High sensitivity of leukemic peripheral blood lymphocytes to triethyllead action

In a previous study we reported that triethyllead (Et3Pb+) inhibits cell proliferation of normal human lymphocytes. To further characterize this interaction, we studied herein the effects of Et3Pb+ on the cell viability of normal and leukemic human lymphocytes and analysed the expression and dynamics of the monomer/polymer equilibrium of tubulin in these cells. Short- and long-term cell culture experiments demonstrated significantly different dose-dependent effects of Et3Pb+ on cell viability of leukemic compared to normal lymphocytes. Indeed, in the presence of increasing concentrations of Et3Pb+ (10(-12)-10(-5) M), primary cultures of chronic lymphocytes (CLL) and acute lymphoblastic (ALL) leukemic human Lymphocytes were much more sensitive to Et3Pb+ treatment when compared to normal peripheral blood lymphocytes (PBL). The IC50 values were approximately 5 x 10(-6) M for PBL and 8 x 10(-10) M for both CLL and ALL respectively, when cells were preincubated for 3 h with this agent. These experiments revealed a 1000-fold higher responsiveness of leukemic cells to Et3Pb+ treatment. Quantitative immunoblot analysis showed that leukemic cells express up to 4-fold higher total tubulin amounts. However, the proportion of polymerized tubulin in leukemic compared to normal lymphocytes increased only slightly (up to 1.4-fold). These findings reveal a significant decrease in the polymeric to total tubulin ratio in leukemic lymphocytes, indicating important modifications in tubulin dynamics and reorganization of the microtubular structures. Our results demonstrate that leukemic cells are much more sensitive than normal lymphocytes to Et3Pb+ action. This effect may be due to the altered monomer/polymer dynamic equilibrium of tubulin shown in leukemic cells. It is, therefore, worthwhile exploring future applied uses of Et3Pb+ as a potential suppressor of leukemic cell growth.     

The effect of alpha4 beta1-integrin binding sequences of fibronectin on growth of cells from human hematopoietic progenitors

Highly regulated interactions between adhesion receptors on progenitor cells and their extracellular matrix ligands are essential for the control of hematopoiesis in bone marrow stroma. We have examined the relationship between alpha4beta1-integrin-mediated adhesion and growth of CD34(+) cells by assessing their adhesive and migratory patterns of proliferation in a mixture of hematopoietic growth factors in the presence of different recombinant fragments of the HepII/IIICS region of fibronectin. CD34(+) cells were isolated from cord blood and placed in culture wells containing serum-free medium and growth factors. Wells were precoated with either the H120 fragment of fibronectin, which contains three alpha4beta1-integrin binding sites, or the H0 fragment, which lacks the two highest affinity alpha4beta1 binding sequences. Proliferation of single cells of CD34(+)38(+)DR+ and CD34(+)38(-)DR+ phenotypes occurred in contact with the H120 substrate and was associated with migration. Larger numbers of cells were used to quantitate proliferative responses. Cells growing in wells coated with H120 formed attachments to the base of the wells throughout the culture period. Higher total cell counts were consistently found in wells coated with H120 compared with H0 and bovine serum albumin controls. The difference was first apparent at day 8 of culture and reached a maximum at days 11 through 13, when expansion with H120 was a mean of 1.8-fold higher than that seen with H0 (P相似文献   

Extensive proliferative capacity of single isolated CD34 human cord blood cells in suspension culture

Nonadherent, low-density T-lymphocyte-depleted (NALT-) CD34 cells from normal human cord blood were assessed in suspension culture for the effects of recombinant cytokines on their proliferation, differentiation, and generation of myeloid progenitor cells. In this cell population, 82% of cells expressed c-kit protein as assessed by in situ hybridization, and their cloning efficiency was 85% when cells were plated at low cell numbers with combinations of growth factors. CD34 cells were sorted as 1, 5, or 10 cell(s) per well and also at 5000 cells per dish to initiate stromal-free suspension cultures in the presence of steel factor (SLF), interleukin (IL)-1 alpha, and IL-3. Forty-eight percent of the wells started with a single CD34 cell were positive for growth after 14 days, and the wells contained greater than 5 x 10(3) cells by 21-28 days. Progenitors were assayed weekly with cultures initiated with 1 or 5000 cells. While the fold expansion of nucleated cells was greater in cultures initiated with one cell per well (> 5000 compared to 791-fold expansion for 5000 cells), the fold expansion of progenitors was greater than 5000 cells were used to initiate cultures. Under optimal conditions, there was, respectively, a 160-, 164-, and 57-fold output of high proliferative potential colony-forming cells, granulocyte-macrophage colony-forming units, and erythroid burst-forming units/granulocyte erythroid macrophage megakaryocyte colony-forming units within 1-3 weeks for cultures initiated with 5000 CD34 cells compared with respective fold increases of 29, 16, and 1, for single-initiated cultures. These results demonstrate the expansion capacity of single CD34 cord blood cells and demonstrate that factors in addition to SLF, IL-1 alpha, and IL-3 are necessary for optimal expansion of progenitors from single isolated CD34 cells.     

A compilation of mutations located in the cytochrome b subunit of the bacterial and mitochondrial bc1 complex

We used a SCID mouse xenograft model to study the in vivo growth patterns of primary leukemic cells from six patients with newly diagnosed B-cell precursor (BCP) acute lymphoblastic leukemia (ALL), including two patients with t(1;19) ALL, two patients with t(4;11) ALL, and two patients with t(9;22) ALL. Leukemic cells from these six patients caused overt leukemia in SCID mice with extensive multiple organ involvement. Leukemic BCP from SCID mice xenografted with leukemic cells from two t(9;22) ALL patients expressed very high levels of both VLA-4 and VLA-5 regardless of the tissue of origin. By comparison, in SCID mice xenografted with leukemic cells from the two patients with t(1;19) ALL and two patients with t(4;11) ALL, leukemic BCP from the bone marrow samples expressed high levels of VLA-4 as well as VLA-5, whereas the vast majority of leukemic BCP in the liver or spleen samples expressed neither of these adhesion molecules at significant levels. These results suggest that the expression of VLA-4 and VLA-5 on t(1;19) or t(4;11) leukemia cells likely determines their binding capacity to bone marrow stroma and may affect their migration to extramedullary tissues. Our findings are in accord with and extend previous studies which demonstrated that extracellular matrix and integrins influence development, compartmentalization, and migration of BCP during B-cell ontogeny. The described SCID mouse model system provides a unique opportunity to study the adhesion receptors which regulate the selective homing of human leukemic BCP to specific SCID mouse organs.     

Hopwood, affirmative action, and deaf education

Leukemic cells of B-lineage acute lymphoblastic leukemia (ALL) are regarded as the malignant counterparts of immature, physiologic B cell precursors (BCPs). To determine whether phenotypic differences exist between these corresponding cell types, we investigated samples of normal pediatric bone marrow (n=30) as well as of B-precursor ALL at diagnosis (n=53; common and pre-B subtype). Using three-color multiparameter flow cytometric analysis, we compared the leukemic populations with the physiologic BCPs of corresponding maturity with respect to the intensity with which they expressed a series of antigens. In some of these antigens, leukemia-associated aberrations were frequently observed. In particular, overexpression of CD10 was displayed by 65% of ALL samples, whereas 58% of leukemic cases aberrantly exhibited very low or no CD45RA expression. Regarding CD11a and CD44, 47% and 35% of ALL populations were aberrant as defined by either the absence or significant overexpression of the antigen. In contrast, antigen densities of CD49d, CD49e, and CD99 on leukemic cells were in the normal range of values for BCPs. Combining the patterns of frequently aberrant markers in a comprehensive analysis, we were able to identify individual phenotypic leukemic cell aberrations in up to 98% of investigated cases. CD10 and/or CD45RA were aberrant in 86% of cases overall, emphasizing the high discriminative potential of these two markers. Using comparative phenotype mapping based on quantitatively aberrant, leukemia-associated antigenic patterns, we were able to detect leukemic blasts among normal bone marrow cells at frequencies as low as 10(-5). We speculate that our approach may have a profound impact on the development of new strategies for minimal residual disease investigations in patients with BCP-ALL.     

Malignant progenitors from patients with acute myelogenous leukemia are sensitive to a diphtheria toxin-granulocyte-macrophage colony-stimulating factor fusion protein

We have previously demonstrated that human granulocyte-macrophage colony-stimulating factor (GM-CSF) fused to a truncated diphtheria toxin (DT388-GMCSF) kills acute myelogenous leukemia (AML) cell lines bearing the GM-CSF receptor. We now report that exposure of malignant cells from 50 different patients with AML for 48 hours in culture to DT388-GMCSF reduces by a median of 1.6 logs (range, 0 to 3.7 logs) the number of leukemic cells capable of forming colonies in semisolid media (leukemic colony-forming cells [CFU-L]) with a median IC50 of 3 x 10(-12) mol/L (range, 5 to >4,000 x 10(-12) mol/L). Furthermore, the cell kill is dependent on the presence of high-affinity GM-CSF receptors on leukemic blasts, because CFU-L from 27 of 28 AML samples expressing > or = 35 GM-CSF receptors per cell were inhibited by the toxin, whereas the colony growth from all 4 leukemic samples (2 AML, 1 acute lymphoblastic leukemia [ALL], and 1 prolymphocytic leukemia [PLL]) that had less than 35 receptors per cell was unaffected by the drug. Sensitivity of CFU-L to DT388-GMCSF was seen regardless of the clinical responsiveness of the patient's leukemia to standard chemotherapy agents. In contrast, clonogenic cells from normal bone marrow formed colonies at near control numbers after exposure to much higher toxin concentrations (4 x 10(-9) mol/L) than those required to kill CFU-L from most patients. Thus, leukemic progenitors isolated directly from the peripheral blood of most AML patients show the same sensitivity to DT388-GMCSF as previously demonstrated for AML cell lines. Under the same conditions of exposure, normal hematopoietic progenitors are relatively unaffected by DT388-GMCSF, suggesting its potential as a therapeutic agent in AML.     

Effect of intraventricular administration of isoprenaline on urinary function in the goat

Normal hematopoetic cells require the presence of a protein (MGI) in the appropriate conditioned medium (CM) for cell viability and growth and for differentiation to mature macrophages and granulocytes. Clones of myeloid leukemic cells have been established in culture (D+ clones) which require CM with this protein for differentiation, but not for cell viability and growth. It has been shown that these leukemic cells can be induced by CM to again require, like normal cells, the presence of CM for cell viability and growth. Induction of this requirement, which will be referred to as RVG, occurred before the D+ cells differentiated to mature granulocytes. Clones of myeloid leukemic cells (D- clones) that could not be induced to differentiate to mature cells, did not show the induction of RVG. The steroid hormones prednisolone and dexamethasone can induce some, but not all the changes associated with differentiation of D+ cells, Incubation with these steroids did not result in the induction of a requirement for these steroids for cell growth and viability. Studies with CM from different sources have shown, that all batches that induced RVG also induced differentiation of D+ cells and that both activities were inhibited after treating the CM with trypsin. It is suggested that the same protein (MGI) may be involved in both activities. Incubation of D+ cells with CM resulted in an increase in agglutinability by concanavalin A and this increase was maintained even in the absence of CM. This suggests, that the induction of RVG in D+ myeloid leukemic cells is associated with a change in the cell surface membrane.     

Characterization of human osteoblastic cells: influence of the culture conditions

Human osteoblastic cells were isolated enzymatically from adult human spongy bone and grown in MEM-Ham F12 1:1 medium supplemented with 2% Ultroser (USM). They were subcultured and examined for osteoblast features by morphological, histological, and biochemical approaches. The cells had a characteristic polyhedral morphology and produced a high level of alkaline phosphatase (ALKP). Confluent cultures were uniformly stained for ALKP and flow cytometry analysis with fluorescein diphosphate gave a single peak signal, reflecting a highly positive population, distinct from cultures of fibroblasts. The ALKP activity was stimulated by 1,25 (OH)2 vitamin D3. CD 44 was strongly expressed in these cultures, although osteoblasts are negative in vivo and osteocytes are positive. The main collagen synthesized was type I collagen and osteocalcin was produced after stimulation by vitamin D3. 10 mM betaGP induced mineralization and microprobe analysis of the crystals showed a composition close to hydroxyapatite. Changing the culture conditions to MEM-10% calf serum acted on cell behavior: it reduced the production of these biochemical markers of osteoblasts and the morphology became fibroblastlike with more rapid cell multiplication. The parameter most affected by the change in culture medium was ALKP, which was selected as the determinant criterion for defining an osteoblast culture. ALKP activity was then used to characterize a culture of cells seeded in a collagen gel.     

In vitro identification of single CD34+CD38- cells with both lymphoid and myeloid potential

Human hematopoietic stem cells are pluripotent, ie, capable of producing both lymphoid and myeloid progeny, and are therefore used for transplantation and gene therapy. An in vitro culture system was developed to study the multi-lineage developmental potential of a candidate human hematopoietic stem cell population, CD34+CD38- cells. CD34+CD38- cells cocultivated on the murine stromal line S17 generated predominantly CD19(+) B-cell progenitors. Transfer of cells from S17 stroma to myeloid-specific conditions ("switch culture") showed that a fraction of the immunophenotypically uncommitted CD19- cells generated on S17 stroma had myeloid potential (defined by expression of CD33 and generation of colony-forming unit-cells). Using the switch culture system, single CD34+CD38- cells were assessed for their lymphoid and myeloid potential. Nineteen of 50 (38%) clones generated from single CD34+CD38- cells possessed both B-lymphoid and myeloid potential. 94.7% of the CD34+CD38- cells with lympho-myeloid potential were late-proliferating (clonal appearance after 30 days), demonstrating that pluripotentiality is detected significantly more often in quiescent progenitors than in cytokine-responsive cells (P = .00002). The S17/switch culture system permits the in vitro assessment of the pluripotentiality of single human hematopoietic cells.     

Injection of apomorphine into the medial prefrontal cortex of the rat increases haloperidol-induced catalepsy

We compared telomere length in donor leukemic cells and corresponding established cell lines from three patients with chronic myeloid leukemia (CML) and three with acute lymphoblastic leukemia (ALL) to study the relation between the immortalization capacity of hematologic neoplasms and telomere length. Six of the seven established leukemia cell lines (four CML and two ALL) carried additional chromosome changes and had shorter telomere repeats than those of the donor patients' leukemic cells; the remaining ALL line showed no significant difference in telomere length between fresh leukemic cells and the corresponding cell line. Thus, most established leukemic cells lose effective telomerase activity during the process of establishment, and reduction in telomere length of established leukemic cells appeared to be associated with the presence of additional chromosome changes.     

Effects of Porphyromonas gingivalis 2561 extracts on osteogenic and osteoclastic cell function in co-culture

This study was undertaken to determine the direct effects of extracts derived from Porphyromonas gingivalis on bone formation and mineral resorption in an osteogenic/osteoclastic cell in vitro co-culture model. Osteogenic bone marrow derived stromal cells were isolated from 18-day old embryonic chickens, while osteoclastic cells were isolated from laying white Leghorn hens on calcium deficient diets. Osteoclastic cells (5 x 10(5)) were seeded onto mineral thin films and suspended above osteogenic cells (1 x 10(4)) already plated on the bottoms of tissue culture plate wells. Sonicated P. gingivalis 2561 extracts were prepared from whole bacterial cells and added in varying proportions (0 to 2 microg/ml) to the co-culture growth medium. These co-cultures, and appropriate mono-culture controls, were incubated for a further 4 days. Parameters of bone forming cell activity including alkaline phosphatase activity, calcium and inorganic phosphate accumulation were performed on the osteogenic cells. Mineral substrate resorption by osteoclastic cells was assessed morphometrically. In their respective mono-cultures, the addition of P. gingivalis sonicate to the culture medium had no effect on osteoclastic mineral resorption, but significantly inhibited osteogenesis (up to 45%; P <0.05). In co-cultures, however, the sonicate induced significant increases in mineral resorption (up to 70%; P <0.05), whereas bone forming cell activity was still inhibited, although to a significantly lesser extent than in mono-cultures (up to 25%; P <0.05). These results suggest that P. gingivalis sonicate induced up-regulation of mineral resorption may be mediated via osteogenic cells.     

Expression of VLA molecules on acute leukemia cells: relationship with disease characteristics

VLA molecules are involved in the adhesion of hematopoietic cells to the bone marrow stroma and play a role in the mediation of cellular interactions and migrations that are potentially important in the biology of acute leukemia (AL). We studied the expression of VLA-2 (CD49b), VLA-4 (CD49d), and VLA-5 (CD49e) by indirect immunofluorescence on leukemic cells from 67 patients with acute myelogenous leukemia (AML) and 40 patients with acute lymphoblastic leukemia (ALL). VLA-2, VLA-4, and VLA-5 were expressed, respectively, on 13 +/- 17%, 33 +/- 29%, and 36 +/- 30% of AML cells with 20, 54 and 61% positive cases and on 22 +/- 27%, 40 +/- 30%, and 39 +/- 29% of ALL cells with 29, 60, and 61% positive cases. Significant difference was neither noted between French-American-British (FAB) subtypes in AML or ALL nor between immunologic subtypes in ALL. There were highly significant correlations between the expression of the three beta 1-integrins tested in both AML and ALL. In AML, expression of both VLA-4 and VLA-5 was associated with that of CD14 (p = 0.003 and p = 0.01, respectively) and CD19 (p = 0.006 and p = 0.009, respectively). Expression of VLA-5 was correlated with that of CD15 (p = 0.004). Expression of VLA-4 was associated with both a high initial blast cell count (p = 0.01) and high percentage of bone marrow blast cell involvement (p = 0.003). In ALL, expression of VLA molecules was correlated neither with differentiation antigen nor with hematologic features. In AML, as in ALL, no significant correlation was noted between expression of VLA molecules and evolution of the disease.     

Primary blasts from infants with acute lymphoblastic leukemia cause overt leukemia in SCID mice

The establishment of an in vivo animal model system for infant acute lymphoblastic leukemia (ALL) would allow the testing of new agents against primary leukemic cells from infant ALL patients. We have demonstrated previously that growth of B-lineage leukemic cells in mice with severe combined immunodeficiency (SCID) was a significant prognostic factor for children with high risk ALL. We now have examined the significance of this prognostic variable for 13 infants with newly diagnosed ALL treated at participating institutions of the Children's Cancer Group (CCG). Chromosomal translocations were detected in 10/12 evaluated cases, including five with t(4;11), one each with t(7;9) and t(7;11), t(1;19), and t(9;22), and two with t(11;19). Twelve of the thirteen infants with ALL achieved remissions following induction chemotherapy. Primary leukemic cells from 8 of the 13 infants caused overt leukemia in SCID mice. Among these 8 SCID+ infants, 7 were CD10- and seven had cytogenetic or molecular evidence of an 11q23 rearrangement. Six of the 8 SCID+ infants have relapsed; only 2 remain in remission following chemotherapy or bone marrow transplant. However, among the 5 SCID- infants there were also two relapses. These data are suggestive of a poorer outcome for SCID+ infants, but larger numbers of patients must be analyzed to assess their statistical significance. In summary, we have established a SCID mouse model for human infant ALL that will be useful for 1) predicting short-term and long-term outcome of patients, 2) testing pharmacokinetics, efficacy, and toxicity of new agents, and 3) elucidating the in vivo mechanisms of chemotherapeutic drug resistance in infant ALL.     

Postoperative stability after sagittal split ramus osteotomy with condylar-positioning appliance and screw fixation: asymmetric versus symmetric cases

BACKGROUND AND OBJECTIVE: Myelodysplastic syndrome progenitor cells can be grown and expanded in long term bone marrow liquid cultures in the presence of multiple cytokines. In this study we investigated the pattern of differentiation and response to growth factors in six cases of myelodysplastic syndrome (MDS) with well-defined cytogenetic abnormalities by means of conventional cytogenetics and fluorescence in situ hybridization (FISH). METHODS: Bone marrow cells were grown in stroma-free liquid cultures in the presence of SCF, IL-3, IL-6 and GM-CSF. RESULTS: IN three cases a CFU-GM expansion comparable to normal controls was observed, together with a decrease or increase of cells with abnormal karyotype. Two cases showed no response to growth factor stimulation, morphological signs of terminal myeloid differentiation and increase (one case) or decrease (one case) in the percentage of abnormal FISH signals along the cultures. In one additional case, while CFU-C expansion was present, clearcut leukemic transformation was observed in the culture, together with a sharp decrease in the percentage of abnormal FISH signals, indicating a leukemic transformation of MDS progenitor cells with a normal karyotype. INTERPRETATION AND CONCLUSIONS: Our data indicate that FISH analysis is generally a poor indicator of clonality in MDS; nevertheless, determining the kinetics of cytogenetically abnormal clones in liquid bone marrow cultures may provide insight as to the growth abnormalities of MDS progenitor cells and may be useful prior to in vivo growth factor administration.     

Effect of a vitamin D3 analog, EB1089, on hematopoietic stem cells from normal and myeloid leukemic blasts

The seco-steroid 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) induces differentiation and inhibits clonal proliferation of HL-60 cells. We analyzed the effect of a novel vitamin D3 analog, EB1089, on normal myeloid and leukemic cells as well as CD34+ cells. EB1089 showed an extraordinary inhibition of clonal growth of HL-60 cells (ED50 = 5 x 10(-11) M) and AML blast cells (ED50 = 9 x 10(-10) M) compared to 1,25(OH)2D3 without suppression of growth of normal human bone marrow CFU-GM. The CD34+ cells from acute myeloid leukemia (AML) blasts were inhibited in a dose-dependent fashion by 1,25(OH)2D3 with an ED50 of 1.2 x 10(-9) M; and even more strikingly, 10(-10) M of EB1089 inhibited all clonal growth of human CD34+ leukemic colony-forming cells. In contrast, both EB1089 and 1,25(OH)2D3 (10(-8) M) showed little or only mild inhibition of CD34+ clongenic hematopoietic cells from normal human peripheral blood (PB); and in liquid culture, EB1089 stimulated the proliferation of normal human CD34+ cells about 2.5 times as compared to control cultures. In order to evaluate the potential use of EB1089 for purging leukemic cells from normal CD34+ progenitor cells for PB stem cell transplantation (PBSCT), normal human PB mononuclear cells (PBMNC) were contaminated with HL-60 cells, and then CD34+ cells purified and treated with EB1089. We found that CD34+ purification and EB1089 purging was able to eliminate approximately 100% of HL-60 leukemic cells with no toxicity to normal CD34+ hematopoietic progenitor cells. These data suggested that purification of CD34+ cells and ex vivo treatment with EB1089 might provide an effective therapeutic approach for PBSCT.     

Establishment of functional human pituitary tumor cell cultures

Five primary human pituitary tumor cell cultures were initiated from adenoma fragments obtained from patients with prolactin-secreting adenomas and acromegaly. Functional cell cultures were maintained and propagated in monolayer or suspension culture for up to 9 months. Optimal cell viability and growth were achieved using Ham's F10 medium enriched with 20% fetal bovine serum, although cells from a patient with acromegaly also grew in serum-free, defined, hormone-containing medium. Bromocriptine (100 ng/ml) did not alter the growth curve of replicating cells derived from a patient with acromegaly. These cells initially secreted 5.5 micrograms human growth hormone/10(6) cells, and hormone production diminished after 6 wk. Prolactin secretion by cells derived from prolactinomas (0.5 to 1.3 micrograms/10(6) cells/24 h) was stimulated by thyrotropin-releasing hormone (10 ng/ml) in two of the cultures. Both dopamine (10 ng/ml) and nickel chloride (1 mM) suppressed PRL secretion. These studies demonstrate that responsive human pituitary tumor cell cultures can be initiated and maintained.     

Growth of single HL60 cells in liquid culture: analysis of the influences of differentiative agents

Treatment of serum-free grown HL60 cells with certain combined amounts of retinoic acid (9-cis or all-trans RA) and 1 alpha 25 dihydroxyvitamin D3 (D3) results in differentiation of 71-77% of cells towards either neutrophils or monocytes. Studies of the differentiation of HL60 cells in flask cultures does not reveal: (i) the extent to which selective growth of cells might have occurred; and (ii) the overall level of cell survival. This information can be obtained by monitoring the effects of differentiative agents on individual cells. Serum-free grown HL60 cells were cultured as single cells in microtitre wells in conditioned medium obtained from exponentially growing and serum-free cultures of HL60. This resulted in a cloning efficiency of 85% and HL60 cells doubled every 24 h. During a period of exponential growth < 0.5 to 2% of the cells generated died. Single HL60 cells were treated with 9-cis and all-trans RA (5 x 10(-7) M) together with a small amount of D3 (3.9 x 10(-14) M) to promote neutrophil differentiation. D3 alone (10(-7) M) and D3 (5 x 10(-9) M) in combination with 9-cis RA (10(-8) M) were used to promote monocyte differentiation. The growth kinetics of HL60 cell cultures that were differentiating to neutrophils and to monocytes were similar. Single-cell experiments have revealed that: (i) differentiating HL60 cells undergo a variable number of divisions (two to five) prior to arresting their growth; and (ii) up to 33% of the cells that are generated (by day 5) die. Seventy to eighty per cent of the cells in each of the wells had matured. These findings have important implications in regard to whether retinoids and D3 provide signals that determine the choice of maturation pathway or that merely facilitate selective survival and/or expansion of cells that have independently determined their differentiation fates.     

Differential CD95 expression and function in T and B lineage acute lymphoblastic leukemia cells

CD95 (Fas/APO-1) is a cell surface receptor able to trigger apoptosis in a variety of cell types. The expression and function of the CD95 antigen on leukemic blasts from 42 patients with B lineage and 53 patients with T lineage acute lymphoblastic leukemia (ALL) were investigated using immunofluorescence staining and apoptosis assays. The CD95 surface antigen was expressed in most ALL cases, with the T lineage ALL usually showing a higher intensity of surface CD95 expression as compared with the B lineage ALL cells (relative fluorescence intensity, RFI: 4.8 +/- 0.47 vs 2.2 +/- 0.23, respectively, P < 0.01). Functional studies disclosed that upon oligomerization by anti-CD95 monoclonal antibodies the CD95 protein was either not able to initiate apoptosis of leukemic cells (75% of cases) or induced low rates of apoptosis (20% of cases). Only in 5% of cases did the apoptosis rate exceed the 20% level of the CD95-specific apoptosis. Most of the CD95-sensitive cases were found among T lineage ALLs (38% of T lineage vs 10% of B lineage ALLs). Overall, the extent of CD95-induced apoptosis did not correlate with the expression level of CD95. Similarly, no significant correlation between expression level and functionality of CD95 in human leukemia cell lines of B and T cell origin could be observed. Bcl-2 protein has been associated with prolonged cell survival and has been shown to block partially CD95-mediated apoptosis, but for ALL cells no correlation between bcl-2 expression and spontaneous or CD95-mediated apoptosis could be found. The results obtained in this study indicate that, despite constitutive expression of CD95, the ALL cells are mainly resistant to CD95-triggering. More detailed investigations of the molecular mechanisms involved in the intracellular apoptotic signal transduction, such as interactions of the bcl-2 and the other members of the bcl-2 family, and functionality of the interleukin-1beta converting enzyme (ICE) like-proteases, may give new insights into key events responsible for the resistance or sensitivity to the induction of apoptosis in acute leukemia.     

Ex vivo culture of CD34+/Lin-/DR- cells in stroma-derived soluble factors, interleukin-3, and macrophage inflammatory protein-1alpha maintains not only myeloid but also lymphoid progenitors in a novel switch culture assay

We have demonstrated that long-term culture initiating cells (LTC-IC) are maintained in a stroma noncontact (SNC) culture where progenitors are separated from stroma by a microporous membrane and LTC-IC can proliferate if the culture is supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein-1alpha (MIP-1alpha). We hypothesize that the same conditions, which result in LTC-IC proliferation, may also maintain lymphoid progenitors. Natural killer (NK) cells are of lymphoid lineage and a stromal-based culture can induce CD34+/Lin-/DR- cells to differentiate along the NK cell lineage. We developed a three-step switch culture assay that was required to demonstrate the persistence of NK progenitors in CD34+/Lin-/DR- cells assayed in SNC cultures supplemented with IL-3 and MIP-1alpha. When CD34+/Lin-/DR- progeny from the SNC culture were plated sequentially into "NK cell progenitor switch" conditions (contact with stromal ligands, hydrocortisone-containing long-term culture medium, IL-2, IL-7, and stem cell factor [SCF]) followed by "NK cell differentiation" conditions (contact with stromal ligands, human serum, no hydrocortisone, and IL-2), significant numbers of CD56+/CD3- NK resulted, which exhibited cytotoxic activity against K562 targets. All steps are required because a switch from SNC cultures with IL-3 and MIP-1alpha directly to "NK cell differentiation" conditions failed to yield NK cells suggesting that critical step(s) in lymphoid commitment were missing. Additional experiments showed that CD34+/CD33- cells present after SNC cultures with IL-3 and MIP-1alpha, which contained up to 30% LTC-IC, are capable of NK outgrowth using the three-step switch culture. Limiting dilution analysis from these experiments showed a cloning frequency within the cultured CD34+/CD33- population similar to fresh sorted CD34+/Lin-/DR- cells. However, after addition of FLT-3 ligand, the frequency of primitive progenitors able to develop along the NK lineage increased 10-fold. In conclusion, culture of primitive adult marrow progenitors ex vivo in stroma-derived soluble factors, MIP-1alpha, and IL-3 maintains both very primitive myeloid (LTC-IC) and lymphoid (NK) progenitors and suggests that these conditions may support expansion of human hematopoietic stem cells. Addition of FLT-3 ligand to IL-2, IL-7 SCF, and stromal factors are important in early stages of NK development.