Bone Marrow CFU-GM Research Articles

Novel Small Molecule, UTS-1401, as a Radioprotector for Total-Body Irradiation.

We report on a new radioprotector, UTS-1401, a small molecule that was synthesized (by one of us, JS) and evaluated here for its radioprotective effect against total-body irradiation (TBI). Female and male NIH Swiss mice were subjected to TBI at doses of 6.5, 7.5 and 8.5 Gy either with or without a 24 h pretreatment of UTS-1401 given ip and observed for 30 days. Survival rates were significantly increased when mice were treated with UTS-1401 compared to those not treated. The radioprotective effect of UTS-1401 was drug-dose dependent for male mice exposed to 8.5 Gy TBI with 150 mg/kg of UTS-1401 as the optimal dose. The radioprotective effect of UTS-1401 on female mice exposed to 8.5 Gy TBI was observed at 50, 100, and 150 mg/kg, with no dose response relationship noted. Female mice were more radioresistant than male mice with LD50/30 values of 7.8 Gy vs. 6.8 Gy, respectively. Weight changes after UTS-1401 alone showed a significant body weight increase at 150 mg/kg. Both the ip and iv route for UTS-1401 were similarly effective for male mice exposed to 8 Gy TBI. Further analysis using an endogenous spleen colony assay demonstrated that pretreatment of UTS-1401 for up to 72h prior to TBI protected both spleen weight and hematopoietic stem cells with a treated/untreated ratio between 2.0 and 3.2 for the latter for times between 0.5 h and 72 h. A separate invivo study showed that pretreatment of UTS-1401 protected bone marrow CFU-GM for mice exposed to TBI. In summary, UTS-1401 is a promising small-molecule radioprotective agent as demonstrated by whole animal, hematopoietic stem cell and bone marrow myeloid progenitor cell survival.

CXCL15/Lungkine has suppressive activity on proliferation and expansion of multi-potential, erythroid, granulocyte and macrophage progenitors in S-phase specific manner

Cytokines/chemokines regulate hematopoiesis, most having multiple cell actions. Numerous but not all chemokine family members act as negative regulators of hematopoietic progenitor cell (HPC) proliferation, but very little is known about such effects of the chemokine, CXCL15/Lungkine. We found that CXCL15/Lungkine−/− mice have greatly increased cycling of multi cytokine-stimulated bone marrow and spleen hematopoietic progenitor cells (HPCs: CFU-GM, BFU-E, and CFU-GEMM) and CXCL15 is expressed in many bone marrow progenitor and other cell types. This suggests that CXCL15/Lungkine acts as a negative regulator of the cell cycling of these HPCs in vivo. Recombinant murine CXCL15/Lungkine, decreased numbers of functional HPCs during cytokine-enhanced ex-vivo culture of lineage negative mouse bone marrow cells. Moreover, CXCL15/Lungkine, through S-Phase specific actions, was able to suppress in vitro colony formation of normal wildtype mouse bone marrow CFU-GM, CFU-G, CFU-M, BFU-E, and CFU-GEMM. This clearly identifies the negative regulatory activity of CXCL15/Lungkine on proliferation of multiple types of mouse HPCs.

EFFICIENCY OF BONE MARROW PRECURSOR CELL COLONY-FORMING AS A PREDICTOR OF DISEASE COURSE IN PLASMA CELL MYELOMA PATIENTS WITH A HISTORY OF RADIATION EXPOSURE.

Assessment of role of the bone marrow colony-forming efficiency in plasma cell myeloma patients at different stages of treatment as a prognostic criterion for the disease course. The colony forming efficiency (CFE) was assayed in stage I-II plasma cell myeloma (PCM)patients (n = 37) aged 42-73, namely in patients survived after the Chornobyl NPP accident (n = 21) and persons notexposed to ionizing radiation (n = 16). There were 11 males exposed to ionizing radiation and having got stage I PCM,9 males and 3 females exposed and having got stage II PCM, 3 males and 3 females not exposed and having got stageI PCM, 6 males and 2 females not exposed and having got stage II PCM. Healthy persons (n = 20) were included in thecontrol group. Number of the bone marrow (BM) granulocyte-macrophage colony-forming units (CFU-GM) in both exposedand not exposed PCM patients depended on a disease stage. CFU-GM was (16.7 ± 1.2) in the stage I PCM patients vs.(11.1 ± 1.1) in the stage II PCM ones both being lower (p < 0.05) compared to control (64.5 ± 2.2). Changes in cluster formation were similar, i.e. (37.7 ± 1.6) and (19.4 ± 1.3) correspondingly in the stage I and stage II PCM patients.Respective values in control were (89.8 ± 3.6). The CFE in stage I and stage II PCM patients at the time of diagnosiswas lower (5.7 ± 1.5 and 2.4 ± 1.1 respectively) vs. control (39.5 ± 1.51, p < 0.05), but has increased in remission upto (29. 6 ± 1.8) and (13.8 ± 1.2) respectively. There was no difference at that between the irradiated and non-irradiated patients. Number of the fibroblast colony-forming units (CFU-F) in the stage I and stage II PCM patients duringdiagnosis, namely (43.9 ± 5.4) and (22.5 ± 3.7), was lower (p < 0.05) vs. control (110.5 ± 4.9). Upon reaching remission the CFU-F value increased significantly (p < 0.05), reaching (87.4 ± 4.2) and (55.6 ± 2.7) correspondingly in thestage I and stage II PCM patients. Dependence of the BM cell CFE on the stage of PCM and presence or absence of remission was established. Prognostic value of the CFE of BM CFU-GM in terms of life span of patients was shown (Ro Spearm = 0.39,p < 0.02), namely in case of CFE > 20 before the polychemotherapy administration the life span of PCM patients wassignificantly longer vs. cases of CFE < 20.

Myeloprotective effects of C-type natriuretic peptide on cisplatin-induced bone marrow granulocytopenia in mice.

Cisplatin is an effective chemotherapeutic agent used to treat a variety of malignant tumors. The major toxicity associated with cisplatin treatment is granulocytopenia. C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, protects against toxicity in many organs, including the heart, blood vessels, lung, and kidney. The objective of this study was to investigate the myeloprotective effects of CNP in a mouse model of cisplatin-induced granulocytopenia. The mice were divided into two groups: cisplatin with vehicle and cisplatin with CNP. CNP (2.5μg/kg/min via osmotic pump, subcutaneously) or vehicle administration was started two day before cisplatin injection, and continued until the mice were killed. At 0, 2, 4, 8, and 14 days after cisplatin injection (16mg/kg, intraperitoneally as a single dose), we counted total and living cells and granulocyte/macrophage colony-forming units (CFU-GM) in bone marrow. In addition, at 0, 1, 2, and 4 days after cisplatin injection, we measured mRNA levels of CXC chemokine receptor 4 (CXCR4) and chemokine CXC ligand 12 (CXCL12) in bone marrow. CNP significantly attenuated the reduction in bone marrow nucleated cell count and CFU-GM in bone marrow at 4 days after cisplatin injection. Four days after cisplatin injection, CNP significantly decreased the CXCR4 mRNA level in bone marrow, but had no effect on the level of CXCL12 mRNA. CNP exerts myeloprotective effects in cisplatin-induced granulocytopenia and decreases CXCR4 expression.

Astragalus Polysaccharide has hematopoietic and thrombopoietic activities in an irradiation mouse model

A herbal decoction (Danggui Buxue Tong, DBT) is a traditional Chinese medicine formula comprising Danggui (Radix Angelicae) and Huang Qi (Radix Astragali), which has been used for promotion of “blood production” for centuries and have shown favorable effects on thrombocytopenia. Previously, we have showed that it has significant effects on promoting hematopoiesis and thrombopoiesis (Yang M et al. J Ethnopharmacol, 2009). Many chemical components have been identified in DBT. Among them is the polysaccharides fraction of Angelica sinensis and Radix Astragali. Our study has demonstrated that Angelica Polysaccharide (APS) promotes hematopoiesis and platelet recovery in a mouse model (Liu et al, BMC Complement Altern Med, 2010). In this present study, we further assess the effect of Astragalus Polysaccharide (ASPS) on hematopoiesis and thrombopoiesis in-vivo and in vitro. A myelosuppression mouse model (4-Gy-irradiated) was treated with ASPS (10 mg/kg/day) and TPO (1 mg/kg/day). Peripheral blood cells from ASPS, TPO and vehicle-treated samples were counted on days 0, 7, 14 and 21. Using the colony-forming unit (CFU) assays, we determine the effects of ASPS on the hematopoietic stem/progenitor cells and megakaryocytic lineages. Analyses of Annexin V, Caspase-3, and Mitochondrial Membrane Potential were also conducted in HL-60 cells and megakaryocytic cell line M-07e. The effects of ASPS on cells treated with Ly294002, a PI3K inhibitor and the effect of ASPS on the phosphorylation of AKT were further studied. Results showed that ASPS, like TPO, significantly enhanced the recovery of WBC and platelet count, and bone marrow CFU-GM and CFU-MK formation (n=8) in this model. Morphological examination of bone marrows also showed that ASPS treatment significantly increased the recovery of hematopoietic stem/progenitor cells and megakaryocytic series. We further analyzed the in vitro effect of ASPS on CFU-MK and CFU-GM formation. ASPS (50-100 ug/ml) enhanced TPO (50 ng/ml) -induced CFU-MK (p=0.05, n=4), and CFU-GM formation (p=0.04, n=4). However, ASPS alone (1-500 ug/ml) did not show significant effect on CFU-MK and CFU-GM proliferation (n=4). Moreover, the anti-apoptotic effect of ASPS in HL-60 and M-07e cells were also demonstrated by using Annexin-V, Caspase-3, and JC-1 assays. Addition of Ly294002 alone increased the percentage of cells undergoing apoptosis. However, additional of ASPS to Ly294002-treated cells reversed the percentage of cells undergoing apoptosis. Furthermore, addition of ASPS significantly increased the phosphorylation of AKT. Here, we showed that ASPS, the polysaccharide from the root of Radix Astragali, has hematopoietic and thrombopoietic activities in a mouse model. This effect is likely to result from the stimulation of cell proliferation through the activation of PI3K/AKT pathway, the activation of which prevents cells from undergoing apoptosis. Disclosures:No relevant conflicts of interest to declare.

Trib2 Pseudokinase Marks Early CMP Progenitors, and Promotes Granulocytic Plus Erythroid Progenitor Cell Formation

Altered expression of Trib2 pseudokinase has been associated with AML (M2, M4 subtypes) (Cancer Cell, 10: 401), and ectopic expression of Trib2 in hematopoietic progenitors drives myeloid leukemogenesis (Blood, 116: 1321). In addition, inhibition of Trib2 expression by EPO in erythroid progenitors has been demonstrated (Blood, 111: 5390). Beyond this, little is known about Trib2's expression profiles and roles during normal myelopoiesis. To critically address functional roles, we have generated and characterized a novel Trib2-KO mouse model. Within bone marrow CFU-GM formation faltered, and Trib2-KO Linneg progenitors exhibited deficit granulocyte production (with a relative increase in monocytes). This was not associated with significant differences in survival or cell cycle features among developing granulomonocytic cells. Instead, Trib2 levels proved to be heightened among Linneg progenitors (and inhibited in GM cells following GMCSF, GCSF or ATRA exposure). This prompted analyses of CMP and early-CMP (eCMP) populations in which RNAseq demonstrated peak Trib2 expression. Furthermore, eCMP and CMP pools were elevated in Trib2-KO mice. Within progenitors of the erythroid lineage, Trib2 expression levels also selectively increased, and in Trib2-KO mice BFUe and CFUe levels were decreased (and RBC plus HGB were diminished). Intriguingly, when Trib2-KO mice were challenged with Flt3L plus GMCSF, BFUe (but not CFU-GM or GEMM) were mobilized to peripheral blood at levels 13.3-fold above wild-type controls. This indicates candidate effects of Trib2 on a proposed BFUe niche. Overall, novel multi-lineage roles for Trib2 are revealed in regulating eCMP pools, GM-CSF populations, balanced granulocytic vs monocytic cell formation, and the production of erythroid progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Anti-apoptotic effect of Astragalus Polysaccharide on myeloid cells

This study was aimed to assess the effect of Astragalus Polysaccharide (ASPS) on in-vitro hematopoiesis. CFU-GM assays were used to determine the effect of ASPS and thrombopoietin (TPO) on granulocytic-monocyte progenitor cells. The CFU assays were also used to investigate the effect of ASPS on the proliferation of HL-60 cells.HL-60 cells were cultured with serum-free RPMI 1640 medium and treated with or without of different concentrations of ASPS. After 72 h incubation, the number of cells were counted.In addition, the caspase-3 and JC-1 expression was determined by flow cytometry with Annexin V/PI double staining. The results showed that ASPS (100, 200 µg/ml) and TPO (100 ng/ml) significantly promoted CFU-GM formation in vitro. Various concentrations of ASPS and TPO also promoted the colony formation of HL-60 cells, the largest effect of ASPS was observed at a concentration of 100 µg/ml. There were no synergistic effects between TPO and ASPS on cellular proliferation. The results also showed that ASPS significantly protected HL-60 cells from apoptosis in condition of serum-free medium culture, suppressed caspase 3 activation, and reduced the cell apoptosis. It is concluded that ASPS can significantly promote the formation of bone marrow CFU-GM and the proliferation of HL-60 cells, the optimal concentration of ASPS is at 100 µg/ml. In the absence of serum inducing apoptosis, ASPS also significantly reduced the apoptosis of HL-60 cells via suppressing the activation of caspase-3.

Analyses of the combination of 6-MP and dasatinib in cell culture.

A major tenet of cancer therapeutics is that combinations of anticancer agents with different mechanisms of action and different toxicities may be effective treatment regimens. Evaluation of additivity/synergy in cell culture may be used to identify drug combination opportunities and to assess risk of additive/synergistic toxicity. The combination of 6-mercaptopurine and dasatinib was assessed for additivity/synergy using the combination index (CI) method and a response surface method in six human tumor cell lines including MCF-7 and MDA-MB-468 breast cancer, NCI-H23 and NCI-H460 non-small cell lung cancer, and A498 and 786-O renal cell cancer, based on two experimental end-points: ATP content and colony formation. Clonal colony formation by human bone marrow CFU-GM was used to assess risk of enhanced toxicity. The concentration ranges tested for each drug were selected to encompass the clinical Cmax concentrations. The combination regimens were found to be additive to sub-additive by both methods of data analysis, but synergy was not detected. The non-small cell lung cancer cell lines were the most responsive among the tumor lines tested and the renal cell carcinoma lines were the least responsive. The bone marrows CFU-GM were more sensitive to the combination regimens than were the tumor cell lines. Based upon these data, it appears that the possibility of enhanced efficacy from combining 6-mercaptopurine (6-MP) and dasatinib would be associated with increased risk of severe bone marrow toxicity, so the combination is unlikely to provide a therapeutic advantage for treating solid tumor patients where adequate bone marrow function must be preserved.

Erufosine, an alkylphosphocholine, with differential toxicity to human cancer cells and bone marrow cells

To investigate the activity and myeloprotective properties of erufosine, a novel alkylphosphocholine (APC), on human malignant cells and normal bone marrow cells. Human or mouse bone marrow cells were exposed to erufosine, miltefosine, perifosine, or edelfosine in CFU-GM assays. Human MDA-MB-231 breast carcinoma, Panc-1 pancreatic carcinoma, and RPMI8226 multiple myeloma cells were exposed to erufosine in colony formation assays. Colony formation of Panc-1 tumor cells and mouse bone marrow cells ex vivo were quantified following intravenous administration of erufosine to tumor-bearing mice. Western blotting methods were applied to human U87 glioblastoma cells exposed to erufosine to investigate Akt inhibition. Erufosine was less toxic to human and mouse bone marrow cells than perifosine, miltefosine, and edelfosine and was equally toxic to human and mouse CFU-GM. The human cancer cells MDA-MB-231 breast, Panc-1 pancreatic, and RPMI8226 MM cells were more sensitive to erufosine in a colony formation assay than were human bone marrow cells generating an approximately tenfold differential in IC(90) values. Erufosine injected intravenously significantly reduced Panc-1 tumor cell colony formation ex vivo but not mouse bone marrow CFU-GM. Erufosine inhibited Akt phosphorylation in human U87 glioblastoma cells. Erufosine offers potential as a novel therapeutic for cancer with a reduced toxicity profile to bone marrow cells compared with other agents in this class. Human cancer cells were more sensitive to erufosine than human or mouse bone marrow cells indicating a favorable therapeutic window for erufosine.

Differential Expression of Alpha2 Integrin In Human Bone Marrow and Cord Blood CD34+CD38- Progenitors and Stem Cells Reconstituting NOD/SCID-IL2Rgammacnull Mice.

AbstractAbstract 2609Human primitive stem cells reside in the CD34+CD38- fraction in cord blood and bone marrow. However, there is a high level of heterogeneity in these cell fractions, and the phenotype of the rare primitive stem cells remains poorly defined. We have studied expression of integrin alpha2 chain, a member of a family of beta1 integrin cell adhesion receptors, in CD34+CD38- cells in adult bone marrow and cord blood. >90% of bone marrow CD34+CD38- cells and >95% of CD34+CD38-CD90+ cells, enriched in long-term in vivo reconstituting stem cells (Majeti et al., Cell Stem Cell 1:635, 2007) expressed the integrin alpha2 chain. In contrast, in cord blood CD34+CD38- and the CD34+CD38-CD90+ subpopulation, the integrin alpha2 chain was expressed only in 37.1+/− 5.3% and 32.2+/− 4.9% of the cells (mean+/− SD), respectively. To determine whether integrin alpha2 expression could be used to identify functionally distinct stem and progenitor cell populations in cord blood and bone marrow, we isolated CD34+CD38- integrin alpha2+ and alpha2- cells by flow cytometry and analyzed these by in vivo transplantation into immunodeficient NOD/SCID-IL2Rgammacnull (NSG) mice and by in vitro progenitor cell assays (long-term culture initiating cell, LTC-IC, and colony assays). Transplantation of cord blood CD34+CD38- integrin alpha2+ cells resulted in significantly higher level of human CD45+ (p<0.05), myeloid (p<0.01) and CD34+ (p<0.05) cell engraftment at 16–18 weeks after transplantation than integrin alpha2- cells (reconstitution/300 cells in age and sex matched recipients, Figure 1). In contrast, there were no differences in reconstitution at 12 weeks in mice transplanted with cord blood CD34+CD38- alpha2+ and alpha2- cells. Because of lower engraftment capacity of adult bone marrow cells in immunodeficient mice, bone marrow CD34+CD38- cells were analyzed after intra-bone transplantation. After 12 weeks only few mice transplanted with the CD34+CD38- integrin alpha2+ cells but none transplanted with the corresponding alpha2- cells were reconstituted with human CD45+ or myeloid cells at a level '0.1%. The LTC-IC progenitors within the CD34+CD38- populations, assayed after 6 weeks culture on stroma, in adult bone marrow were highly enriched in the alpha2+ as compared with alpha2- fraction (mean +/− SD 96.7+/− 57.0 and 0.2+/− 0.6 colonies/50 cells, respectively), whereas in cord blood they resided equally in both integrin alpha2+ and alpha2- cell fractions (mean +/− SD 77.4+/− 59.4 and 108.8+/− 96.0 colonies/50 cells, respectively). The lineage committed CFU-GM and BFU-E progenitors in adult bone marrow were within integrin CD34+CD38- alpha2+ and alpha2- fractions (mean +/− SD 8.5+/− 5.3 and 14.9+/− 16.2 colonies/100 cells, respectively), whereas in cord blood they were significantly enriched in the alpha2- fraction (mean +/− SD 9.3+/− 5.5 and 22.0+/− 6.7 colonies/100 cells in alpha2+ and alpha2- fractions, respectively). Taken together, our results show expression of integrin alpha2 receptor in most of the primitive cord blood long-term in vivo reconstituting stem cells, with a gradual loss of the integrin alpha2 receptor during maturation to short term in vivo reconstituting stem cells, LTC-IC and lineage committed progenitors. Furthermore, our findings show distinct ontogeny-related differences in the expression of the integrin alpha2 receptor in the functionally defined primitive and lineage-committed CD34+CD38- progenitors, indicating differences in cellular interactions of cord blood and bone marrow progenitors with the hematopoietic niches. [Display omitted] Disclosures:Ekblom:Bristol-Myers Squibb: Honoraria.

Bone marrow lymphoid and myeloid progenitor cells are suppressed in 7,12-dimethylbenz(a)anthracene (DMBA) treated mice

In this study we used colony forming unit (CFU) assays to demonstrate rapid suppression (within 6 h) of lymphoid (CFU-preB) and myeloid (CFU-GM) progenitor cells in DMBA-treated mice. The duration of these changes were consistent with the blood levels of DMBA and its metabolites that were achieved by either IP or oral DMBA administration. CFU-GM and CFU-preB activities returned to control levels by 2 and 7 days after oral DMBA exposure, respectively, but remained suppressed through 7 days after IP DMBA administration. The continued presence of low levels of DMBA in the bloodstream following IP administration was associated with sustained suppression of CFU-preB, total bone marrow lymphoid cells and peripheral blood lymphocytes. The changes noted above were not observed in Cyp1b1 null mice, demonstrating the need for local DMBA metabolism in the bone marrow by Cyp1b1 to impair bone marrow CFU-preB and CFU-GM. Furthermore, these data provide evidence that myeloid-lineage cells are restored more quickly than lymphoid-lineage cells after DMBA exposure.

Bone marrow CFU-GM and human tumor xenograft efficacy of three antitumor nucleoside analogs

Nucleoside analogs are rationally designed anticancer agents that disrupt DNA and RNA synthesis. Fludarabine and cladribine have important roles in the treatment of hematologic malignancies. Clofarabine is a next generation nucleoside analog which is under clinical investigation. The bone marrow toxicity, tumor cell cytotoxicity and human tumor xenograft activity of fludarabine, cladribine and clofarabine were compared. Mouse and human bone marrow were subjected to colony forming (CFU-GM) assays over a 5-log concentration range in culture. NCI-60 cell line screening data were compared. In vivo, a range of clofarabine doses was compared with fludarabine for efficacy in several human tumor xenografts. The IC90 concentrations for fludarabine and cladribine for mouse CFU-GM were >30 and 0.93 microM, and for human CFU-GM were 8 and 0.11 microM, giving mouse to human differentials of >3.8- and 8.5-fold. Clofarabine produced IC90s of 1.7 microM in mouse and 0.51 microM in human CFU-GM, thus a 3.3-fold differential between species. In the NCI-60 cell line screen, fludarabine and cladribine showed selective cytotoxicity toward leukemia cell lines while for clofarabine there was no apparent selectivity based upon origin of the tumor cells. In vivo, clofarabine produced a dose-dependent increase in tumor growth delay in the RL lymphoma, the RPMI-8226 multiple myeloma, and HT-29 colon carcinoma models. The PC3 prostate carcinoma was equally responsive to clofarabine and fludarabine. Bringing together bone marrow toxicity data, tumor cell line cytotoxicity data, and human tumor xenograft efficacy provides valuable information for the translation of preclinical findings to the clinic.

Bone marrow CFU-GM and human tumor xenograft efficacy of three tubulin binding agents

The dynamic instability of microtubules in cells is one of the key targets of anticancer therapeutics. Microtubule-disrupting agents such as vinca alkaloids and microtubule-stabilizing agents such as taxanes are important antitumor agents. The bone marrow toxicity and human tumor xenograft activity of three tubulin-binding compounds, vincristine, paclitaxel, and tasidotin were compared. Mouse and human bone marrow were subjected to colony-forming (CFU-GM) assays over a 5-log concentration range in culture. In vivo, a range of tasidotin doses was compared with vincristine, paclitaxel, and docetaxel for efficacy in several human tumor xenografts. The IC(90) concentrations for vincristine and paclitaxel for mouse CFU-GM were 30 and 27 nM, and for human CFU-GM were 3 and 9 nM, giving mouse to human differentials of ten- and threefold. Tasidotin produced IC(90)s of >300 nM in mouse and 65 nM in human CFU-GM, thus a >4.6-fold differential between species. In vivo, tasidotin resulted in a dose-dependent increase in tumor growth delay in the RL lymphoma, the RPMI 8226 multiple myeloma, and MX-1 breast carcinoma models. Vincristine and tasidotin were also very effective against these tumors. The PC-3 prostate carcinoma was very responsive to full-dose paclitaxel and docetaxel while tasidotin generated a dose dependent effect. Bringing together bone marrow toxicity data, pharmacokinetic parameters, and human tumor xenograft efficacy provides valuable information for the translation of preclinical findings to the clinic.

Transplanted CD34+ Cells Stimulated with Flt3 Ligand and Granulocyte-Colony Stimulating Factor Display An Impaired Early Reconstitution Capacity

Flt3/flk2 belongs to the class III hematopoietic growth factor receptors with intrinsic kinase activity, ligand-activated tyrosine phosphorylation and a central role in early stages of hematopoiesis. Flt3-ligand (FL) has been shown to enhance the effects of several growth factors, including Granulocyte-colony stimulating factor (G-CSF). In this study the reconstitution ability of FL+G-CSF primed CD34+ cells was evaluated in a nonhuman primate model of radiation-induced myeloablative treatment followed by transplantation of stimulated autologous bone marrow (BM) and/or peripheral blood (PB) cells. Rhesus monkeys (n=9) were subjected to subcutaneous injections with FL (100 μg/kg/day) and G-CSF (100 μg/kg/day) for 7 to 14 days and subsequently irradiated with a lethal dose of total body irradiation (TBI) followed by transplantation with primed stem cells or with a midlethal dose of TBI (n=2) without transplantation. White blood cell counts (WBC) in PB showed a steep increase and reached a mean level of 98.9×109/L (71.5–131.7×109/L) after 7 days (P<0.01). BM cellularity increased from 35.2×109/L before treatment to 193.8×109/L after priming (95.0–348.2×109/L) (P<0.01). CD34+ cells displayed a 1-log increase (P<0.01) in BM and a 2-log increase in PB (P<0.01). Both total nucleated and CD34+ cell numbers reached after 7 days a steady state level, which was maintained until cessation of growth factor treatment. Treatment with FL+G-CSF resulted in a moderate increase (2-fold) of CFU-GM in BM after 7 days (P<0.05) but did not affect BFU-E, consistent with the predilection of FL for myeloid and lymphoid cells, in contrast to SCF, which predominantly influences the erythroid and thrombopoietic lineage. The effect of treatment with FL+G-CSF on WBC was significantly greater than treatment with either G-CSF at day 7 (P<0.05) or FL at day 7 and 14 (P<0.01). Lethally irradiated monkeys, transplanted with FL+G-CSF stimulated cells displayed a much slower regeneration of WBC, platelets and reticulocytes relative to the cell number transplanted and were consequently in need of prolonged antimicrobial prophylaxis and transfusions. Furthermore, these monkeys displayed regeneration patterns consistent with a 1-log depletion of repopulating cells relative to non-stimulated BM cells. Mid-lethal irradiated FL+G-CSF treated monkeys were not transplanted to study the intrinsic regeneration capacity of primed BM. These monkeys displayed normal regeneration of reticulocytes, platelets and WBC. Thus, stimulation with FL+G-CSF leads to a steep and synergistic increase of nucleated and CD34+ cells in both BM and PB. Priming with FL+G-CSF does not affect the intrinsic regeneration capacity of BM after radiation; but when transplanted, primed CD34+ cells do not per se contribute to accelerated short-term engraftment after transplantation, rather the reconstitution patterns were consistent with on average one order of magnitude stem cell depletion relative to the numbers of CD34+ cells transplanted.

Angelica Polysaccharides Promotes Thrombopoiesis through the Phosphatidylinositol 3-Kinase/Akt Pathway.

Angelica Polysaccharide is from the root of Radix Angelicae Sinensis (Chinese Danggui). Danggui has been used for centuries to treat blood-deficiency related diseases. The hematopoietic effect of Danggui may be related to its constituent, polysaccharide. However, the effects of angelica polysaccharide on hematopoietic stem/progenitor cells and megakaryocyte/platelet lineage have not been well studied. In this study, we specifically investigate the thrombopoietic effect of Angelica polysaccharide (APS) in a mouse model and its molecular mechanism. A myelosuppression mouse model (4-Gy-irradiated) was treated with APS (10 mg/kg/day) and thrombopoietin (TPO) (1 mg/kg/day). Peripheral blood cells from APS, TPO and vehicle-treated samples were counted on days 0, 7, 14 and 21. Then CFU assays were used to determine the effects of APS on the megakaryocytic progenitor cells and other lineages. Analyses of Annexin V, Caspase-3, and Mitochondrial Membrane Potential were conducted in megakaryocytic cell line M-07e. The effects of APS on cells treated with Ly294002, a Phosphatidylinositol 3-Kinse inhibitor and the effect of APS on the phosphorylation of AKT were also studied. APS as well as TPO significantly enhanced the recovery of platelet and WBC count, and bone marrow CFU-MK and CFU-GM formation (n=6). Morphological examination of bone marrows showed that APS treatment significantly increased the recovery of the megakaryocytic series. APS also increased the recovery of the body weight and organ weight (liver and spleen). However, APS didn't have a up-regulating effect on TPO production by a ELISA assay. We further analyzed the in vitro effect of APS on CFU-MK and CFU-GM formation. APS (50 ug/ml) enhanced TPO (50 ng/ml) -induced CFU-MK (p=0.06, n=4), and CFU-GM formation (p=0.032, n=6). However, APS alone (00 ug/ml) did not show significant effect on CFU-MK and CFU-GM proliferation (n=6). The effect of APS (1–500 ug/ml) on the growth of bone marrow stromal cells was further investigated using CFU-F (fibroblastoid) assay. APS (50 ug/ml) alone showed a promoting effect on CFU-F growth (p=0.049, n=3). APS (50 ug/ml) also significantly enhanced PDGF, bFGF and VEGF-induced CFU-F formation (n=4). Moreover, the anti-apoptotic effect of APS in M-07e cells was also demonstrated by using Annexin-V, Caspase-3, and JC-1 assays. Addition of Ly294002 alone increased the percentage of cells undergoing apoptosis. However, additional of APS to Ly294002-treated cells reversed the percentage of cells undergoing apoptosis. Furthermore, addition of APS significantly increased the phosphorylation of AKT. APS promotes thrombopoiesis in a mouse model. This effect is likely to be mediated by the PI3K/AKT pathway.

Bcl-2 expression and apoptosis induction in human HL60 leukaemic cells treated with a novel organotellurium(IV) compound RT-04

Organotellurium(IV) compounds have been reported to have multiple biological activities including cysteine protease-inhibitory activity, mainly cathepsin B. As cathepsin B is a highly predictive indicator for prognosis and diagnosis of cancer, a possible antitumor potential for these new compounds is expected. In this work, it was investigated the effectiveness of organotellurium(IV) RT-04 to produce lethal effects in the human promyelocytic leukaemia cell line HL60. Using the MTT tetrazolium reduction test, and trypan blue exclusion assay, the IC 50 for the compound after 24 h incubation was 6.8 and 0.35 μM, respectively. Moreover, the compound was found to trigger apoptosis in HL60 cells, inducing DNA fragmentation and caspase-3, -6, and -9 activations. The apoptsosis-induced by RT-04 is probably related to the diminished Bcl-2 expression, observed by RT-PCR, in HL60-treated cells. In vivo studies demonstrated that the RT-04 treatment (2.76 mg/kg given for three consecutive days) produces no significant toxic effects for bone marrow and spleen CFU-GM. However, higher doses (5.0 and 10 mg/kg) produced a dose-dependent reduction in the number of CFU-GM of RT-04-treated mice. These results suggest that RT-04 is able to induce apoptosis in HL60 cells by Bcl-2 expression down-modulation. Further studies are necessary to better clarify the effects of this compound on bone marrow normal cells.

Hematopoietic Recovery after Transplantation CD117+B220- (lacZ+) Bone Marrow Cells in Lethally Irradiated Mice

Experiments presented here were aimed at the description of hematopoiesis repair and in vivo homing of transplanted separated CD117+B220- bone marrow cells after whole-body lethal irradiation at LD 9Gy. ROSA 26 mice were used as donors of marrow cells for transplantation [B6;129S/Gt (ROSA)26Sor] and were tagged with lacZ gene, and F2 hybrid mice [B6129SF2/J] were used as recipients of bone marrow transplanted cells. Hematopoiesis repair was provided by transplantation, both suspension of whole bone marrow cells (5x106) and isolated CD117+B220- cells (5x10(4)). Mice survived up to thirty days after irradiation. We demonstrated that transplantation of suspension of whole bone marrow cells led to faster recovery of CFU-GM (Granulocyte-macrophage colony forming units) in bone marrow and in the spleen too. It is not clear what the share of residential and transplanted cells is in the repair process. Our results demonstrate that sufficient hematopoietic repair occurs after transplantation of CD117+B220- (lacZ+) in lethally irradiated mice, and the difference in CFU-GM numbers in the bone marrow and spleen found on day 8 posttransplant has no influence on the survival of lethally irradiated mice (30 days follow-up).

Short and Long Term Efficacy of Autologous Bone Marrow Cells To Support Intensive Chemotherapy in Patients Failing Peripheral Blood Stem Cell Mobilization.

Introduction: Most autologous hematopoietic cell transplants (AHCT) are performed using peripheral blood mobilized progenitor cells (PBSC). However a small but significant proportion of patients (pts) are unable to mobilize adequate numbers of PBSC. The use of G-CSF-stimulated bone marrow (BM) derived progenitor cells is one method to circumvent this problem and enable AHCT to be performed. However, the long and short-term ability to engraft and the impact of this method on overall and progression free survival (OS and PFS) have not been established.Methods: We reviewed 52 pts (17 AML, 24 NHL, 11 HL) who failed PBSC collection between Jan 1999-Dec 2006 (<2×5106 CD34+cells/kg) and underwent G-CSF stimulated BM harvest to permit high dose therapy (etoposide 60 mg/kg + melphalan 160–180 mg/m2, + TBI 500 or 1200 cGy for pts with AML). 23 pts were supported by harvested BM (44%) and 29 (56%) with a mixture (Mx) of BM and PBSC. Overall and PFS was compared to pts undergoing AHCT using chemotherapy-mobilized PBSCs (n=440) in the same time period (AML n=32, NHL n=273, HL=135) who had a similar median age, pre-transplant induction, salvage therapy and intensive therapy regimens. Kaplan-Meier curves and the Log-rank test were used to compare the OS and PFS between cases and controls. Due to skewed data, the non-parametric Wilcoxon Rank Test was used for simple comparisons of clinical factors.Results: Median age of the cohort at AHCT was 43 years (range 24–68, 67% females). Status prior to AHCT was CR 44%, PR 54%, and SD 2%. Number of chemotherapy regimens (median):AML: 3; NHL: 3; HL: 2. Median BM CFU-GM infused was 4.4×104/kg and median PBSC infused was 0.1×106/kg (n=29). Median follow up times for AML/NHL/HL was 20, 18 and 50 months respectively. Twenty-three pts died:18 from disease progression, 4 treatment-related (TRM) and one of second cancer. Median engraftment time for neutrophils (>0.5×109/L) was 14 days and platelets (>20×109/L) was 27 days and was significantly longer compared to pts who received PBSC alone (10 and 11 days, respectively, p<0.0001). Pts receiving Mx grafts had significantly faster engraftment vs those receiving BM alone (p<0.001). Transfusion requirements (median) during the ASCT admission were 4 units for RBC and 25 units for platelets. Median 12 month Hb, platelet and neutrophil counts were 116 g/L (70–149), 113×109/L (21–238) and 2.4×109/L (0.3–6.2). Two year OS of patients who received harvested BM versus PBSC alone was: 63%/71% (AML, p=0.51), 57%/70% (NHL, p=0.04) and 71%/94% (HL, p=0.04). There was no significant difference in PFS between BM recipients and controls. Non relapse mortality was 5/52 (9.6%) for pts receiving BM compared to 18/440 (4%) supported by PBSCs.Conclusion: Performing an AHCT with BM in patients failing PBSC collection is feasible. Although engraftment is significantly delayed it appears sustained at 12 months post AHCT. Overall survival is inferior compared with patients receiving PBSC alone. Reasons for inferior disease-specific outcomes for pts with lymphoma receiving BM need to be determined.

Regulation of myeloid progenitor cell proliferation/survival by IL-31 receptor and IL-31

Interleukin (IL)-31 is a recently discovered helical cytokine. Its receptor consists of a ligand-specific IL-31 receptor (IL-31R) subunit and a receptor chain that is shared with Oncostatin M (OSM), called OSM-Rbeta. Because OSM-Rbeta-deficient animals have reduced hematopoietic progenitor cells (HPC) and OSM has effects on and is involved in homeostasis of HPC, we studied whether IL-31 and IL-31R play a role in hematopoiesis. IL-31R(-/-) mice and their littermate wild-type (WT) controls were assessed for absolute numbers and cycling status of bone marrow and spleen HPC (colony-forming unit granulocyte macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], colony-forming unit granulocyte, erythrocyte, macrophage, megakaryocyte). Recombinant IL-31 was evaluated for stimulation, enhancement, or inhibition of colony formation by HPC, and for survival-enhancing effects on HPC subjected to growth-factor withdrawal and delayed addition of grown factors. Hematopoietic stem cells (HSC) from WT and IL-31R(-/-) mice were compared for competitive repopulating capacity in lethally irradiated congenic mice. IL-31R(-/-) mice demonstrated significantly decreased absolute numbers and cycling status of immature subsets of HPC in bone marrow bone and spleen compared to WT mice. There were no significant differences in absolute numbers of more mature subsets of WT and IL-31R(-/-) bone marrow CFU-GM. WT but not IL-31R(-/-) bone marrow CFU-GM responded to synergistic stimulation by combinations of cytokines. While IL-31 had neither colony-stimulating, -enhancing, or -inhibiting activity for bone marrow HPC, it did enhance survival of these HPC in the context of delayed addition of growth factors. No significant differences were detected in competitive repopulating HSC activity between WT and IL-31R(-/-) bone marrow cells. IL-31R is involved in positive regulation of absolute numbers and cycling status of immature subsets of HPC in vivo. While IL-31 in vitro does not modulate proliferation of HPC, it does enhance their survival, which may contribute to effects on cycling and numbers of HPC in vivo. Under steady-state conditions, loss of IL-31R on HPC does not appear to influence the activity of competitive repopulating HSC. These results with HPC may be of future utility for manipulation of hematopoiesis in a preclinical setting.

Telintra™ (TLK199) Enhances Granulocyte Colony-Stimulating Factor Production.

TLK199 is a novel glutathione analog inhibitor of the enzyme GST P1-1. GST P1-1 has been shown to be a negative regulator of c-Jun N-terminal kinase, which has been implicated in the control of cellular growth and differentiation. Studies in rodents have shown that TLK199 treatment increases the levels of circulating white blood cells and stimulates the production of CFU-GM in bone marrow. In a rodent model of 5-FU-induced neutropenia, treatment with TLK199 accelerated the recovery of neutrophil levels at a rate similar to that observed with G-CSF. In a recently reported Phase 2 clinical trial in patients with refractory MDS involving multilineage blood cell dysfunction, treatment with TLK199 resulted in a 65% Hematological Improvement of neutrophils, erythrocytes and/or platelets, confirming the myelorestorative effect of TLK199 in humans. The present study was designed to determine if the myelostimulative effects of TLK199 on neutrophils and their precursors are due in part to increased levels of endogenous G-CSF. TLK199 was tested in normal and neutropenic rodents for its effects on G-CSF production. A single administration of TLK199 (p.o. or i.p.) to normal mice resulted in up to 500% increase in G-CSF serum levels. In a rat model of 5-FU-induced neutropenia, TLK199 administration accelerated the recovery of circulating neutrophils, which corresponded to maximal G-CSF serum levels being observed earlier than vehicle control. The ability of TLK199 to enhance G-CSF production in primary human bone marrow stromal cells was also examined. Treatment of these cells with TLK199 potentiated IL-1b-induced production of G-CSF, GM-CSF and IL-6 from 200 to 400%. These data suggest that the enhancement of G-CSF production may contribute to the accelerated recovery of neutrophils observed with TLK199 treatment under neutropenic conditions. Together with the results from the Phase 2 clinical trial in MDS, these data support further clinical assessment of TLK199 treatment for chemotherapy-induced cytopenia.