Growth Of Myeloid Progenitor Cells Research Articles

Deletion of Bcl11a Gene Reveals Roles in Lymphoid, Myeloid, and Leukemic Cell Development.

B-cell lymphoma/leukemia 11a (Bcl11a) gene, encodes a zinc finger protein that is expressed in normal B- and T-lymphocytes, monocytes, megakaryocytes, and physically interacts with the human B-cell proto-oncogene, BCL6. Bcl11a is activated by fusion of IG the locus in human B-cell lymphomas carrying the (2;14)(p13:q32.3) translocation. To determine the function of Bcl11a in normal hematopoiesis and proliferation of leukemic cells, we generated a Bcl11a knockout mouse model. This showed that Bcl11a is essential for postnatal development and normal lymphopoiesis. While Bcl11a+/− mice are fertile, Bcl11a−/− die a few hours after birth from unknown causes. To evaluate the role of Bcl11a in hematopoiesis, fetal liver (FL) cells were analyzed by FACS for the expression of lineage specific cell surface markers. B-cell maturation was blocked prior to the development of pro-B cells (B220+/CD43+), while myeloid and erythroid lineages were normal. The Bcl11a−/− FL also showed defects in T-cell maturation (reduced CD4+ CD8− and increased CD4− CD8+ fetal thymocytes). Lethally irradiated mice transplanted with Bcl11a−/− FL cells showed normal myeloid and erythroid development, while B- and T-cell development was blocked. Thus, defects in lymphoid development were intrinsic to the hematopoietic stem cells (HSC). We established that this defect in lymphopoiesis was not due to homing to the thymus, as shown by failure of T-cell repopulation when Bcl11a−/− FL cells were injected into the thymus. Greater than 80% of the mice transplanted with Bcl11a−/− FL cells died between 15 and 27 weeks, while 100% mice transplanted with Bcl11a+/− survived. Histopathology of moribund mice showed enlarged thymus with lymphoblasts infiltrating the bone marrow, liver, lung, heart, and kidney. Furthermore, analysis by real time-PCR of thymocytes from the sick mice showed increased expression of Notch1, a negative regulator of lymphoid development. Leukemic cells were tumorogenic when injected into SCID-NOD mice, and were immortalized by co-culturing on OP9 stromal cells. Immortalized leukemic cell lines showed high expression of T-cell markers CD3e, CD4, CD8, and B-cell marker CD43. Finally, myeloid progenitor cell growth (CFU-c, 3H-Thymidine incorporation, and viable cell growth) was inhibited by over-expression of Bcl11a in bone marrow cells from 5-FU treated mice. Thus, these data show that Bcl11a is required for B and T cell differentiation and may also function as a T-cell tumor suppressor gene. Furthermore, these results are the first indication of a possible suppressive role for Bcl11a during normal myeloid progenitor development and differentiation. This project has been funded in part from NCI, NIH Contract # NO1-CO-56000, with SAIC-Fredrick, Inc.

Inverse relationship between myeloid maturation and leukotriene C4 synthase expression in normal and leukemic myelopoiesis-consistent overexpression of the enzyme in myeloid cells from patients with chronic myeloid leukemia.

Leukotriene (LT) C(4) synthase (LTC(4)S) is the key enzyme in the biosynthesis of LTC(4), which has been reported to stimulate the growth of human myeloid progenitor cells and is specifically overproduced in chronic myeloid leukemia (CML). The aim of this study was to clarify the expression of LTC(4)S during normal and leukemic myelopoiesis and to investigate the correlation between abnormal LTC(4)S expression in CML myeloid cells and the activity of the disease-specific tyrosine kinase p210 BCR-ABL. Immature and mature myeloid cell subpopulations were isolated with magnetic cell sorting from healthy volunteer bone marrow (n = 11) and CML patient peripheral blood (n = 8), respectively. The cells were subjected to analysis of LTC(4)S protein expression and activity. Expression of LTC(4)S was investigated in CD16(+) neutrophils from CML patients before and after 1 month of medication with imatinib mesylate (STI571), which is a specific inhibitor of p210 BCR-ABL. Among normal cells, the highest enzyme activity was observed in the most immature, CD34(+) progenitor cell-enriched and CD15(+) myelocyte-enriched fractions. Subsequently, LTC(4)S activity decreased with increasing maturity, with only negligible amounts of LTC(4) produced in CD16(+) neutrophils. LTC(4)S was expressed at the protein level in the immature myeloid cell fractions but not in CD16(+) cells. In CML cells, LTC(4)S activity and expression were consistently elevated. Thus, the CML CD34(+) and CD15(+) cell fractions, as well as the CD11b(+) myelocyte/metamyelocyte-enriched fractions, produced 6 to 10 times as much LTC(4) as the corresponding normal cells. Again, enzyme expression was highest in the most immature cells, although evident LTC(4)S expression and activity remained in CML CD16(+) neutrophils. Interestingly, treatment of five CML patients with imatinib mesylate down-regulated the abnormal neutrophil LTC(4)S expression and activity. Expression of LTC(4)S in immature myelopoid cells is in line with a role for this enzyme in myelopoiesis. In addition, consistent overexpression of LTC(4)S in CML and the correlation to p210 BCR-ABL activity suggests that LTC(4)S may be involved in leukemic pathogenesis.

Immune responses in 4-1BB (CD137)-deficient mice.

The 4-1BB (a TNFR superfamily member) is an inducible costimulatory molecule that can exert regulatory effects on T cells independently of CD28 stimulation. The in vitro expression of 4-1BB (CD137) is induced following activation of T cells with various stimuli, including anti-TCR mAbs, lectins, and a combination of PMA and ionomycin. To delineate further the physiological role of 4-1BB in immunity, mice deficient in this receptor were generated. These mutant mice developed normally, and were viable and fertile. Humoral responses to vesicular stomatitis virus were comparable with those seen in wild-type mice, whereas the IgG2a and IgG3 isotype responses to keyhole limpet hemocyanin were somewhat reduced in the mutant mice. The 4-1BB-deficient mice demonstrated enhanced T cell proliferation in response to mitogens or anti-CD3 even in the environment of reduced ability to secrete growth-supporting cytokines (IL-2 and IL-4). Although T cells from 4-1BB-deficient mice showed enhanced proliferation, the T cell immune responses of these animals, such as cytokine production and CTL activity, were diminished. In addition, 4-1BB deletion appears to play a role in the regulation of myeloid progenitor cell growth, leading to an increase in these precursor cells in peripheral blood, bone marrow, and spleen.

Investigating the platelet-sparing mechanism of paclitaxel/carboplatin combination chemotherapy

AbstractPaclitaxel and carboplatin chemotherapy is reported to be a platelet-sparing drug combination. This study investigated potential mechanisms for this observation by studying the effects of paclitaxel and carboplatin on (1) normal donor and chemotherapy patient-derived erythroid (burst-forming units-erythroid [BFU-E]), myeloid (colony-forming units-granulocyte/macrophage [CFU-GM]), and megakaryocyte (CFU-Meg) progenitor cell growth; (2) P-glycoprotein (P-gp) protein and glutathione S-transferase (GST) messenger RNA (mRNA) expression; (3) serum thrombopoietin (Tpo), stem cell factor (SCF), interleukin-6 (IL-6), IL-11, IL-1β, IL-8, and tumor necrosis factor-α levels in patients treated with paclitaxel and carboplatin; and (4) stromal cell production of Tpo and SCF after paclitaxel and carboplatin exposure. CFU-Meg were more resistant to paclitaxel alone, or in combination with carboplatin, than CFU-GM and BFU-E. Although all progenitors expressed P-gp protein and GST mRNA, verapamil treatment significantly, and selectively, increased the toxicity of paclitaxel and carboplatin to CFU-Meg, suggesting an important role for P-gp in megakaryocyte drug resistance. Compared to normal controls, serum Tpo levels in patients receiving paclitaxel and carboplatin were significantly elevated 5 hours after infusion and remained elevated at day 7 (287% ± 63% increase,P < .001). Marrow stroma was shown to be the likely source of this Tpo. It is concluded here that P-gp–mediated efflux of paclitaxel, and perhaps GST-mediated detoxification of carboplatin, results in relative sparing of CFU-Meg, which may then respond to locally high levels of stromal cell–derived Tpo. The confluence of these events might lead to the platelet-sparing phenomenon observed in patients treated with paclitaxel and carboplatin chemotherapy.

Investigation of the platelet sparing mechanism of paclitaxel and carboplatin chemotherapy

Abstract Paclitaxel (P) and carboplatin (C) chemotherapy is reported to be a platelet-sparing drug combination. We investigated possible mechanisms for this by studying P & C's effects on: 1] normal human, and P- and C- treated patient-derived erythroid (BFU-E), myeloid (CFU-GM), and megakaryocyte (CFU-Meg) progenitor cell growth; 2] P-glycoprotein (P-gp) protein and glutathione S-transferase (GST) mRNA expression; 3] P & C patient serum levels of TPO, SCF, IL-6, IL-11, IL-1b, IL-8 and TNFa; and 4] marrow stromal cell production of TPO and SCF after P & C exposure. We found that CFU-Meg were more resistant to P alone, or in combination with C, than CFU-GM and BFU-E. We also found that CFU-Meg express P-gp protein and GST mRNA. That the former might play a role in drug resistance is suggested by the finding that verapamil treatment significantly increased P & C's toxicity to CFU-Meg (colony formation decreased ∼70%, p

Regulation of Jak2 tyrosine kinase by protein kinase C during macrophage differentiation of IL-3–dependent myeloid progenitor cells

Differentiation of macrophages from myeloid progenitor cells depends on a discrete balance between cell growth, survival, and differentiation signals. Interleukin-3 (IL-3) supports the growth and survival of myeloid progenitor cells through the activation of Jak2 tyrosine kinase, and macrophage differentiation has been shown to be regulated by protein kinase C (PKC). During terminal differentiation of macrophages, the cells lose their mitogenic response to IL-3 and undergo growth arrest, but the underlying signaling mechanisms have remained elusive. Here we show that in IL-3-dependent 32D myeloid progenitor cells, the differentiation-inducing PKC isoforms PKC-alpha and PKC-delta specifically caused rapid inhibition of IL-3-induced tyrosine phosphorylation. The target for this inhibition was Jak2, and the activation of PKC by 12-O-tetradecanoyl-phorbol-13-acetate treatment also abrogated IL-3-induced tyrosine phosphorylation of Jak2 in Ba/F3 cells. The mechanism of this regulation was investigated in 32D and COS7 cells, and the inhibition of Jak2 required catalytic activity of PKC-delta and involved the phosphorylation of Jak2 on serine and threonine residues by the associated PKC-delta. Furthermore, PKC-delta inhibited the in vitro catalytic activity of Jak2, indicating that Jak2 was a direct target for PKC-delta. In 32D cells, the inhibition of Jak2 either by PKC-delta, tyrosine kinase inhibitor AG490, or IL-3 deprivation caused a similar growth arrest. Reversal of PKC-delta-mediated inhibition by the overexpression of Jak2 promoted apoptosis in differentiating 32D cells. These results demonstrate a PKC-mediated negative regulatory mechanism of cytokine signaling and Jak2, and they suggest that it serves to integrate growth-promoting and differentiation signals during macrophage differentiation. (Blood. 2000;95:1626-1632)

Granulocyte Colony-Stimulating Factor Deficiency in a Rottweiler with Chronic Idiopathic Neutropenia

Journal of Veterinary Internal MedicineVolume 13, Issue 1 p. 72-75 Open Access Granulocyte Colony-Stimulating Factor Deficiency in a Rottweiler with Chronic Idiopathic Neutropenia Anne Lanevschi, Corresponding Author Anne Lanevschi Department of Pathology and Microbiology (Lanevschi), and the Department of Clinical Sciences (Daminet), Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Quebec, Canada 3200 Sicotte, CP 5000, Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, QC J2S 7C6, Canada; e-mail: [email protected]Search for more papers by this authorSylvie Daminet, Sylvie Daminet Department of Pathology and Microbiology (Lanevschi), and the Department of Clinical Sciences (Daminet), Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Quebec, CanadaSearch for more papers by this authorGlenn P. Niemeyer, Glenn P. Niemeyer Scott Ritchey Research Center (Niemeyer, Lothrop), and the Department of Small Animal Surgery and Medicine (Lothrop), College of Veterinary Medicine, Auburn University, Auburn, AL.Search for more papers by this authorClinton D. Lothrop Jr., Clinton D. Lothrop Jr. Scott Ritchey Research Center (Niemeyer, Lothrop), and the Department of Small Animal Surgery and Medicine (Lothrop), College of Veterinary Medicine, Auburn University, Auburn, AL.Search for more papers by this author Anne Lanevschi, Corresponding Author Anne Lanevschi Department of Pathology and Microbiology (Lanevschi), and the Department of Clinical Sciences (Daminet), Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Quebec, Canada 3200 Sicotte, CP 5000, Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, QC J2S 7C6, Canada; e-mail: [email protected]Search for more papers by this authorSylvie Daminet, Sylvie Daminet Department of Pathology and Microbiology (Lanevschi), and the Department of Clinical Sciences (Daminet), Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Quebec, CanadaSearch for more papers by this authorGlenn P. Niemeyer, Glenn P. Niemeyer Scott Ritchey Research Center (Niemeyer, Lothrop), and the Department of Small Animal Surgery and Medicine (Lothrop), College of Veterinary Medicine, Auburn University, Auburn, AL.Search for more papers by this authorClinton D. Lothrop Jr., Clinton D. Lothrop Jr. Scott Ritchey Research Center (Niemeyer, Lothrop), and the Department of Small Animal Surgery and Medicine (Lothrop), College of Veterinary Medicine, Auburn University, Auburn, AL.Search for more papers by this author First published: 28 June 2008 https://doi.org/10.1111/j.1939-1676.1999.tb02168.xCitations: 6AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 Mishu L., Callahan G., Alleban Z., et al. Effects of recombinant canine granulocyte colony-stimulating factor on white blood cell production in clinically normal and neutropenic dogs. J Am Vet Med Assoc 1992; 200: 1957– 1964. 2 Warren DJ, Maniatis M., Moore MAS, et al. Cyclic production of granulocyte colony-stimulating factor in cyclic hematopoietic dogs. In: DW Golde, SC Clark, eds. Hematopoiesis. UCLA Symposia on Molecular and Cellular Biology. New York , NY : Wiley-Liss; 1990; 120: 237. 3 Bravo L., Legendre AM, Hahn KA, et al. Serum granulocyte colony-stimulating factor (G-CSF) and interleukin-1 (IL-1) concentrations after chemotherapy-induced neutropenia in normal and tumor-bearing dogs. Exp Hematol 1996; 24: 11– 17. 4 Kyle RA. Natural history of chronic idiopathic neutropenia. N Engl J Med 1980; 302: 908– 909. 5 Bernini JC. Diagnosis and management of chronic neutropenia during childhood. Review. Pediatr Clin North Am 1996; 43: 773– 792. 6 Morikawa K., Yamamori S., Oiwa K., et al. Impaired G-CSF production at post-transcriptional level in a patient with chronic idiopathic neutropenia. Br J Haematol 1993; 85: 200– 202. 7 Lieschke GJ, Grail D., Hodgson G., et al. Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization. Blood 1994; 84: 1737– 1746. 8 Morikawa K., Oiwa K., Oseko F. et al. Distinct effect of G-CSF on the growth and differentiation of myeloid progenitor cells from chronic idiopathic neutropenia. Jpn J Med (Intern Med) 1995; 34: 384– 387. 9 Shitara T., Ijima H., Yugami S., et al. Increased cytokine levels and abnormal response of myeloid progenitor cells to G-CSF in a case of severe congenital neutropenia. Am J Pediatr Hematol/Oncol 1994; 16: 167– 172. 10 Boxer LA, Hutchison R., Emerson S. Recombinant human granulocyte colony-stimulating factor in the treatment of patients with neu-tropenia. Review. Clin Immunol Immunopathol 1992; 62: 39– 46. 11 Dale DC, Hammond WE Cyclic neutropenia: A clinical review. Blood Rev 1988; 2: 178– 185. 12 Kalra R., Dale D., Freedman M., et al. Monosomy 7 and activating RAS mutations accompany malignant transformation in patients with congenital neutropenia. Blood 1995; 86: 4579– 4586. 13 Lothrop CD Jr, Warren DJ, Souza LM, et al. Correction of canine cyclic hematopoiesis with recombinant human granulocyte colony-stimulating factor. Blood 1988; 72: 1324– 1328. Citing Literature Volume13, Issue1January 1999Pages 72-75 ReferencesRelatedInformation

Expansion of Philadelphia Chromosome–Negative CD3+CD56+ Cytotoxic Cells From Chronic Myeloid Leukemia Patients: In Vitro and In Vivo Efficacy in Severe Combined Immunodeficiency Disease Mice

AbstractWe have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMNCs) by the timed addition of interferon-γ (IFN-γ), interleukin-2 (IL-2), and the monoclonal antibody (MoAb) OKT3. These cells, termed cytokine-induced killer (CIK) cells, are composed primarily of T cells, and the population of cells with the greatest cytotoxic activity is an otherwise rare population of CD3+CD56+ cells that expand dramatically under these culture conditions. CIK cells were expanded from PBMNCs from 13 patients with chronic myeloid leukemia (CML). These cultures contained a variable number of T cells at the start of the culture (median 44%, range 1% to 64%), yet after 21 to 28 days of culture, virtually all of the cells were CD3+ T cells (median 97%, range 90% to 99%). The CD3+CD56+subset of cells expanded significantly (median 25-fold, range 2.2- to 525-fold). CIK cells from all patients showed cytotoxicity against the tumor cell lines OCI-LY8 and K562. In four patients the expanded CIK cells suppressed colony growth of autologous CML blast cells and myeloid progenitor cells. Allogeneic CIK cells from normal donors also suppressed CML colony growth but did not inhibit growth of normal hematopoietic colonies. Twelve of the 13 cultures were exclusively composed of Philadelphia (Ph)-negative cells and one culture had 1 out of 20 Ph-positive metaphases after 4 weeks in culture. Intracellular cytokine production was assayed by fluorescence-activated cell sorter (FACS), and the expanded T-cell cultures produced IL-2, IFN-γ, and tumor necrosis factor-α (TNF-α), but not IL-4. Both the CD4+ and CD8+ subsets secreted this cytokine profile. To test the in vivo activity of the expanded CIK cells, CML was engrafted into severe combined immunodeficiency disease (SCID) mice using matrigel. After 4 weeks, 4 × 107autologous CIK cells were injected intravenously by tail vein injection into groups of mice, and the animals were sacrificed after a total of 18 weeks. Bcr-abl was detected in the bone marrow or spleen of 5 out of 6 control mice and only 2 out of 13 mice who received the autologous CIK cells (P = .02). In an additional series of animals, the mice did not engraft with CML but instead developed large human Epstein-Barr virus–associated lymphomas by 12 weeks. The mice who received autologous CIK cells at 4 weeks had either no tumor (5) or small tumors (5), whereas all 10 mice that received CIK cells at week 8 developed lymphomas; however, these were not as large as in the 10 control mice who did not receive CIK cells (P = .03). This study shows that CIK cells, which are Ph chromosome–negative, can be expanded from patients with CML and have potent in vitro and in vivo efficacy against autologous tumor cells.© 1998 by The American Society of Hematology.

Kinetics of hematopoiesis in Dexter-type long-term cultures established from human umbilical cord blood cells.

In the present study, we have established Dexter-type long-term cultures (D-LTC) from human umbilical cord blood (UCB) and followed the kinetics of different hematopoietic progenitor cells (HPCs)--including multipotent (colony forming unit [CFU]-Mixture), erythroid (CFU-erythroid, BFU-E), and myeloid (CFU-granulocyte, CFU-macrophage, CFU-granulocyte/marcophage) progenitors as well as of morphologically recognizable erythroid, myeloid and lymphoid cells--during a nine-week culture period. D-LTC were also established from adult bone marrow (BM) as controls. On day 0, both UCB and BM showed similar total numbers of HPCs (about 310/10(5) cells), however, UCB showed a higher proportion of primitive HPCs (i.e., CFU-Mixture, CFU-granulocyte/macrophage and BFU-E). A poor adherent cell layer, consisting almost exclusively of macrophages, was developed in UCB D-LTC and this correlated with a continuous decline in HPC numbers throughout the culture period. In contrast, adherent cell numbers in BM D-LTC, including fibroblasts and macrophages, were two- to fourfold higher than in UCB cultures, and the numbers of HPCs were also significantly higher, reaching plateau levels between weeks 6 and 9. In both types of cultures, erythroid and multipotent progenitors declined relatively fast, reaching undetectable levels after five weeks of culture. Myeloid progenitors, on the other hand, were sustained longer (always at higher levels in BM cultures) and were still detected by week 9. Among myeloid progenitors, a shift towards the predominance of macrophage HPCs was observed, both in UCB and BM D-LTC, and this correlated with an increase in the proportion of mature monocytes and macrophages. Taken together, our results indicate that myeloid progenitor cell growth is deficient in UCB D-LTC and suggest that this is due to the impaired development of an adherent cell layer, unable to provide the factors and conditions required for their growth. Interestingly, throughout the culture period the total numbers of multipotent and erythroid progenitors were similar both in UCB and BM cultures regardless of the number and types of adherent cells present; this suggests that the stroma developed in D-LTC is not sufficient for the proliferation of these progenitor cells.

GCSF augments post-progenitor proliferation in serum-free cultures of myelodysplastic marrow while ATRA enhances maturation

All-trans retinoic acid (ATRA) and granulocyte colony stimulating factor (GCSF) are potential inducers of myeloid progenitor cell growth and neutrophil differentiation in myelodysplasia (MDS). We have compared the effects of ATRA and GCSF on the colony growth of 10 MDS marrows, in semi-solid and liquid serum-free mononuclear cell (MNC) cultures, supplemented with a mixture of stem cell factor (SCF), interleukin 3 (IL-3) and granulocyte-monocyte colony stimulating factor (GmCSF) (SIGm mix), which is fully-supportive for myeloid and erythroid (with erythropoietin (EPO)) colony formation in normal marrow. Only 1 10 MDS patients produced normal granulocyte-macrophage colony-forming cell (GmCFC) numbers, under SIGm conditions and erythroid colonies (ECFC) were subnormal in all patients. ATRA (10 −7 M) increased GmCFC numbers ( P = 0.05) in semi-solid cultures of normal, but not MDS marrow MNC and decreased erythroid colonies in cultures of marrow from either source ( P = 0.008 and P = 0.0001 for normal and MDS, respectively). ATRA enhanced neutrophilic maturation in liquid cultures of both normal and myelodysplastic CD34 + ve cells, as detected by conventional morphology and acquisition of CD15. In contrast to ATRA, GCSF increased Gm colony size but not numbers in semi-solid cultures of normal marrow MNC, which suggests the cytokine augments post-progenitor amplification. This would explain why GCSF increased cell yields in liquid cultures of normal and MDS MNC while GmCFC accumulation remained unchanged. GCSF, though, increased Gm colony numbers in semi-solid cultures of MDS marrow MNC ( P = 0.014) so that 4 10 patients now grew colonies within the normal range. This was again probably due to increasing clone size, so that some clusters, the numbers of which may be elevated in MDS, were now scored as colonies. Overall, these data indicate that ATRA can enhance the maturation of the progeny of MDS GmCFC whilst GCSF can augment their amplification.

Differentiation of Myeloid Cells and 1, 25-Dihydroxyvitamin D3

It is well established that1, 25(OH)2D3 induces monocyte/macrophage (Mo/Mø) colonies when added to culture of granulocyte/macrophage progenitors. Recently, we demonstrated that one of the target cells of1, 25(OH)2D3 in Mo/Mø differentiation is the neutrophilic promyelocyte that is believed to belong to the neutrophilic lineage. This fact overthrows the established theory that normal hematopoietic precursors are committed to respective cell lineages and do not deviate from their own lineage. The lineage switching from the promyelocyte to Mo/Mø was suggested to be operating in vivo because1, 25(OH)2D3 is a physiological substance produced by Mø. More recently, we have shown that transient exposure (24 h) of promyelocytes to1, 25(OH)2D3 causes Mo/Mø differentiation. This strategy could be useful for examining the effects of1, 25(OH)*D3 on the growth and differentiation of normal myeloblasts and myeloid progenitor cells. Recent advances in molecular biology have enabled investigators to identify a number of genes involved in Mo/Mø differentiation induced by1, 25(OH)2D3. Some of these may be the determinant genes for Mo/Mø differentiation; however, further studies are required to determine the underlying mechanisms of Mo/Mø differentiation.

Regulatory roles of the ligand for Flk2/Flt3 tyrosine kinase receptor on human hematopoiesis.

The biological activities of the ligand for the Flk2/Flt3 receptor tyrosine kinase (FL) on human hematopoietic cells are reviewed. In in vitro studies, FL shows relatively few effects by itself on the proliferation and differentiation of hematopoietic cells, but exhibits a potent costimulatory activity in enhancing the proliferation of progenitor cells of multiple lineages. FL promotes the growth of clonogenic myeloid progenitor cells in the presence of other cytokines known to be active on myeloid progenitors, including GM-CSF, interleukin 3 (IL-3), kit ligand (KL), M-CSF and G-CSF. In addition, FL synergizes with IL-7 in inducing the proliferation of pro-B cells, whereas FL has little effect on the growth of clonogenic erythroid progenitors. Furthermore, FL induces the in vitro expansion of the high proliferative potential colony-forming cells (HPP-CFC) and stimulates the proliferation of long-term culture-initiating cells (LTC-IC), suggesting an activity on the proliferation of putative stem cells. Thus, FL plays important roles in regulating the proliferation of hematopoietic progenitor cells and, therefore, may have therapeutic applications.

2′,2′‐Difluorodeoxycytidine (Gemcitabine) Induces Apoptosis in Myeloma Cell Lines Resistant to Steroids and 2‐Chlorodeoxyadenosine (2‐CdA)

The paucity of effective cytotoxic agents for the treatment of steroid resistant multiple myeloma explains the ongoing search for alternative substances for chemotherapy of this disease. In the present study, the purine antagonist 2-chlorodeoxyadenosine (2-CdA, cladribine) and the pyrimidine antagonist 2',2'-difluorodeoxycytidine (gemcitabine) were tested on four myeloma cell lines (i.e., U 266, OPM 2, RPMI 8226, IM 9), one plasma cell leukemia cell line (HS Sultan) and a myeloid control cell line (HL 60), all of which are resistant to 10-6 M dexamethasone. Gemcitabine has been found to be promising in the chemotherapy of other tumors with low proliferative activity, but its effectiveness against myeloma cells has not been analyzed so far. In our tests, gemcitabine induced a significant degree of apoptosis in all cell lines investigated. After incubation for 48 h with 10 microM gemcitabine, the median numbers of apoptotic cells were in the range of 45% in the OPM 2 and 79% in the U 266 cell line. All of the investigated cell lines were responsive to concentrations of 10 microM gemcitabine even after an exposure of only 30 min, three of them (U 266, HS Sultan, IM 9) also responded to a concentration of 10 nM. Higher concentrations and longer exposure times were necessary to suppress the growth of normal hematopoietic bone marrow progenitor cells. In contrast to gemcitabine, standard concentrations of 2-CdA (i.e., 30 and 300 nM) failed to induce a significant degree of apoptosis in the cell lines investigated but inhibited the growth of myeloid progenitor cells. The results suggest that gemcitabine induces apoptosis in myeloma and plasma cell leukemia lines resistant to steroids and 2-CdA. The fact that tumor cell apoptosis was achieved at concentrations clinically achievable and tolerable, which at the same time do not inhibit the growth of normal CFU-GM progenitor cells, favors the initiation of phase I trials with this drug for the treatment of multiple myeloma.

The RAR-RXR as well as the RXR-RXR pathway is involved in signaling growth inhibition of human CD34+ erythroid progenitor cells

Previous studies have shown that retinoic acid (RA), similar to tumor necrosis factor-alpha (TNF-alpha), can act as a bifunctional regulator of the growth of bone marrow progenitors, in that it can stimulate granulocyte-macrophage colony-stimulating factor (GM-CSF)- or interleukin-3 (IL-3)-induced GM colony formation, but potently inhibit G-CSF-induced growth. The present study, using highly enriched human CD34+ as well as Lin- murine bone marrow progenitor cells, demonstrates a potent inhibitory effect of 9-cis-RA on burst-forming unit-erythroid (BFU-E) colony formation regardless of the cytokine stimulating growth. Specifically, 9-cis-RA potently inhibited the growth of BFU-E response to erythropoietin (Epo) (100%), stem cell factor (SCF) + Epo (92%), IL-3 + Epo (97%), IL-4 + Epo (88%), and IL-9 + Epo (100%). Erythroid colony growth was also inhibited when CD34+ progenitors were seeded at one cell per well, suggesting a direct action of RA. Using synthetic ligands to retinoic acid receptors (RARs) and retinoid X receptors (RXRs) that selectively bind and activate RAR-RXR or RXR-RXR dimers, respectively, we dissected the involvement of the two retinoid response pathways in the regulation of normal myeloid and erythroid progenitor cell growth. Transactivation studies showed that both the RAR (Ro 13-7410) and RXR (Ro 25-6603 and Ro 25-7386) ligands were highly selective at 100 nmol/L. At this concentration, Ro 13-7410 potently inhibited G-CSF-stimulated myeloid as well as SCF + Epo-induced erythroid colony growth. At the same concentration, Ro 25-6603 and Ro 25-7386 had little or no effect on G-CSF-induced colony formation, whereas they inhibited 75% and 53%, respectively, of SCF + Epo-stimulated BFU-E colony growth. Thus, the RAR-RXR response pathway can signal growth inhibition of normal bone marrow myeloid and erythroid progenitor cells. In addition, we demonstrate a unique involvement of the RXR-RXR pathway in mediating growth inhibition of erythroid but not myeloid progenitor cells.

The RAR-RXR as Well as the RXR-RXR Pathway Is Involved in Signaling Growth Inhibition of Human CD34+ Erythroid Progenitor Cells

Abstract Previous studies have shown that retinoic acid (RA), similar to tumor necrosis factor-alpha (TNF-alpha), can act as a bifunctional regulator of the growth of bone marrow progenitors, in that it can stimulate granulocyte-macrophage colony-stimulating factor (GM-CSF)- or interleukin-3 (IL-3)-induced GM colony formation, but potently inhibit G-CSF-induced growth. The present study, using highly enriched human CD34+ as well as Lin- murine bone marrow progenitor cells, demonstrates a potent inhibitory effect of 9-cis-RA on burst-forming unit-erythroid (BFU-E) colony formation regardless of the cytokine stimulating growth. Specifically, 9-cis-RA potently inhibited the growth of BFU-E response to erythropoietin (Epo) (100%), stem cell factor (SCF) + Epo (92%), IL- 3 + Epo (97%), IL-4 + Epo (88%), and IL-9 + Epo (100%). Erythroid colony growth was also inhibited when CD34+ progenitors were seeded at one cell per well, suggesting a direct action of RA. Using synthetic ligands to retinoic acid receptors (RARs) and retinoid X receptors (RXRs) that selectively bind and activate RAR-RXR or RXR-RXR dimers, respectively, we dissected the involvement of the two retinoid response pathways in the regulation of normal myeloid and erythroid progenitor cell growth. Transactivation studies showed that both the RAR (Ro 13– 7410) and RXR (Ro 25–6603 and Ro 25–7386) ligands were highly selective at 100 nmol/L. At this concentration, Ro 13–7410 potently inhibited G- CSF-stimulated myeloid as well as SCF + Epo-induced erythroid colony growth. At the same concentration, Ro 25–6603 and Ro 25–7386 had little or no effect on G-CSF-induced colony formation, whereas they inhibited 75% and 53%, respectively, of SCF + Epo-stimulated BFU-E colony growth. Thus, the RAR-RXR response pathway can signal growth inhibition of normal bone marrow myeloid and erythroid progenitor cells. In addition, we demonstrate a unique involvement of the RXR-RXR pathway in mediating growth inhibition of erythroid but not myeloid progenitor cells.

A Functional Analysis of Protooncogene Vav's Role in Adult Human Hematopoiesis

The Vav protooncogene is expressed almost exclusively in hematopoietic cells, but its role in regulating adult human hematopoietic cell development remains uncertain. To analyze Vav function in adult blood cell formation, we used antisense (AS) oligodeoxynucleotides (ODN) to disrupt its expression in normal and malignant human hematopoietic cells. Bone marrow or peripheral blood mononuclear cells (MNC) were obtained from consenting normal donors and patients with acute or chronic myelogenous leukemia (AML and CML, respectively) and polycythemia vera (PV). Adherent and T-cell-depleted (A-T-) MNC or CD34+ MNC were exposed to unmodified sense, antisense, or scrambled sequence ODN corresponding to codons 2-7 of Vav's mRNA sequence. Cells were then assayed for Vav mRNA expression by reverse transcription-polymerase chain reaction and Vav protein expression by Western binding. After showing that Vav-targeted AS ODN could specifically diminish Vav mRNA and protein expression, we assessed the ability of Vav-deficient cells to form myeloid and erythroid colonies in methyl-cellulose cultures. When normal CD34+ MNC were exposed to Vav AS ODN, no effect on colony-forming unit-granulocyte-macrophage (CFU-GM) or CFU-megakaryocyte colony formation was observed. In contrast erythroid colony growth was inhibited by a mean +/- SD of 62% +/- 16%. In patients with hematopoietic malignancies. Vav-targeted AS ODN inhibited CFU-GM colony formation in a sequence-specific and dose-dependent manner in 1 of 3 AML, 13 of 17 CML, and 2 of 2 PV patients. At the highest concentration used, the Vav AS ODN inhibited CFU-GM colony formation from 66% to 81% when compared with control cell colony growth. Burst-forming unit-erythroid (BFU-E) colony-formation was also assessed in 7 PV patients. The Vav-targeted AS ODN inhibited BFU-E colony formation in all by a mean +/- SD of 81% +/- 4%. These findings suggest that Vav function may not be easily complemented in a significant subset of normal adult erythroid progenitor cells and may also be necessary for myeloid progenitor cell growth in a variety of hematopoietic malignancies.

Growth inhibition of follicular small-cleaved-cell lymphoma cells in short-term culture by interleukin-3

The role of interleukin-3 (IL-3) in stimulating the growth of early myeloid progenitor cells is very well established. Therefore, IL-3 has been incorporated into many post-bone-marrow transplantation and intensive chemotherapy programs for the treatment of solid tumors and hematologic malignancies, including lymphomas. However, the effect of IL-3 on normal and malignant lymphocytes has not been well studied. The purpose of this study was to evaluate the in vitro effect of IL-3 on the growth of follicular small-cleaved-cell lymphoma (FSCCL). IL-3 receptor expression on the surface of CD19+ cells was determined by two-color flow cytometry measuring the receptor-binding of biotinylated IL-3 to CD19+ B-cells. Seven cases of FSCCL were compared to six normal controls. Cell proliferation was evaluated by [3H]thymidine incorporation into cells grown in suspension cultures. All seven cases of FSCCL expressed the IL-3 receptor on the surface of CD19+ cells, whereas all six cases of CD19+ cells isolated from the peripheral blood of normal donors did not express IL-3 receptors. IL-3 had antiproliferative activity against FSCCL as manifested by a decrease in [3H]thymidine incorporation and a decrease in the total number of cells after 72 hours of culture. IL-3 inhibits the growth of FSCCL cells in vitro. Clinical trials to evaluate the in vivo effect of IL-3 in patients with FSCCL are warranted.

Stem cell factor and basic fibroblast growth factor are synergistic in augmenting committed myeloid progenitor cell growth

Stem cell factor (SCF) and basic fibroblast growth factor (bFGF) are hematopoietic cytokines produced by bone marrow stromal cells. It is known that, although SCF and bFGF have limited clonogenic activity on their own, they can augment colony-stimulating factor (CSF)-mediated progenitor cell growth. Because these factors are both sequestered by stromal cells, we examined their interaction on progenitor cell growth in conjunction with granulocyte-macrophage-CSF (GM-CSF). In this study, we show that clonogenic growth derived from low-density bone marrow cells stimulated by GM-CSF is significantly augmented (P &lt; .001) in the presence of maximal (100 ng/mL) concentrations of SCF in combination with 100 ng/mL of bFGF. When CD34+ cells are used, the synergistic effect of bFGF and SCF for GM-CSF-mediated progenitor cell growth is further increased, resulting in as much as a sevenfold increase in detectable colony-forming units granulocyte-macrophage (P &lt; .001). These data suggest that the synergistic activity of bFGF and SCF is mediated directly on hematopoietic precursors. These observations suggest that bFGF and SCF, concentrated locally on stromal cell surfaces, might interact in concert with other hematopoietic cytokines to regulate stem cell proliferation and differentiation in hematopoietic niches in the bone marrow.

Stem Cell Factor and Basic Fibroblast Growth Factor Are Synergistic in Augmenting Committed Myeloid Progenitor Cell Growth

Stem cell factor (SCF) and basic fibroblast growth factor (bFGF) are hematopoietic cytokines produced by bone marrow stromal cells. It is known that, although SCF and bFGF have limited clonogenic activity on their own, they can augment colony-stimulating factor (CSF)-mediated progenitor cell growth. Because these factors are both sequestered by stromal cells, we examined their interaction on progenitor cell growth in conjunction with granulocyte-macrophage-CSF (GM-CSF). In this study, we show that clonogenic growth derived from low-density bone marrow cells stimulated by GM-CSF is significantly augmented (P < .001) in the presence of maximal (100 ng/mL) concentrations of SCF in combination with 100 ng/mL of bFGF. When CD34+ cells are used, the synergistic effect of bFGF and SCF for GM-CSF-mediated progenitor cell growth is further increased, resulting in as much as a sevenfold increase in detectable colony-forming units granulocyte-macrophage (P < .001). These data suggest that the synergistic activity of bFGF and SCF is mediated directly on hematopoietic precursors. These observations suggest that bFGF and SCF, concentrated locally on stromal cell surfaces, might interact in concert with other hematopoietic cytokines to regulate stem cell proliferation and differentiation in hematopoietic niches in the bone marrow.

Variable effects of tamoxifen on human hematopoietic progenitor cell growth and sensitivity to doxorubicin.

To determine the influence of tamoxifen on the drug sensitivity of normal human hematopoietic progenitor cells, T-cell- and adherent-cell depleted human bone marrow mononuclear cells (T-, Ad-) were exposed in vitro to 5 microM tamoxifen for 24 h. The effects of tamoxifen were highly variable, as exposure to tamoxifen produced an increase (97% +/- 12.3%) in the growth of day-12 committed myeloid progenitors (CFU-GM) in only four of ten experiments utilizing bone marrow from different donors. When T-, Ad- myeloid progenitor cells treated with tamoxifen were subsequently exposed to doxorubicin, 7 of 14 experimental samples studied demonstrated a net increase in the number of surviving clonogenic cells as compared with cells exposed to doxorubicin alone. Tamoxifen also stimulated the growth of a more purified (CD34(+)-selected) progenitor cell population in four of four experiments (by 62.5% +/- 4.9%) but did not increase the survival of these cells upon exposure to doxorubicin; in fact, in five of ten experimental samples, tamoxifen enhanced cell sensitivity to doxorubicin. Taken together, these observations indicate that tamoxifen produces variable stimulation of committed myeloid progenitor cell growth in vitro. Furthermore, while under some circumstances, tamoxifen appears to have the capacity to enhance CFU-GM survival in the presence of doxorubicin, this drug combination may also result in enhanced toxicity to normal bone marrow progenitors.