| Description: |
A monoclonal antibody, named CAMAL-1, was raised previously in our laboratory to a common antigen of acute myeloid leukemia (CAMAL), and was shown to be highly specific in its recognition of cells from patients with acute (AML) or chronic (CML) myelogenous leukemia. CAMAL was also reported to be prognostic of disease, in that patients whose numbers of CAMAL-1 reactive cells were high, or rose over time, had poorer prognoses and shorter survival times than patients whose CAMAL values were low or decreased. This correlation between CAMAL and disease prognosis led to the discovery that CAMAL-1immunoaffinity-purified leukemic cellular lysates contained a selective growth inhibitory activity for normal myeloid progenitor cells, since the growth of CML progenitors was not inhibited. The work described in this thesis focused primarily on the purification and characterization of the myelopoietic activity present in the CAMAL preparations, and its relationship to the leukemic marker (CAMAL). Initial purifications involved CAMAL-1immunoaffinity chromatography of leukemic cellular lysates, followed by FPLC molecular size fractionation and/or preparative SDS-PAGE. The myelopoietic activity was located within a30-35 kDa molecular weight fraction (P30), and the P30 fraction was consistently found to be selective in its inhibition of normal myeloid progenitors, since the growth of CML progenitors was not inhibited but was, in fact, stimulated. Antibodies were raised to P30 and used in the subsequent purification and characterization of the myelopoietic activity. Amino acid sequence analysis of the N-terminus and P30 tryptic peptides strongly suggested that P30 belonged to the serine protease family of enzymes, and the results obtained from protease assays indicated thatP30 preparations did possess enzyme activity. Prior to the completion of P30 molecular cloning experiments, however, the cDNA sequence for azurocidin/CAP37 was reported, and its predicted amino acid sequence was found to be identical to those obtained from the ... |