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Characterization of the effects of P30-35 CAMAL on normal and leukemic myelopoiesis

University of British Columbia

P30-35 CAMAL is a component of material immunoaffinity enriched from lysates of myeloid leukemia leucocytes using the monoclonal antibody CAMAL- 1. Reactivity with CAMAL- 1 is diagnostic of myeloid leukemias. CAMAL- 1 enriched material inhibited myelopoiesis by normal progenitor cells in vitro. This study demonstrates that inhibition is associated with P30-35 CAMAL. Neutrophilic granulocyte colonies were preferentially inhibited by P30-35 CAMAL while higher concentrations affected all colony types. Nonadherent progenitor cell numbers were reduced in P30-35 CAMAL-treated long term cultures of normal marrow cells whereas adherent cell numbers were increased, suggesting a block in differentiation. In addition, colony formation by murine cells was inhibited by P30-35 CAMAL. As in cultures of human cells, neutrophilic granulocyte colonies were preferentially inhibited, indicating that downregulation of normal myelopoiesis by P30-35 CAMAL crosses species barriers, and might be an important regulatory mechanism. Using highly enriched P30-35 CAMAL, a stimulatory effect on CML colony formation was defined. Stimulation of colony formation occurred at low and high concentrations of P30-35 CAMAL, but was reduced at intermediate concentrations. At low concentrations of P30-35 CAMAL primitive colonies were increased, whereas high concentrations affected all colony types. Colony formation by several myeloid leukemiaderived cell lines was increased by P30-35 CAMAL. P30-35 CAMAL prepared using protease inhibitors lacked effects on colony formation. Alterations in normal and CML colony formation were similar whether cells were preincubated or cocultured with P30-35 CAMAL. Activity was retained in the supematant of treated cells, indicating that effects were immediate and irreversible. Alterations in normal and CML colony formation were fully blocked using either phenyl methyl sulfonyl fluoride or a chloro-methyl ketone-linked peptide, both inhibitors of serine protease activity, suggesting that the alterations in myelopoiesis require serine protease activity. Experiments using characterized neutrophil proteins suggested protease activity in P30-35 CAMAL preparations which might be unique. Reactivity of the monoclonal antibodies a-P30/35, raised against P30-35 CAMAL, and CAMAL- 1 was compared. Both antibodies reacted more extensively with CML than with normal leucocytes, and cc-P30/35 reacted with several myeloid leukemia-derived cell lines, suggesting that the antigen recognized by CAMAL- 1 and the component which alters myelopoiesis might form an association.

Thesis/Dissertation

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2008-12-17

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http://hdl.handle.net/2429/3072

Microbiology and Immunology

University of British Columbia

UBCV

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