusual profile of MHC class II bound peptides and the low level of CLIP bound to HLA-DR suggest that the antigen-processing pathway of KG-1 is different from that characterized in professional antigen-presenting cells and that exogenous antigen-processing may be a developmentally acquired characteristic in the myeloid lineage.

Description: The promyelocytic leukemia cell line, NB4, carries the t(15; 17) translocation and undergoes limited maturation in response to differentiation agents. Growth on laminin enhanced the ability of all- trans retinoic acid (ATRA) to promote morphologic maturation of these cells. Although exposure to ATRA in suspension yielded minimal maturation beyond the myelocyte stage, after 72 hours of exposure to ATRA on laminin the cells acquired the histologic appearance of metamyelocytes, band forms, and segmented neutrophils. After 96 hours, some cells acquired a spindle shape and became tightly adherent. Growth on collagen types I, III, IV, or fibronectin did not have this effect, although some cells did adhere to fibronectin. NB4 cells treated with ATRA in suspension or on laminin acquired the equivalent ability to reduce nitroblue tetrazolium or cytochrome C. Despite the improved morphologic maturation on laminin, the cells did not express secondary granule proteins such as lactoferrin or neutrophil collagenase. In addition, growth on laminin abolished cell proliferation in the presence of ionomycin. Growth on laminin and/or with ATRA induced new expression of alpha 6 integrin, a laminin receptor, as assessed by reverse transcription-polymerase chain reaction. Different conditions of growth (laminin or differentiation agent) resulted in specific patterns of expression of the alpha 6A and alpha 6B isoforms. Treatment with ATRA also resulted in the acquisition of high-level surface expression of alpha 6 integrin, as assessed by flow cytometry. Thus, treatment of NB4 promyelocytic leukemia cells with ATRA induced expression of alpha 6 integrin (a laminin receptor alpha-chain) and enabled more advanced maturation when the cells were grown on the extracellular matrix component, laminin, compared with tissue culture plastic.

Description: Hereditary macrothrombocytopenia and prolonged bleeding times are associated with the recessive mouse pigment dilution gene gunmetal (gm). Other platelet abnormalities include a mild storage pool deficiency and abnormal expression of two low-molecular-weight guanosine triphosphate binding proteins. These studies were designed to further elucidate the cause of the macrothrombocytopenia. The life span of gunmetal mouse platelets was not significantly different from normal. However, rates of platelet synthesis, measured by sulfate incorporation, were decreased to 25% of normal values. Bone marrow transplantation of normal marrow cells corrected the thrombocytopenia. Furthermore, direct morphologic analysis of mature mutant marrow megakaryocytes by transmission electron microscopy showed reductions in the normal cytoplasmic demarcation membrane system, areas of abnormal membrane complexes, and an increased incidence of emperipolesis. Mutant platelets were relatively more heterogeneous in size and contained unusual elongated and striated inclusions. Mutant megakaryocyte numbers were increased threefold to fivefold over normal numbers in marrow and spleen. Thus, the efficiency of platelet production from gunmetal megakaryocytes is reduced by an order of magnitude. Mutant marrow had a greater proportion of 32N and a smaller proportion of 8N megakaryocytes. Collectively, the results indicate that the gunmetal gene acts intrinsically in megakaryocytes and that an abnormality in this gene causes significant qualitative and quantitative effects on platelet production.

Description: Stage I and II follicular non-Hodgkin's lymphoma (NHL) is clinically defined as a localized disease. To study the possibility that this disease is in fact disseminated, we used the sensitive polymerase chain reaction (PCR) method using translocation (14;18) as marker. Samples from 21 patients who were clinically diagnosed with stage I or II follicular NHL were analyzed for the presence of t(14;18)-positive cells using PCR. We analyzed (1) the diagnostic lymph node biopsy and (2) the peripheral blood or bone marrow samples from these patients. Translocation (14;18) cells were detected in the diagnostic lymph node biopsies of 12 patients. In 9 of these patients, t(14;18)-positive cells were detected in peripheral blood and/or bone marrow samples at diagnosis and/or after therapy. Thus, in 75% of the follicular NHL patients carrying the t(14;18) as a marker for lymphoma cells, t(14;18)- positive cells were detected in peripheral blood and bone marrow at diagnosis and after therapy. Our results show that t(14;18)-positive cells can be detected in the circulation of patients with stage I and II follicular NHL, indicating that, although diagnosed as localized, the disease is disseminated.

Description: L-leucine methyl ester (Leu-OMe), Leu-Leu-OMe, Phe-OMe, and Glu-(OMe)2 are toxic to mononuclear phagocytes (M phi) and neutrophils. In the present studies, the mechanism of this toxicity was examined. A concentration of NH4Cl known to neutralize lysosomal pH and to block conversion of Leu-OMe to the dipeptide condensation product Leu-Leu-OMe inhibited Leu-OMe- or Glu-(OMe)2- but not Leu-Leu-OMe-mediated M phi toxicity. Leu-OMe-, Glu-(OMe)2-, or Leu-Leu-OMe-mediated killing of M phi was prevented by Gly-Phe-CHN2, a specific inhibitor of the thiol protease, dipeptidyl peptidase I (DPPI). Neither NH4Cl nor Gly-Phe-CHN2 prevented Phe-OMe-mediated M phi toxicity. In contrast, inhibition of M phi serine esterase activity prevented Phe-OMe- but not Leu-OMe- or Glu- (OMe)2-mediated killing of M phi. The myeloid tumor lines U937, HL60, and THP-1 were found to be uniformly enriched in DPPI and susceptible to Leu-Leu-OMe but not Leu-OMe toxicity. Whereas HL60 were resistant to Phe-OMe, THP-1 cells were killed by this agent. Incubation of peripheral blood mononuclear cells with Leu-OMe resulted in loss of natural killer (NK) functions and cytotoxic T lymphocytes (CTL) precursors, a process that requires the DPPI-dependent generation of membranolytic polymerization products. Phe-OMe had no toxic effects on NK cells or CTL precursors. These results indicate that Leu-OMe and Glu- (OMe)2 toxicity for M phi is related to the production of higher molecular weight hydrophobic polymerization products via the sequential action of two nonserine esterase lysosomal enzymes. In contrast, Phe- OMe toxicity for myeloid cells was found to correlate with serine esterase-mediated intracellular trapping of high concentrations of the free amino acid Phe. These distinct enzymatic mechanisms may provide a unique means of targeting agents capable of selectively deleting cells of myeloid lineage.

Description: Rats of the Wistar Furth (WF) strain have hereditary macrothrombocytopenia with decreased platelet alpha-granule proteins. The autosomal recessive pattern of inheritance of the large mean platelet volume (MPV) phenotype and platelet alpha-granule protein deficiencies suggest that a component common to both formation of platelet alpha-granules and subdivision of megakaryocyte cytoplasm into platelets is quantitatively or qualitatively abnormal in WF megakaryocytes and platelets. We examined WF platelets for such an abnormality using electrophoretic and immunologic analyses. Rabbit antiserum prepared against WF rat platelets and absorbed with Wistar rat platelets recognized a major 235-Kd band, and minor bands of WF rat platelets ranging from 200 to 130 Kd, not present in immunoblots of Wistar, Sprague-Dawley, or Long-Evans rat platelets. The minor bands were labeled with affinity-isolated antibody to the 235-Kd band, indicating that all bands contained the same unique antigenic site. The 235-Kd antigen had the same mobility as rat platelet talin identified with a platelet antitalin antibody. Activation of calcium-dependent proteases during Triton X-100 extraction caused conversion of the 235- Kd antigen into a major fragment of 200 Kd and minor fragments ranging to 115 Kd, identical in mobility to fragments of rat platelet talin produced in the same samples. The absorbed anti-WF platelet antiserum also detected a 235-Kd antigen in WF lung, kidney, and small intestine by immunoblotting. Finally, the 235-Kd antigen unique to WF rats was immunoprecipitated from Triton X-100 supernatants of WF platelets with an antitalin monoclonal antibody (MoAb). These data indicate that the unique antigenic site is on WF talin. Examination of talin distribution in Wistar megakaryocytes showed localization beneath the plasma membrane, on the cytosolic face of demarcation membranes, associated with alpha-granule membranes, and diffusely throughout the cytoplasm. Although WF megakaryocytes showed the same general distribution pattern, some differences were apparent. In contrast to membrane systems of the Wistar rat, the large membrane complexes in WF megakaryocytes contained little or no talin. In addition, approximately half of WF megakaryocytes showed an increased peripheral localization of talin, often associated with membrane blebs, with decreased talin in the cytoplasmic interior. The association of the unique talin antigenic determinant and anomalous megakaryocyte talin distribution with abnormal platelet formation in WF rats suggests that talin is abnormal in this rat strain and that talin plays an important role in subdivision of megakaryocyte cytoplasm into platelets.

Description: We have restudied two kindreds that formed the basis of the original report of autosomal recessive chronic granulomatous disease (CGD) associated with leukocyte glutathione peroxidase deficiency. Case 1 from the original study and the surviving brother of the originally reported case 2 both have severe CGD, with no detectable respiratory burst activity in purified intact neutrophils. However, their leukocytes exhibit normal glutathione peroxidase enzyme activity and gene expression. Examination of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase components known to be defective in CGD reveals no detectable cytochrome b558 nor any membrane activity in a cell-free NADPH oxidase assay system. Molecular analysis of the genes encoding cytochrome b558 subunits shows, in case 1, a C--〉T substitution at nucleotide 688 of the gene encoding the gp91-phox subunit of cytochrome b558, resulting in a termination signal in place of Arginine-226. Levels of gp91-phox mRNA are markedly decreased despite normal levels of gene transcription, indicating a post- transcriptional effect of the nonsense mutation on mRNA processing or stability. The X-linked form of CGD developed in this cytogenetically normal female due to the uniform inactivation of the normal X chromosome in her granulocytes, indicated by the expression in her granulocyte mRNA of only one allele of a glucose-6-phosphate dehydrogenase polymorphisms for which she is heterozygous in genomic DNA. Case 2 (of the present study) has distinct mutations in each allele of the p22-phox gene.(ABSTRACT TRUNCATED AT 250 WORDS)