Structural and partial amino acid sequence analysis of the human hemopoietic progenitor cell antigen CD34.

Monoclonal antibodies of the CD34 class all recognize a monomeric cell surface antigen of approximately Mr 110,000 which is selectively expressed on human hemopoietic progenitor cells. This structure can be readily surface-labeled with [125I]actoperoxidase and by periodate-[3H]borohydride, but it labels only weakly with [35S]methionine, [35Sl]cysteine, 3H-amino acids, or 3H-mannose, even after prolonged labeling periods. However, the antigen is more efficiently labeled by [3H]glucosamine. Lectin binding studies, sensitivity to certain glycosidases, and gel filtration analysis of glycans released by alkaline hydrolysis indicate that this glycoprotein contains several complex-type N-linked glycans as well as several highly sialylated O-linked glycans. Western blotting experiments show that various CD34 antibodies fail to efficiently detect desialylated and/or de-N-glycosylated forms of the antigen. Experiments involving the use of tunicamycin, together with metabolic labeling studies, strongly suggest that this structure "turns over" very slowly in vivo. The CD34 antigen is not detectably labeled by 32P-phosphate in vivo, nor are immune complexes containing it associated with phosphokinase activity in vitro. Sequential immunoprecipitation and Western blotting studies indicate that this antigen is not a member of the leukosialin/sialophorin family despite the fact that these molecules share several structural similarities. Partial amino acid analysis of highly purified CD34 antigen revealed no significant sequence similarity with any previously described structures.

The correlation of breakpoint cluster region rearrangement and p210 phl/abl expression with morphological analysis of Ph-negative chronic myeloid leukemia and other myeloproliferative diseases.

The chromosome 22 derivative, the Philadelphia (Ph) chromosome, results from a reciprocal translocation t(9;22) (q34;q11) and is associated with chronic myeloid leukemia (CML). The translocation can be identified at the DNA level in Ph-positive CML by using a probe to the breakpoint cluster region (bcr). In addition, as a result of this translocation an abl-related 210-kd protein with protein tyrosine kinase (PTK) activity is produced. We analyzed 28 cases of Ph-negative CML for rearrangement of the chromosome 22 sequences and found that eight of the 28 show rearrangement of the bcr. When 12 of the Ph-negative cases were independently reviewed, five were indistinguishable from Ph-positive CML on the basis of morphology, peripheral blood film and clinical details. These five also showed bcr rearrangement. The other seven were reclassified as six atypical CML (aCML) and one chronic myelomonocytic leukemia (CMML). None of these seven showed bcr rearrangement. In addition 11 cases of bcr- CML were assayed for abl-related PTK, and no detectable activity was present, whereas p210 phl/abl PTK was observed both in Ph-positive (three cases examined) and Ph-negative, bcr + (four cases examined) CML. Therefore, bcr + CML, whether or not the Ph chromosome is cytogenetically apparent, involves a similar molecular alteration and produces the 210-kd protein with enhanced PTK activity. Furthermore, these cases can be distinguished from Ph-negative bcr- CML by careful evaluation of clinical and hematologic data.

The heterogeneity of arylsulphatase-A and arylsulphatase-B in normal human urine and urine from cancer patients.

Arylsulphatase-A and arylsulphatase-B heterogeneity in normal and cancer patient urine was investigated using high resolution agarose isoelectricfocusing. Normal urine contained up to nine forms of arylsulphatase-A activity with isoelectric points from 4.45 to 5.43 and at least 5 forms of arylsulphatase-B between 8.58 and 9.15 along with a broad zone of activity between pH 6.5 and 7.6. Although cancer patients had significantly higher levels of arylsulphatase-A and arylsulphatase-B activity, their pattern of activity was essentially the same as for the normals with only minor quantitative differences in some peaks.

Detection of a multidrug resistant phenotype in acute non-lymphoblastic leukaemia.

Most adult patients with acute non-lymphoblastic leukaemia (ANLL) relapse with drug resistance. Overexpression of a plasma membrane protein, P-glycoprotein, correlates with multidrug resistance in human and animal cell lines. We have detected a multidrug resistance phenotype in two patients with drug resistant ANLL by an immunocytochemical assay using a monoclonal antibody to P-glycoprotein. Sequential analysis of peripheral blood samples from both patients showed a progressive increase in both the intensity of staining and the proportion of leukaemic cells that bound antibody as the disease progressed. The assay is simple, and may have prognostic and therapeutic implications.

Characterization of a p43 human thymocyte antigen.

Two murine monoclonal antibodies (MoAbs), designated WM-25 and WM-35, have been prepared which react with human thymocyte cell surface membrane antigens. Both antigens were found to be expressed by normal and leukaemic immature human thymocytes. However, comparative examination by tissue section staining, immunoprecipitation, and electrophoresis, as well as competitive binding studies, demonstrated clear differences in the antigens detected by these two MoAbs. Antibody WM-35 identified a 48 kilodalton (kD) protein expressed on epidermal Langerhans cells as well as thymocytes, and appears similar to the previously reported Mabs OKT-6 and NA1-34. In contrast, MoAb WM-25 immunoprecipitated a 43 kD antigen, non-covalently associated with a 12 kD light chain, which was not present on Langerhans cells. Competitive binding and sequential immunoprecipitation studies confirmed that the 48 and 43 kD proteins are antigenically distinct. These results support the concept of at least two forms of thymocyte antigens within Cluster of Differentiation One (CD-1).