Biomedical subjects

M J Fackler

Publications and source records attributed to M J Fackler.

5 recordsLinked to original sources

PubMed1992

Up-regulation of surface CD34 is associated with protein kinase C-mediated hyperphosphorylation of CD34.

CD34 is a transmembrane sialoglycoprotein expressed by early hematopoietic progenitor cells as well as endothelial cells. Previously we found that CD34 is rapidly and stoichiometrically phosphorylated by activated protein kinase C (PKC) (Fackler, M.J., Civin, C.I., Sutherland, D.R., Baker, M.A., and May, W.S. (1990) J. Biol. Chem. 265, 11056-11061). In the present study, we find dose-dependent up-regulation of CD34 surface expression following treatment of normal human CD34+ bone marrow progenitor cells, cord blood-derived KMT-2, or KG1 a myeloid leukemia cells with the PKC activator 12-O-tetradecanoylphorbol-13-acetate. Up-regulation begins within 1 min of treatment, is maximal by 30 min, is maintained for at least 3 h, and is associated with CD34 hyperphosphorylation. A specific inhibitor of PKC, 2,6-diamino-N-(1[1-(1-oxotridecyl)-2-piperadinyl]methyl)h exan-amide (NPC 15437), blocks both up-regulation and hyperphosphorylation of CD34. CD34 up-regulation is independent of transcription and/or translation and results from the recruitment of preformed intracellular CD34. The endocytosis rate of surface CD34 is unaltered by 12-O-tetradecanoylphorbol-13-acetate. Thus, activation of PKC mediates increased surface expression of the CD34 molecule possibly as a result of phosphorylation of CD34.

Antigens, CD↗

PubMed1992

Activated protein kinase C directly phosphorylates the CD34 antigen in acute lymphoblastic leukemia cells.

The precursors of all blood cell lineages are contained within the 1-3% of bone marrow cells which express the CD34 antigen, and this population can reconstitute the hematopoietic system of lethally irradiated animals and humans. A potential regulatory role for the CD34 antigen in progenitor cell function and differentiation was indicated by our recent findings that the CD34 antigen can be phosphorylated in vivo to high stoichiometry in primitive CD34+ cell-lines by activated protein kinase C. To exclude the possibility that these effects were restricted to cell-lines, we have performed similar experiments on fresh cells from a patient with drug-resistant acute lymphoblastic leukemia. Similar to our previous findings, we found the CD34 antigen to be hyperphosphorylated in lymphoblasts labeled in the presence of active phorbols. The same peptides which were hyperphosphorylated in phorbol-stimulated cell-lines were also phosphorylated in phorbol-stimulated lymphoblasts. These data indicate that CD34 is a substrate molecule for PKC in fresh CD34+ lymphoblasts and underline the role of modulators of PKC activity in the biology of primitive leucocytes.

Adult↗

PubMed1990

Activated protein kinase C directly phosphorylates the CD34 antigen on hematopoietic cells.

The CD34 antigen is a human leukocyte membrane protein expressed specifically by lymphohematopoietic progenitor cells. We found that CD34 is a phosphoprotein and therefore examined the regulation of its phosphorylation. Activation of protein kinase C (PKC) enhanced CD34 phosphorylation. The PKC activators, 12-O-tetradecanoylphorbol-13-acetate and bryostatin-1, induced rapid, stoichiometric hyperphosphorylation of CD34 protein in cells, resulting in a 5-fold increase in CD34 phosphorylation. In vitro kinase studies revealed that purified PKC could directly phosphorylate purified CD34. Only serine phosphorylation was detected in the CD34 molecule. Two-dimensional phosphopeptide mapping experiments indicated that PKC induces the phosphorylation of identical serine residue(s) in vitro and in vivo (in KG1 cells). These are newly phosphorylated serine residue(s), which are not detectably phosphorylated in CD34 from exponentially growing KG1 cells. These data indicate that the developmental stage-specific molecule, CD34, is a phosphorylation target for activated PKC. Furthermore, these findings raise the possibility that PKC activation and phosphorylation of the CD34 molecule may play a role in signal transduction during early lymphohematopoiesis.

Antigens, CD↗

PubMed1990

Positive stem cell selection--basic science.

Immunologic strategies for removal of malignant cells from autologous marrow grafts by "negative selection" (i.e., "purging") requiring multiple specific monoclonal antibodies for each tumor type. "Positive selection" of marrow stem cells for grafting is a possible alternative strategy, using a monoclonal antibody which selectively recognizes lymphohematopoietic stem cells. The human hematopoietic progenitor cell antigen, CD34, is an integral cell membrane glycoprotein of approximately 115 kD, which has been molecularly cloned and sequenced. Although its function has not been determined, the glycoprotein has been characterized biochemically, including preliminary epitope mapping. Collective results from several laboratories indicate that CD34 monoclonal antibodies (My10, BI-3C5, 12.8, etc.) have the appropriate specificity to warrant testing their utility in positive selection for autologous bone marrow transplantation. First, precursors for all human hematopoietic lineages assayed (including most CFU-GM, BFU-E, CFU-MEG, CFU-EO, CFU-MIX or CFU-GEMM, pre-CFU, CFUBLAST, and terminal transferase+ B [and probably T] lymphoid precursors) are CD34+. Second, only 1.5% (mean) of low density human marrow mononuclear cells express CD34; mature human blood and marrow cells are CD34-. Endothelial cells are the only fixed tissue cells which express CD34. Third, the expression of CD34 in malignancies appears to parallel normal cellular expression: of hematopoietic malignancies, some acute leukemias and chronic myelogenous leukemia blasts are CD34+, but chronic lymphois leukemias, lymphomas, myelomas and non-hematopoietic malignancies are uniformly CD34-. Fourth, it appears feasible to isolate CD34+ cells from clinical marrow harvest samples in large scale, using either columns or immunomagnetic microspheres. Fifth, recent studies in very small numbers of non-human primates and human patients suggest that isolated CD34+ cells include the true hematopoietic stem cell, since transplantation of CD34+ cells, into myeloblated recipients results in at least short-term hematopoietic engraftment. It is anticipated that transplantation of CD34+ marrow cells may have broad applicability in clinical bone marrow transplantation.

Animals↗

PubMed1990

Lymphohematopoiesis: role of growth factors in leukemogenesis and therapy.

The interrelationship between proliferation, differentiation, and activation responses of hematopoietic progenitor cells and mature blood cells is complex. Therefore, we are only now learning what role colony-stimulating factors play in the regulation of normal hematopoiesis in vivo and in the dysregulation of hematopoiesis in leukemia. Recent advantages in molecular hematology have opened the door to the therapeutic administration of recombinant growth factors. Through continued preclinical trials in animals and by clinical trials in humans, a better understanding of the precise target cells and mechanisms of action of hematopoietic growth factors will improve the therapeutic index of administering colony-stimulating factors. Better understanding of hematopoietic growth factors will, in turn, suggest novel approaches to therapy of acute lymphoblastic leukemia.

Colony-Stimulating Factors↗