Differential expression of bcl-2 homologs in human CD34(+) hematopoietic progenitor cells induced to differentiate into erythroid or granulocytic cells.

The Bcl-2 family of proteins has been shown to play a central role in the regulation of apoptosis. We have examined the expression of several Bcl-2 homologs upon stimulation of CD34(+) human hematopoietic progenitor cells. CD34(+) cells were induced to differentiate into predominantly erythroid cells in the presence of erythropoietin (Epo) and stem cell factor (SCF), while the addition of G-CSF and SCF led to differentiation predominantly into granulocytic cells, as demonstrated by immunophenotyping and morphological examination of cultured cells. In Epo- and SCF-stimulated cells, we found a marked increase in the level of Bcl-x(L) protein expression and downregulation of Bax expression, apparent from day 4 and more pronounced on days 8 and 21. In contrast, Bcl-x(L) protein expression was downregulated in G-CSF- and SCF-stimulated cells compared with cells cultured in medium alone, whereas there was no sign of change in the level of Bax. Mcl-1 expression showed a biphasic expression pattern in both early erythropoiesis and early granulopoiesis, but with an inverse regulation. Thus, Mcl-1 levels initially decreased in granulocytic progenitor cells and increased in erythroid progenitor cells. Finally, Bcl-2 expression was significantly downregulated in both Epo and SCF and G-CSF- and SCF-stimulated cells. The role of the distinct upregulation of Bcl-x(L) in early erythroid differentiation was further examined by use of specific ribozymes against Bcl-x(L). Addition of Bcl-x(L) ribozymes promoted a clear increase in cell death of Epo- and SCF-stimulated cells, while erythroid differentiation was not affected. In conclusion, we found a distinct regulation of several Bcl-2 family members in CD34(+) cells dependent on the cytokine stimulation given. The use of Bcl-x(L)-specific ribozymes suggested that Bcl-x(L) is important for survival but not for differentiation of erythroid progenitor cells.

Protein kinase C-alpha isoform is involved in erythropoietin-induced erythroid differentiation of CD34(+) progenitor cells from human bone marrow.

Protein kinase C (PKC) is a family of serine/threonine protein kinases involved in many cellular responses. Although the analysis of PKC activity in many systems has provided crucial insights to its biologic function, the precise role of different isoforms on the differentiation of normal hematopoietic progenitor cells into the various lineages remains to be investigated. The authors have assessed the state of activation and protein expression of PKC isoforms after cytokine stimulation of CD34(+) progenitor cells from human bone marrow. Freshly isolated CD34(+) cells were found to express PKC-alpha, PKC-beta2, and PKC-epsilon, whereas PKC-delta, PKC-gamma, and PKC-zeta were not detected. Treatment with erythropoietin (EPO) or with EPO and stem cell factor (SCF) induced a predominantly erythroid differentiation of CD34(+) cells that was accompanied by the up-regulation of PKC-alpha and PKC-beta2 protein levels (11.8- and 2.5-fold, respectively) compared with cells cultured in medium. Stimulation with EPO also resulted in the nuclear translocation of PKC-alpha and PKC-beta2 isoforms. Notably, none of the PKC isoforms tested were detectable in CD34(+) cells induced to myeloid differentiation by G-CSF and SCF stimulation. The PKC inhibitors staurosporine and calphostin C prevented EPO-induced erythroid differentiation. Down-regulation of the PKC-alpha, PKC-beta2, and PKC-epsilon expression by TPA pretreatment, or the down-regulation of PKC-alpha with a specific ribozyme, also inhibited the EPO-induced erythroid differentiation of CD34(+) cells. No effect was seen with PKC-beta2-specific ribozymes. Taken together, these findings point to a novel role for the PKC-alpha isoform in mediating EPO-induced erythroid differentiation of the CD34(+) progenitor cells from human bone marrow. (Blood. 2000;95:510-518)

Relation between S-phase fraction of myeloma cells and anemia in patients with multiple myeloma.

In an attempt to define the relation among anemia, tumor mass, and proliferative activity of tumor cells in vivo, we measured the proportion and cell cycle distribution of erythropoietic cells and myeloma cells in the bone marrow of patients with multiple myeloma using four-parameter flow cytometry. Forty-three bone marrow samples from 33 patients with stage II or III disease and normal renal function at diagnosis (n = 9), in partial remission (n = 9), and in progression or relapse after chemotherapy (n = 25) were evaluated. Early and late erythropoietic cells were discriminated based on published light scatter properties in combination with CD71 expression. Myeloma cells were detected by exploiting their strong CD38 positivity and light scatter characteristics. Cell cycle distribution of the three cell populations was determined by propidium iodine staining. In the whole group of patients, hemoglobin (Hb) concentration was inversely correlated with beta2-microglob-ulin (p = 0.03), percentage of marrow CD38++ cells (p = 0.008), and percentage of CD38(++) cells in S phase (S-CD38++; p < 0.001). Partial correlation analysis revealed S-CD38++ to be the only independent predictor of Hb concentration (p < 0.001). No correlation was found between Hb concentration and the S-phase fraction of erythropoietic cells. In the subgroup of patients with moderate to severe anemia, defined as Hb concentration <11 g/dL, Hb level correlated negatively only with S-CD38++ (p < 0.001) but not with beta2-microglobulin and percentage of marrow CD38++ cells. In addition, Hb and the S-phase proportion of early erythropoietic cells correlated positively (p = 0.029). The strong inverse correlation between Hb concentration and percentage of myeloma cells in S phase suggests that in multiple myeloma, tumor proliferative activity may have a more important impact on the development of anemia than tumor mass. The S-phase fraction of tumor cells appears to be the most important pathogenic factor, especially in anemic patients. In these patients, the positive relation between Hb concentration and the S-phase fraction of erythropoietic progenitors indicates that development of anemia is associated with inhibition of erythropoiesis.

Fas ligand promotes cell survival of immature human bone marrow CD34+CD38- hematopoietic progenitor cells by suppressing apoptosis.

Fas (CD95, APO-1) is a member of the TNF receptor family, and engagement of Fas by its ligand, Fas ligand (FasL), can induce apoptotic death of Fas expressing cells. Signaling through Fas has previously been shown to induce apoptosis of CD34+ human hematopoietic progenitor cells after exposure to IFN-gamma or TFN-alpha. In contrast, we found that FasL promoted a significantly increased viability of primitive CD34+CD38- cells. Thus, incubation with FasL for 48 hours reduced cell death from 46 to 29% compared to cells cultured in medium alone as measured by propidium iodide (PI) incorporation (n = 8, p < 0.02). Inhibition of apoptosis was confirmed by morphological analysis and by the Nicoletti technique. Furthermore, by using a delayed addition assay at the single cell level we found that sFasL treatment had a direct viability-promoting effect on CD34(+)CD38(-) cells. The effect of sFasL was completely blocked by NOK-1, a neutralizing mAb against FasL. In agreement with previous reports, FasL alone slightly increased cell death of more mature CD34(-)CD38+ cells, indicating an interesting shift in the responsiveness to FasL during early hematopoiesis.

Activation of the cAMP signaling pathway increases apoptosis in human B-precursor cells and is associated with downregulation of Mcl-1 expression.

During B- and T-cell ontogeny, extensive apoptosis occurs at distinct stages of development. Agents that increase intracellular levels of cAMP induce apoptosis in thymocytes and mature B cells, prompting us to investigate the role of cAMP signaling in human CD10+ B-precursor cells. We show for the first time that forskolin (which increases intracellular levels of cAMP) increases apoptosis in the CD10- cells in a dose-dependent manner (19%-94% with 0-1,000 microM forskolin after 48 hours incubation, IC50 = 150 microM). High levels of apoptosis were also obtained by exposing the cells to the cAMP analogue 8-chlorophenylthio-cAMP (8-CPT-cAMP). Specific involvement of cAMP-dependent protein kinase (PKA) was demonstrated by the ability of a cAMP antagonist, Rp-isomer of 8-bromo-adenosine- 3', 5'- monophosphorothioate (Rp-8-Br-cAMPS), to reverse the apoptosis increasing effect of the complementary cAMP agonist, Sp-8-Br-cAMPS. Furthermore, we investigated the expression of Bcl-2 family proteins. We found that treatment of the cells with forskolin or 8-CPT-cAMP for 48 hours resulted in a fourfold decline in the expression of Mcl-1 (n = 6, P = 0.002) compared to control cells. The expression of Bcl-2, Bcl-xL, or Bax was largely unaffected. Mature peripheral blood B cells showed a smaller increase in the percentage of apoptotic cells in response to 8-CPT-cAMP (1.3-fold, n = 6, P = 0.045) compared to B-precursor cells, and a smaller decrease in Mcl-1 levels (1.5-fold, n = 4, P = 0.014). Taken together, these findings show that cAMP is important in the regulation of apoptosis in B-progenitor and mature B cells and suggest that cAMP-increased apoptosis could be mediated, at least in part, by a decrease in Mcl-1 levels.

Generation and characterization of gp100 peptide-specific NK-T cell clones.

MHC-restricted cytotoxic T lymphocytes (CTLs) specific for antigens expressed by malignant cells are important components of immune responses against human cancer. Peripheral blood monocytes of HLA-A2+ healthy donors were used to induce dendritic cells (DCs) by granulocyte-macrophage colony-stimulating factor and interleukin-4 and loaded with a gp100 peptide (YLEPGPVTA). By applying these peptide-loaded DCs, a CTL line that displayed high cytotoxic reactivity with peptide-loaded target cells was generated. A total of 11 gp100 peptide-specific CTL clones were generated from this cell line. Several of these CTL clones were studied in detail. Of particular interest was clone CTL-45, which, contrary to the parental cell line, displayed strong NK activity and, by flow-cytometric analysis, revealed a CD3+, TCR BV17, CD8+ and CD56+ phenotype. This clone was strictly peptide-specific and effectively killed a panel of melanoma cells expressing HLA-A2 and gp100. Tumor-specific T cells with this kind of dual function are potentially of great clinical importance as they have a backup mechanism that may go into action when tumor cells escape specific killing by losing their HLA-class I molecules.