Expression of rhombotin 2 in normal and leukaemic haemopoietic cells

The congenital disorder of erythropoiesis Diamond Blackfan anaemia (DBA) exhibits a defect in the stem/progenitor cell compartment, located at the erythroid progenitor level (CFU-GEMM, BFU-E, CFU-E). Treatment of DBA with interleukin-3 (IL-3) has had limited effect, despite in vitro studies suggesting that progenitor cells were capable of responding to IL-3. Whether IL-3 is not reaching the appropriate defective target cell, the cells cannot respond, or the marrow humoral inhibitory system is overriding it, is not clear. To investigate humoral inhibitory activities we examined the response of 15 DBA bone marrows in vitro to the inhibitory chemokine macrophage inflammatory protein 1-α (MIP1-α) in the presence of the stimulatory cytokines erythropoietin, granulocyte-macrophage colony-stimulating factor, IL-3, and stem cell factor. In vitro data agreed with our previous work showing that our patients formed three statistically different groups in response to stimulatory cytokines (type I DBA erythroid colony numbers≈ normal > type II DBA > type III DBA). Addition of MIP1-α to cultures caused average erythroid and myeloid suppression, which sequentially increased with DBA type (type I inhibition < type II < type III). The differential level of inhibition shown by MIP1-α in these DBA patients lends further evidence for the presence of distinct subgroups in this disorder.     

Expression and modulation of IL-3 and GM-CSF receptors in human growth factor dependent leukaemic cells

From the interleukin-3 (IL-3) responsive human myeloid cell line M-07 we derived a subclone, named M-07e, which is fully dependent for growth and survival on either granulocyte-macrophage colony stimulating factor (GM-CSF) or IL-3. In this paper the expression, specificity and modulation of GM-CSF and IL-3 receptors on M-07e cells are described. The specificity of the binding was demonstrated by the failure of other cytokines to compete, at 4 degrees C, with GM-CSF or IL-3 receptors. In addition, IL-3 was found to compete as well as GM-CSF for GM-CSF receptors while GM-CSF was a weak competitor for IL-3 receptors. Quantitative binding studies and Scatchard plot analysis revealed the presence of a single class of high-affinity GM-CSF binding sites (405 +/- 27.4 sites per cell, dissociation constant at the equilibrium 52 +/- 20 pM) and the presence of high and low-affinity regions for IL-3 binding sites (27 +/- 12 and 416 +/- 92 sites per cell for the high and low affinity regions respectively, dissociation constant at the equilibrium, 58 +/- 22 pM and 5.7 +/- 2.0 nM respectively). Finally, in agreement with the hierarchical down-modulation model, we found that IL-3 can down-modulate both IL-3 and GM-CSF receptors while GM-CSF can down-modulate only its own receptors. The present results suggest that M-07e cells, in spite of their neoplastic nature, share, with murine bone marrow cells, similar growth factor receptor regulatory mechanisms. This cell line may be regarded as a candidate model to investigate the physiological events triggered by growth factors binding to human haemopoietic cells.     

Effect of the protein tyrosine kinase inhibitor genistein on normal and leukaemic haemopoietic progenitor cells

Receptor and nonreceptor protein tyrosine kinases (PTKs) play a key role in the control of normal and neoplastic cell growth. The availability of PTK inhibitors prompted us to evaluate the effects of genistein, a natural inhibitor of PTKs, on in vitro colony formation by normal multilineage colony-forming units (CFU-Mix), erythroid bursts (BFU-E), granulocyte-macrophage colony-forming units (CFU-GM), long-term culture-initiating cells (LTC-IC) and acute myelogenous leukaemia colony-forming units (CFU-AML). Continuous exposure of normal marrow and blood mononuclear non-adherent cells, blood CD34⁺CD45RA^? cells, and leukaemic blasts to increasing doses of genistein (1–100 μM ) resulted in a statistically significant (P ≤ 0.05) dose-dependent suppression of CFU-Mix, BFU-E, CFU-GM and CFU-AML growth. Regression analysis showed that growth inhibition was linearly related to genistein concentration. Genistein dose causing 50% inhibition (ID₅₀) of CFU-AML was significantly lower compared to CFU-GM ID₅₀ for marrow (19 v 32 μM P ≤0.017), unseparated blood (19 v 44 μM P ≤ 0.028) or CD34⁺CD45RA^? blood (19 v 36, P ≤ 0.04). Preincubation of leukaemic blasts with genistein (200 μM ) for 1–2 h confirmed that CFU-AML were significantly more sensitive than normal marrow and blood CFU-GM to genistein. Preincubation conditions which maximally suppressed leukaemic and normal colony growth spared a substantial percentage of marrow (29 ± 4%) and blood (40 ± 3%) LTC-IC. In conclusion, our data demonstrate that: (a) genistein strongly inhibits the growth of normal and leukaemic haemopoietic progenitors; (b) growth inhibition is dose- and time-dependent; (c) leukaemic progenitors are more sensitive than normal progenitors to genistein-induced growth inhibition; (d) genistein exerts a direct toxic effect on haemopoietic cells while sparing a substantial proportion of LTC-IC. The potent CFU-AML growth inhibition associated with the relative resistance of normal LTC-IC strongly supports the use of genistein for marrow purging.     

Differences in the distribution of CD34 epitopes on normal haemopoietic progenitor cells and leukaemic blast cells

The CD34 molecule expressed on haemopoietic progenitor cells contains a large number of epitopes whose expression may be related to the maturation or function of the cells. Monoclonal antibodies specific for different epitopes have been reported to detect different numbers of CD34⁺ leukaemic blast cells. We wanted to confirm this observation and study whether parallel findings could be observed for normal haemopoietic progenitor cells. The cells were immunophenotyped by flow cytometry with a series of monoclonal antibodies reactive with different CD34 epitopes. Class III epitopes (resistant to enzymatic cleavage with neuraminidase, chymopapain and a glycoprotease from Pasteurella haemolytica) showed a broader distribution on normal haemopoietic progenitor cells and leukaemic blast cells than class I epitopes (sensitive to cleavage with all three enzymes) and class II epitopes (sensitive to degradation with glycoprotease and chymopapain, and resistant to neuraminidase). The subpopulation of normal progenitor cells which exclusively expressed class III epitopes had flow cytometric characteristics compatible with mature myeloid progenitor cells, whereas class I, II and III epitopes were equally expressed on cells enriched for immature subsets. No discordant epitope expression could be observed for the more immature leukaemias (AML-M0/1) and a higher percentage of the more mature leukaemic blast cells (AML-M3 and AML-M4/5) expressed class III epitopes compared to the percentage expressing class I and II epitopes. These data indicate that CD34 class III epitopes are more broadly distributed on normal haemopoietic progenitor cells and leukaemic blast cells than class I and II epitopes, and that class I and II epitopes may be down-regulated prior to class III epitopes during normal haemopoietic progenitor cell differentiation. These findings should be considered when selecting CD34 mabs for quantification and positive selection of haemopoietic progenitor cells for research and clinical purposes.     

Surface antigens on normal and leukaemic human cells detected by monoclonal antibodies

Surface antigens were analyzed on normal human marrow and chronic myeloid leukaemic cells using 4 monoclonal mouse anti-human antibodies. The fluorescence-activated cell sorter was used to quantify the binding of each antibody to different subpopulations of cells, and sorted fractions were cultured in agar-medium to assay for granulocyte-macrophage and eosinophil precursors. All cells in the granulocyte series including colony-forming cells bound a similar quantity of an antibody to the human leucocyte common antigen. This antibody did not bind to cells in the erythroid series. A monoclonal antibody to antigen present on brain, lymphocytes and granulocytes (and almost certainly homologous to the W3/13 antigen of the rat) bound to the cells in the order: blast greater than promyelocytes and myelocytes greater than granulocytes. The third monoclonal antibody was directed against a determinant of the leucocyte common antigen present predominantly on B lymphocytes and absent from the myeloid series. The fourth antibody, directed against the human homologue of Thy-1, reacted with less than 1% of marrow cells, none of which appeared to be granulocyte or eosinophil progenitors. The leucocoyte common antigen and the brain-lymphocyte-granulocyte-antigen were also present on colony- and cluster-forming cells from a patient with chronic myeloid leukemia. Using the low angle and wide angle light scatter properties of CML blood cells, 7-fold enrichment was obtained for progenitor cells from chronic myeloid leukaemia. With the monoclonal antibodies up to 4-fold enrichment was obtained.     

Differential action of diffusible molecules in long-term marrow culture on proliferation of leukaemic and normal haemopoietic cells

Summary. The capacity of diffusible molecules in the fluid phase of long-term human bone marrow culture (LTMC) to exert preferential adverse effects on leukaemic relative to normal haemopoietic cells has been investigated. Responses of isolated cell populations were assessed in diffusion chamber inserts which permitted contact with fluid phase molecules but not with the adherent stromal cell layer of the LTMC system. Growth of AML cells in diffusion chambers was inhibited during co-culture with LTMC of autologous leukaemic bone marrow, and the same effect was produced during co-culture with normal LTMC. No inhibitory action was exerted on growth of normal haemopoietic precursors under the same conditions. Comparable responses were observed with human leukaemic cell lines and patient leukaemic cells, and studies on cell lines demonstrated inhibition of growth was induced by molecules generated in LTMC which caused accumulation in G1 phase of leukaemic cells of both myeloid and lymphoid lineage. The inhibitory effect was not reproduced by TGFβ, IFNγ, IFNα, TNFα, LIF, SCF or II-6, and was not impaired by inhibitors of nitric oxide or PGE production in the LTMC. These observations suggest the action of diffusible molecules of undefined constitution contributes to the preferential loss of leukaemic cells in LTMC.     

Lactoferrin receptors in normal and leukaemic human blood cells

The binding of 125I-lactoferrin to a variety of peripheral blood cells was examined. In the concentration range from 10(-9) mol/l to 10(-6) mol/l a receptor-like binding of lactoferrin was observed in monocytes as well as in polymorphonuclear leucocytes. At the low concentrations of lactoferrin in plasma (about 10(-9) mol/l) the cellular binding to monocytes was about 10 times higher than the binding to polymorphonuclear leucocytes and lymphocytes. A major result from a kinetic analysis was a lower apparent binding affinity to polymorphonuclear leucocytes (KD about 2 X 10(-7) mol/l) than to lymphocytes and monocytes (KD about 2 X 10(-8) mol/l). Studies in leukaemic cells showed that lymphocytes from patients with chronic lymphocytic leukaemia bound lactoferrin to the same small extent as normal lymphocytes. In contrast, a larger component of binding with high affinity (KD about 2 X 10(-8) mol/l) could be demonstrated to lymphoblasts as well as to myeloblasts.     

Expression of an antigen defined by a monoclonal antibody (SA-1) on normal and malignant cells

A monoclonal antibody (SA-1) has been characterized. The antibody was derived from the fusion of the X-63 myeloma cell-line with splenocytes from a mouse immunized with human acute megakaryoblastic leukaemia cells. The antibody reacted with blast cells from 65% of patients with AML without correlation to morphologic classification. The antibody further reacted with a subset of myeloid cells from normal bone marrow, and with peripheral neutrophil granulocytes. In lymph nodes the antibody showed reactivity with subsets of dendritic reticulum cells. In skin biopsies the antibody reacted with subsets of Langerhans cells, subsets of indeterminate cells and activated T-lymphocytes. The antigen was not expressed on non-activated T-lymphocytes. Neither was it expressed on B-lymphocytes, erythrocytes, platelets nor blast cells from patients with ALL. The antigenic target for the antibody SA-1 was a surface exposed polypeptide (mol. wt. 15 000 D).     

Expression of the CD117 antigen (C-Kit) on normal and myelomatous plasma cells

Summary. The surface expression of CD117 on plasma cells (PCs) from normal individuals and patients with multiple myeloma (MM) has been analysed using triple-stained cells for flow cytometry. In addition, the clinical significance of CD117 expression in MM patients and its possible value for the evaluation of minimal residual disease was explored. A total of 11 healthy volunteers and 56 untreated MM patients were included in the study. The expression of CD117 was analysed by flow cytometry, using simultaneous staining with the MAbs BB4, CD117 and CD38. Cell acquisition was performed in two consecutive steps using a live gate drawn on SSC/CD38⁺⁺⁺ cells and a total of 300000 events were acquired. For data analysis, the Paint-a-Gate Plus software (Becton Dickinson) was used. PCs were identified according to their strong reactivity for CD38 and their positivity for BB4, as well as by their light scatter distribution. Dilution experiments of CD117⁺ myelomatous PCs with normal bone marrow (BM) cells were performed in order to assess the sensitivity level of the technique for detection of CD117⁺ residual PCs. None of the PCs from normal BM samples showed reactivity for the CD117 antigen. In contrast, CD117 antigen was present in 18/56 MM patients (32%), the proportion of positive cells in these cases being as high as 92·1 ± 9%. Therefore, within PC lineage the c-Kit antigen would be restricted to the myelomatous population and thus could be considered as a “tumour-associated marker” for monitoring minimal residual disease in about one third of MM patients. Dilution experiments indicate that the detection limit with this marker would be 10^?4 (one myelomatous PC/10⁴ normal BM cells). Upon comparing the clinical and haematological disease characteristics of CD117-positive and CD117-negative cases, no significant differences were found.     

Different sensitivity of normal and leukaemic progenitor cells to Ara-C and IL-3 combined treatment

In the present study the effects of a combined treatment with cytosine-arabinoside (Ara-C) and interleukin-3 (IL-3) on acute myeloblastic leukaemia clonogenic cells and on normal haemopoietic progenitors was investigated, with the aim of improving the tumoricidal effect of cycle specific drugs. Blast cells from 24 acute myeloblastic leukaemia (AML) patients were screened with a short-term proliferative assay based on 3H-thymidine (3H-TdR) uptake for their response to IL-3. To evaluate the synergism between the growth factor and Ara-C, the cells were pretreated for 3 d in liquid culture in the presence or absence of IL-3 (10 U/ml) and for the last 24 h with Ara-C (3 micrograms/ml). The cells were then washed and seeded in semisolid media to assess their clonogenic ability. The results showed that, in those cases which were good responders to IL-3 in the 3H-TdR uptake assay (19 out of 24), Ara-C exposure eliminated a greater proportion of clonogenic cells if pretreated with IL-3 than if untreated (P less than 0.001), while in cases unresponsive to IL-3 this effect was not significant. Moreover, when the same protocol was applied to bone marrow cells from normal donors, it was found that IL-3 pretreatment did not significantly enhance the toxic effect of Ara-C on day 14 granulocyte-macrophage colony forming units (CFU-GM) and erythroid burst forming units (BFU-E). Finally IL-3 pretreatment was also able to increase the cytotoxic effect of Ara-C on leukaemic cells co-cultured, to simulate clinical AML remission, with normal bone marrow cells. The results indicate that IL-3 may improve the therapeutic index of cycle-specific drugs in AML therapy.     

Immunocytochemical and flow cytometric detection of proteinase 3 (myeloblastin) in normal and leukaemic myeloid cells

Summary. Proteinase 3 (P3) is a serine proteinase present in the primary granules of neutrophils. We have investigated the expression of this protein in samples of bone marrow from healthy individuals and patients with different types of leukaemias by using immunocytochemical staining and flow cytometric quantitation. In normal bone marrow the enzyme was found in promyelocytes, myelocytes, metamye-locytes, band forms and polymorphonuclear neutrophils, correlating with the synthesis of neutrophil serine proteinases during myeloid maturation. No staining was found within the lymphoid, erythroid and megakaryocytic lineage. In the leukaemic samples, only those of acute myeloid and chronic myeloid leukaemia patients were labelled with the antiproteinase 3 antibody. Cases of acute lymphoblastic and chronic lymphocytic leukaemia, as well as other malignant lymphomas, were consistently negative, indicating that P3 may be used as a specific marker for the discrimination between myeloid and lymphoid leukaemias. In addition. immunoreactivity of myeloperoxidase (MPO) was investigated and the expression of P3 and MPO correlated with the French-American-British (FAB) classification. P3 was not detected in minimally differentiated MO and Ml cases but was in predominantly labelled cells of M2 and M3 subtypes plus half of the M4 and one out of six M5 cases but not those of M6. These findings correspond to the differentiation stage in which P3 is expressed and stored in the primary granules. Therefore the enzyme may also be used as an adjunct to the classic morphological and cytochemical methods to elucidate further the stage at which the differentiation arrest of the leukaemic clone has occurred.     

Expression of the multidrug resistance-associated protein (MRP) mRNA and protein in normal peripheral blood and bone marrow haemopoietic cells

We studied the expression of multidrug resistance-associated protein (MRP) in normal haemopoietic cells from peripheral blood and bone marrow. The MRP mRNA levels were estimated by RT/PCR and in situ hybridization (ISH) assay, and the protein levels by flow cytometry. 21 samples of peripheral blood and 21 samples of bone marrow (11 normal bone marrow donors, 10 patients in complete remission after chemotherapy for large cell lymphoma or acute myeloid leukaemia) were analysed. In peripheral blood the mean MRP mRNA level in CD3⁺ cells was statistically higher than in the other cells (3-fold by the methods used). The levels of MRP in CD3⁺ varied from one individual to another (4.5–34.8 units by RT/PCR and 5–23 grains/cell by ISH); however, this was proportional to the variation in all the cell lineages of same individual ( r  = 0.84). In bone marrow the mean MRP levels of the various cell lineages (including CD34⁺) were similar to the basal level in HL60 cells. Individual expression levels were again variable; however, there was no difference between untreated normal bone marrow and post chemotherapy normal bone marrow. MRP protein expression was determined by flow cytometry with the monoclonal antibody MRPm6. The CD4⁺ lymphocytes exhibited a higher MRP protein expression than the other cell lineages, including CD8⁺ cells. There was a good correlation between the three methods used (RT/PCR and ISH, P  = 0.0001, r  = 0.87; RT/PCR and flow cytometry, P  = 0.0001, r  = 0.85; ISH and flow cytometry, P  = 0.002, r  = 0.67).     

Expression of the human OX40 (hOX40) antigen in normal and neoplastic tissues

Summary. The Ber-ACT35 mAb was raised against the human T-cell lymphotrophic virus (HTLV) 1-transformed, CD4⁺ HUT 102 cell line and recognizes the human homologue of the 0X40 (hOX40) antigen. The analysis of the expression of hOX40 by immunohistochernical techniques in malignant lymphomas, carcinomas and non-malignant tissues of different organs shows that hOX40 expression was almost completely restricted to T lymphocytes. Besides T cells only a small subpopulation of macrophages in Langerhans'cell histiocytosis and a few blasts in B-cell non-Hodgkin's lymphoma (B-NHL) revealed a faint immunostaining with the Ber-ACT35 mAb. Furthermore, most of the hOX40⁺ T-cells are CD4⁺.     

Expression and factor-dependent modulation of the interleukin-3 receptor subunits on human hematopoietic cells

Interleukin-3 (IL-3) regulates growth and differentiation of multipotential as well as lineage-committed progenitor cells. The human IL-3 receptor (IL-3R) consists of the alpha and common beta (beta c) subunits. The alpha subunit (IL-3R alpha) is specific for IL-3 and binds IL-3 with low affinity. In contrast, the beta c subunit does not bind any cytokine by itself, but forms a high-affinity receptor with IL- 3R alpha. As the same beta c subunit also forms high-affinity receptors for IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) with the respective cytokine-specific alpha subunit, the expression of the alpha subunits is responsible for specificity of cytokines. To examine the expression of IL-3R alpha, we have developed a monoclonal antibody (MoAb), N3A. N3A specifically bound to cells expressing IL-3R alpha and immunoprecipitated a 75 Kd glycoprotein, which became 43 Kd on N-glycosidase digestion. N3A and an anti-beta c antibody, CRS1, were used in double color fluorescence-activated cell sorter (FACS) staining with several lineage markers to see the IL-3R expression pattern in peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells. Both IL-3R subunits were expressed on myeloid cell lineages (CD13+, CD14+, CD15Lo, or CD33+). To further study the IL-3R expression on hematopoietic progenitor cells, the CD34+ populations were isolated from both BM and CB cells. Those populations showed positive staining profiles with the N3A MoAb and were weakly stained with the CRS1 MoAb. Furthermore, anti c-kit antibody staining of the CD34+ fraction from CB, but not from BM, showed two intensities and the IL-3R alpha expression seemed to be higher in a fraction of low c-kit expression. Because IL-1, IL-6, G-CSF, stem cell factor (SCF), interferon (IFN)- gamma, and tumor necrosis factor (TNF)-alpha are known to enhance IL-3- dependent colony formation, we have examined whether this enhancement could be correlated with upregulation of the IL-3R expression. Incubation of CD34+ cells with TNF-alpha for 2 days significantly increased the level of beta c and G-CSF increased the number of cells with high level expression of alpha, while other factors did not affect the IL-3R expression. Thus, different cytokines appear to have different mechanisms for enhancement of IL-3-dependent proliferation.     

Expression and functional role of urokinase-type plasminogen activator receptor in normal and acute leukaemic cells

Urokinase-type plasminogen activator receptor (UPA-R-CD87) is a GPI-anchored membrane protein which promotes the generation of plasmin on the surface of many cell types, probably facilitating cellular extravasation and tissue invasion. A flow cytometric quantitative analysis of expression levels for UPA-R was performed on fresh blast cells from patients with acute myeloid leukaemia (AML, n = 74), acute lymphoblastic leukaemia (ALL, n = 24), and biphenotypic leukaemia (BAL, n = 3) using two CD87 monoclonal antibodies (McAbs) (3B10 and VIM5). Peripheral blood and bone marrow (BM) cells from 15 healthy adults served as controls. Using 3B10 McAb, UPA-R was expressed (>99%) by blood monocytes, neutrophils, and BM myelomonocytic precursors in controls, whereas resting T and B lymphocytes, and CD34⁺ cells were UPA-R negative. We also attempted to clarify whether UPA-R has a role in mediating neutrophil functions. Oriented locomotion induced by different chemotaxins and lysozyme release by granules stimulated with fMLP or PMA were significantly decreased when UPA-R was neutralized by CD87 McAb. In contrast, the anti-UPA-R McAb had no effect on superoxide anion generation of normal neutrophils. Blasts from AML showed a heterogenous pattern of expression for the UPA-R McAbs, with reactivity strictly dependent on FAB subtype. The highest UPA-R expression was seen in the M5 group: all patients tested (n = 20) showed strong positivity for the UPA-R McAb whereas only 12% (3/24) of ALL patients were CD87 positive, and 2/3 of BAL patients showed a dim expression for CD87. The number of receptors expressed by blast cells in 6/74 (8.1%) AML patients was higher than those of normal samples; in addition, since co-expression of UPA-R and CD34 was not found in normal haemopoietic cells, it may be postulated that CD87 can be used alone (when overexpressed) or in combination with CD34 for the detection of minimal residual disease. Results also indicated that patients with UPA-receptors >12 × 10³ ABC/cell, irrespective of FAB subtype, had a greater tendency for cutaneous and tissue infiltration and a higher frequency of chromosome abnormalities, thus suggesting the concept that cellular UPA-R content positively correlates with the invasive potential of AML cells. The combination of higher UPA-R positivity, abnormalities of chromosome 11, and M5 FAB morphology may identify a peculiar subset of AML, characterized by a more aggressive clinical course.     

Comparative quantitative expression of bcl-2 by normal and leukaemic myeloid cells

Summary. Expression of the bcl-2 oncoprotein by AML blasts has previously been demonstrated to be heterogenous with high levels of bcl-2 expression being associated with a low complete remission rate and poor survival. We have quantified bcl-2 expression in AML blasts in relation to expression of the CD34 antigen and in comparison to CD34-positive cells from normal bone marrow. When expressed as molecules of equivalent soluble fluorochrome (MESF) per cell, AML blast cell bcl-2 expression varied from 11.1 to 99.9 × lO³ (median 39.4 × 10³, n=56) with 28.5% of patients expressing high MESF values (>50 × 10³) and 16% of patients expressing low MESF values (<20 × 10³), the remainder expressing intermediate values. There was no significant difference between intensity of bcl-2 expression and FAB classification in the de novo AML cases, and there was no significant differences between de novo and secondary AML cases. Blasts from CD34⁺ AML patients expressed significantly higher levels of bcl-2 (mean MESF 43.6 × 10³, n =36) than CD34^? AML patients (mean MESF 31.7 × 10³, n=19). In five cases of CD34⁺ AML, bcl-2 expression was determined on purified CD34⁺ and CD34^? blast cell populations. In all cases CD34⁺ blasts were found to express significantly higher bcl-2 MESF values compared to the CD34^? fraction. Purified CD34⁺ cells from normal bone marrow consistently expressed high levels of bcl-2 (MESF >75 × 10³ n = 4), which was comparable to that found on CD34⁺ AML cells. Our results suggest that the poor prognosis previously associated with AML blasts expressing the CD34 antigen may in part be related to high expression of bcl-2. Also the ability to measure bcl-2 in AML blasts quantitatively by flow cytometry and to categorize patients into discrete groups may be of value as a prognostic indicator in AML.     

Expression of adhesion molecules LFA-3 and N-CAM on normal and malignant human plasma cells

Normal and malignant plasma cells were investigated for the expression of seven cellular adhesion molecules by immunofluorescence microscopy. The antigens investigated were CD2 and its ligand, LFA-3 (CD58). LFA-1 alpha (CD11a) and LFA-1 beta (CD18) and their ligand ICAM-1 (CD54), H-CAM (lymphocyte homing receptor; CD44) and N-CAM (CD56). Marrow from 18 patients with myeloma, two with plasma cell leukaemia (PCL), four with monoclonal gammopathy of uncertain significance (MGUS) and 10 normal allogeneic bone marrow donors was studied. All plasma cells from normals and multiple myeloma patients were negative for CD2, CD11a and CD18. All normal and myeloma marrow plasma cells were positive for ICAM-1. 16/18 myeloma cases tested, and all other samples (normal, MGUS and PCL), contained plasma cells positive for H-CAM. Only one normal, but 12/16 myelomas tested were positive for N-CAM (P less than 0.02). One of four MGUS cases was moderately positive and one other weakly positive for N-CAM. Both PCLs were N-CAM negative. 12/18 myelomas were positive for LFA-3, but only two normals (P less than 0.05). All MGUS cases were negative for LFA-3, as was one PCL, the other being weakly positive. Three cases were negative for both adhesion molecules, three cases expressed only N-CAM or LFA-3 and 10 cases expressed both. LFA-3 and N-CAM are expressed significantly in myeloma rather than normal plasma cells. Cases of MGUS may express N-CAM but not, in this small series, LFA-3. Plasma cells in the peripheral blood (PCL) and plasma cell lines express little or no LFA-3 or N-CAM.     

Expression of CD9 (p24) antigen on hematopoietic cells following treatment with phorbol ester

Expression of the leukemia-associated cell surface antigen p24 (CD9) on human hematopoietic cell lines and B-cell chronic lymphocytic leukemia (B-CLL) cells was analyzed before and after treatment with the phorbol ester 12-o-tetradecanoyl-phorbol 13-acetate (TPA). Little or no expression of CD9 was detected in any of the cell lines used or in B-CLLs before treatment with TPA. After exposure to TPA, HL-60, Epstein-Barr virus-immortalized B-cell lines, Molt-3, MT-2 and B-CLLs showed markedly augmented CD9 expression. U937 and K562 showed slight increases of CD9 expression. However, no expression of CD9 was induced in CCRF-CEM or HUT-102. Although CD9 is known to be one of the most useful markers of pre-B-cell common acute lymphoblastic leukemia, the expression of CD9 does not seem to be restricted to any specific cell lineage and can be induced in various hematopoietic cell lineages by treatment with TPA.