Search

Summary The effect of CMC-544, a calicheamicin-conjugated anti-CD22 monoclonal antibody, was analysed in relation to CD22 and P-glycoprotein (P-gp) in B-cell chronic lymphocytic leukaemia (CLL) and non-Hodgkin lymphoma (NHL) in vitro. The cell lines used were CD22-positive parental Daudi and Raji, and their P-gp positive sublines, Daudi/MDR and Raji/MDR. Cells obtained from 19 patients with B-cell CLL or NHL were also used. The effect of CMC-544 was analysed by viable cell count, morphology, annexin-V staining, and cell cycle distribution. A dose-dependent, selective cytotoxic effect of CMC-544 was observed in cell lines that expressed CD22. CMC-544 was not effective on Daudi/MDR and Raji/MDR cells compared with their parental cells. The MDR modifiers, PSC833 and MS209, restored the cytotoxic effect of CMC-544 in P-gp-expressing sublines. In clinical samples, the cytotoxic effect of CMC-544 was inversely related to the amount of P-gp (P = 0·003), and to intracellular rhodamine-123 accumulation (P

Using a non-isotopic ligand binding assay, we quantitatively examined the amount of human thrombopoietin (TPO) receptor (TPO-R) on leukaemia cells from 128 patients with acute myeloid leukaemia (AML) or acute lymphoblastic leukaemia (ALL). The TPO-R was expressed in 53 (47%) of 114 AML cases, and an in vitro treatment with TPO led to proliferation (stimulation index >1 5) of leukaemia cells in 13 (20%) of 66 AML cases examined. The TPO levels had no relation to the FAB classification except for FAB-M7 AML. All five FAB-M7 cases expressed TPO-R, and one of three FAB-M7 examined showed in vitro proliferative response to TPO. Although there was no significant correlation (r = 0.3125) between the amount of TPO-R and the proliferative response, all of the AML cases which showed the in vitro response had TPO-R expression. There was no relationship between TPO-R amount and CD phenotypes, or the amount of granulocyte-colony stimulating factor (G-CSF) receptor. TPO-R was also expressed in two (14%) of 14 cases of ALL, and only these two cases had in vitro proliferative response to TPO. One had only lymphoid antigens, and the other had both lymphoid and myeloid antigens. Our results suggest that some leukaemia cells express functionally active TPO-R.

Using a non-isotopic ligand binding assay using multi-colour flow cytometry, we quantitatively examined the amount of mpl in megakaryocyte-platelet lineage cells. Firstly, we quantified the amount of mpl on cell lines. Mpl gene-transfected BaF3 cells expressed a large amount of mpl, whereas original BaF3, K562, HL-60 and NOMO-1 cells showed no mpl. In bone marrow cells from healthy volunteers, mpl was expressed on CD34 + cells from the very early stage of differentiation when they had no CD38 antigen. The amount of mpl increased with differentiation to CV34 + CD41 + cells, but decreased with further differentiation to CD34 + CD41 + cells. In CD34 + CD41 + cells the amount of mpl varied according to cell size: abundant in large cells, moderate in medium-size cells and a little in small cells. In bone marrow cells from patients with refractory anaemia (RA), the amount of mpl was decreased compared with that in bone marrow cells from healthy volunteers. When analysed by the same CD phenotype and same cell size, the amount of mpl was less in RA patients compared with that in healthy volunteers in all phenotypes and sizes tested. The proportion of large CD34 + CD41 + cells was less in RA patients than in normal volunteers.

In order to develop a non-isotopic quantitative assay of erythropoietin (Epo) receptor (EpoR) on human cells, we devised a flow-cytometric assay using cells stained with biotin-labelled and a streptavidine-RED670 conjugate. For quantification, we applied the Kolmogorov-Smirnov test and calculated the D value. The D value was evaluated from the degree of shift in two profiles according to the increase of fluorescence intensity due to the specific binding of biotin-labelled Epo to EpoR. A good correlation was observed between the number of EpoR calculated by 125I-Epo binding assay and the D value. Then, EpoR expression on bone marrow cells from normal individuals was studied by three-colour flow cytometry. In normal bone marrow, the number of EpoR on cells was highest in CD34+CD38 cells (approximately 1600 sites/cell), and decreased in the following order: CD34+CD38- cells > CD34+CD38+ cells > CD34-CD38+ cells. Glycophorin A (GpA) positive erythroid cells also expressed EpoR, and their CD34+ fraction expressed more EpoR than their CD34- fraction. However, the expression levels of EpoR of these fractions were lower than CD34+CD38- cells. These results indicated that EpoR was highly expressed on CD34+ haemopoietic progenitors from very early stages of differentiation without expression of CD38 antigen, and that the level of expression decreased with erythroid differentiation as well as with various lineage commitment in human bone marrow cells.

We analysed the relationship between all-trans retinoic acid (ATRA) resistance and P-glycoprotein (P-gp)-associated multidrug resistance (MDR) in acute promyelocytic leukaemia (APL). There was no difference in the intracellular ATRA accumulation between NB4 cells and an MDR1 cDNA-transduced NB4 subline and between ATRA-resistant NB4 cells (NB4/RA) and an MDR1 cDNA-transduced NB4/RA subline. PSC833, a MDR modifier, did not increase the intracellular accumulation of ATRA or affect the expression of CD11b, the nitroblue tetrazolium (NBT) reduction activity, the proportion of apoptotic cells or the morphology of these four ATRA-treated cell lines. Similar results were obtained in the analysis of APL cells from five patients relapsed after ATRA-induced complete remission.

Expression of erythropoietin (EPO) receptor (EPO-R) was analysed in leukaemia cells from 150 patients with acute myeloid leukaemia (AML) or acute lymphoblastic leukaemia (ALL). EPO-R was expressed in 81 (60%) out of 136 AML, and in vitro treatment with EPO led to proliferation of leukaemia cells in 13 (16%) out of 81 AML examined. EPO-R expression and in vitro response to EPO were observed in all subtypes of AML according to the French-American-British (FAB) classification. All eight patients with FAB-M6 expressed EPO-R, and one out of four showed an in vitro response to EPO. Although there was no significant correlation (r = 0.2522) between the amount of EPO-R and the in vitro response to EPO, all of the AML patients who showed in vitro response expressed EPO-R. Stem cell factor significantly enhanced both EPO-R expression and in vitro response to EPO. Interleukin-3 tended to increase in vitro response to EPO. CD phenotypes, the amount of granulocyte colony-stimulating factor (G-CSF) receptors and the amount of TPO receptors had no significant relationship with the amount of EPO-R. Patients with both EPO-R expression and in vitro response to EPO had shorter duration of complete remission than those without EPO-R (P = 0.0053). EPO-R was expressed in four (29%) out of 14 ALL, and none out of five ALL showed in vitro response to EPO.

We examined the multidrug resistant P-glycoprotein (P-gp) on normal bone marrow (BM) cells and acute myeloid leukaemia (AML) cells, using newly devised flow cytometric multi-parameter analysis with CD33, CD34 and MRK16 monoclonal antibodies. In both normal BM cells and AML cells, CD34 + CD33 - cells expressed P-gp strongly, CD34 + CD33 + cells moderately, and CD34 - CD33 + cells weakly. Acute promyelocytic leukaemia, mainly expressing CD34 - CD33 + but not CD34 + CD33 - at diagnosis, expressed less P-gp. P-gp expression of AML cells at diagnosis was increased as compared with normal cells of the same phenotype. P-gp expression was more increased in relapsed cases, especially in immature subpopulations.