William S. Kloetzer

The human cellular abl gene product in the chronic myelogenous leukemia cell line K562 has an associated tyrosine protein kinase activity

Three antisera against the mouse v-abl gene product were used to identify two potential human c-abl gene products in the chronic myelogenous leukemia cell line K562. Two antipeptide sera were generated in rabbits using the predicted amino acid sequence of the mouse v-abl gene product. One antiserum was made against a polypeptide overlapping the in vivo tyrosine phosphorylation site of murine P120gag-abl and what is believed to be a homologous tyrosine phosphorylation site of the predicted normal human c-abl gene product (v-abl 263-280). The second antipeptide serum, abl 389-403, was generated against a predicted hydrophilic peptide of the v-abl gene product. Immunoprecipitation from K562 cells metabolically labeled with [32P]orthophosphate by a mouse tumor regressor and abl 389-403 antipeptide sera detected two proteins of 190,000 and 240,000 Da. Both proteins were labeled primarily at serine and, to a much lesser extent, at tyrosine residues. Immune complex kinase assays using conditions that allow the tyrosine phosphorylation of P120gag-abl showed that in vitro phosphorylation of P190 and P240 occurs primarily at tyrosine residues. The detection of these enzymatically active human c-abl gene products is a rare observation which may be in part attributed to the c-abl gene translocation from chromosomes 9 to 22 occurring in the vast majority of chronic myelogenous leukemia patients.

Rearrangement in the Breakpoint Cluster Region and the Clinical Course in Philadelphia-Negative Chronic Myelogenous Leukemia

We have followed one patient with Philadelphia (Ph)-negative chronic myelogenous leukemia and identified an additional four patients from the literature who showed the rearrangement in the breakpoint cluster region (bcr) on chromosome 22 characteristic of Ph-positive chronic myelogenous leukemia. The clinical course of these five patients was similar to that of Ph-positive patients, with easily controlled leukocyte counts, a prolonged benign phase, and prolonged survival. Furthermore, we have shown, for the first time, that bcr rearrangement in Ph-negative chronic myelogenous leukemia can result in expression of the aberrant 210-kilodalton bcr-abl fusion protein, which has been strongly implicated in Ph-positive leukemogenesis. Research data pertaining to possible cytogenetic mechanisms leading to production of p210bcr-abl in the absence of the Ph chromosome are reviewed. Molecular analysis provides an important tool for classifying and predicting prognosis of some patients with Ph-negative chronic myelogenous leukemia.

In vitro IgE inhibition in B cells by anti-CD23 monoclonal antibodies is functionally dependent on the immunoglobulin Fc domain.

CD23, the low affinity receptor for IgE (FcvarepsilonRII), is involved in regulation of IgE synthesis by B-lymphocytes. Five monoclonal antibodies to human CD23 were generated from cynomolgus macaques immunized with purified soluble CD23 (sCD23). Four of the five primate antibodies blocked the binding of IgE complexes to CD23 positive cells and also inhibited the production of IgE in vitro by IL-4 induced human peripheral blood mononuclear cells (PBMC). The variable domains of several primate antibodies were utilized to construct chimeric macaque/human (PRIMATIZED((R))) monoclonal antibodies. PRIMATIZED((R)) p5E8G1, containing human gamma 1 constant region, inhibited IgE production in vitro as efficiently as the parent primate antibody, but the human gamma 4 constant version, PRIMATIZED((R)) p5E8G4, was not as effective in IgE inhibition. An F(ab)(2) of p5E8G1 did not inhibit IgE production but did interfere with IgE inhibition by the intact anti-CD23 antibody in a dose dependent fashion. The murine monoclonal antibody MHM6 recognizes human CD23 at a different epitope than primate antibody 5E8, and inhibits IgE production by IL-4 induced PBMC. As with the F(ab)(2) of p5E8G1, the F(ab)(2) of MHM6 also failed to inhibit IgE production. These data imply that the mechanism by which anti-CD23 antibodies inhibit IgE production requires cross-linking of CD23 to an IgG receptor. These data also imply that neither bivalent cross-linking of CD23 alone or inhibition of CD23 binding to its natural ligands is sufficient to inhibit IgE production.

Anti-CD23 monoclonal antibody inhibits germline Cε transcription in B cells

Abstract A chimeric macaque/human (PRIMATIZED®) anti-CD23 antibody, p6G5G1, demonstrated a strong inhibitory effect on IL-4- and anti-CD40 antibody-stimulated IgE production by human peripheral blood mononuclear cells (PBMCs). RNA analysis by both reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot showed that p6G5G1 inhibited germline Ce RNA synthesis, but had no effect on CD23 mRNA levels. These data suggest that p6G5G1 may inhibit immunoglobulin class switching to IgE through the inhibition of germline Ce RNA synthesis. Early addition of p6G5G1 after stimulation by IL-4 and anti-CD40 was critical for IgE inhibition. In contrast, later addition of p6G5G1 still showed inhibition of increased levels of surface CD23, which is normally upregulated by stimulation with IL-4 and anti-CD40.

Binding of retrovirus-associated protein kinase and proteins to Staphylococcus aureus.

Formalin-fixed Staphylococcus aureus strain Cowan, bearing protein A, routinely used for the absorption of antigen-antibody complexes, was found to bind protein kinase activity from disrupted Moloney murine leukaemia virus (Mo-MuLV). The Wood strain of S. aureus lacking protein A also bound the kinase with similar efficiency. About 50% of the bound kinase activity, as detected by phosphorylation of casein using [gamma-32P]ATP, could be eluted from the bacterial preparation with buffer containing 0 X 5 M-KC1. Similar results were obtained with Moloney murine sarcoma virus (Mo-MuSV) strain 349 and ts110 MuSV(MuLV). The bacterial preparation was also found to bind casein kinase activity from cellular extracts of uninfected, Rauscher murine leukaemia virus (R-MuLV)-infected and Mo-MuLV-infected cells. Analysis of [3H]leucine-labelled proteins from purified virus showed selective binding to S. aureus of only two major labelled virus proteins. One virus component bound to S. aureus had the relative mobility of p15; the other polypeptide co-migrated with virus p10. Upon exposure to increased salt concentration, most of the p10 but very little of the p15 proteins were released. The S. aureus-binding proteins from ts110 Mo-MuSV and MuSV-349 revealed similar binding and elution patterns of p10 and p15 molecules. The p10 and protein kinase activity eluted from Mo-MuLV-absorbed bacteria were separated by gel filtration into a high molecular weight species, containing p10 and kinase activity, and a low molecular weight p10 monomer lacking enzymic activity.