搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Individual variation in fetal hemoglobin (HbF,alpha(2)gamma(2)) response underlies the remarkable diversity in phenotypic severity of sickle cell disease and beta thalassemia. HbF levels and HbF-associated quantitative traits (e.g.,F cell levels) are highly heritable. We have previously mapped a major quantitative trait locus (QTL) controlling F cell levels in an extended Asian-Indian kindred with beta thalassemia to a 1.5-Mb interval on chromosome 6q23,but the causative gene(s) are not known. The QTL encompasses several genes including HBS1L,a member of the GTP-binding protein family that is expressed in erythroid progenitor cells. In this high-resolution association study,we have identified multiple genetic variants within and 5' to HBS1L at 6q23 that are strongly associated with F cell levels in families of Northern European ancestry (P = 10(-75)). The region accounts for 17.6% of the F cell variance in northern Europeans. Although mRNA levels of HBS1L and MYB in erythroid precursors grown in vitro are positively correlated,only HBS1L expression correlates with high F cell alleles. The results support a key role for the HBS1L-related genetic variants in HbF control and illustrate the biological complexity of the mechanism of 6q QTL as a modifier of fetal hemoglobin levels in the beta hemoglobinopathies. View Publication

The mechanisms that regulate the growth and survival of acute myeloid leukemia (AML) cells are largely unknown. We hypothesized that constitutive activation of phosphatidyl-inositide 3 kinase (PI3 kinase) could regulate survival in primary cells from patients with AML. Here we demonstrate that Akt,a critical substrate of PI3 kinase,is activated in AML blasts. In a short-term culture system,most AML patient samples showed a dose-dependent decrease in survival after incubation with the PI3 kinase inhibitor LY294002. This decrease in survival was partially due to the induction of apoptosis. Furthermore,we have shown that p70 S6 kinase and 4EBP-1,downstream mediators of Akt signaling,also are phosphorylated in AML blasts. Phosphorylation of these proteins is inhibited by the mTOR inhibitor RAD001. Incubation of AML blasts with RAD001 induces only a small decrease in survival of the cells; however,when combined with Ara-C,RAD001 enhances the toxicity of Ara-C. These results demonstrate that constitutive activation of the PI3 kinase pathway is necessary for the survival of AML blasts and that targeting of this pathway with pharmacologic inhibitors may be of clinical benefit in treatment of AML. View Publication

We studied the actions of geldanamycin (GA) and herbimycin A (HMA),inhibitors of the chaperone proteins Hsp90 and GRP94,on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses,T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA,but not HMA,showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase,a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL. View Publication

Identification of agents that target human leukemia stem cells is an important consideration for the development of new therapies. The present study demonstrates that rocaglamide and silvestrol,closely related natural products from the flavagline class of compounds,are able to preferentially kill functionally defined leukemia stem cells,while sparing normal stem and progenitor cells. In addition to efficacy as single agents,flavaglines sensitize leukemia cells to several anticancer compounds,including front-line chemotherapeutic drugs used to treat leukemia patients. Mechanistic studies indicate that flavaglines strongly inhibit protein synthesis,leading to the reduction of short-lived antiapoptotic proteins. Notably though,treatment with flavaglines,alone or in combination with other drugs,yields a much stronger cytotoxic activity toward leukemia cells than the translational inhibitor temsirolimus. These results indicate that the underlying cell death mechanism of flavaglines is more complex than simply inhibiting general protein translation. Global gene expression profiling and cell biological assays identified Myc inhibition and the disruption of mitochondrial integrity to be features of flavaglines,which we propose contribute to their efficacy in targeting leukemia cells. Taken together,these findings indicate that rocaglamide and silvestrol are distinct from clinically available translational inhibitors and represent promising candidates for the treatment of leukemia. View Publication

Proliferation in the nonpregnant human breast is highest in the luteal phase of the menstrual cycle when serum progesterone levels are high,and exposure to progesterone analogues in hormone replacement therapy is known to elevate breast cancer risk,yet the proliferative effects of progesterone in the human breast are poorly understood. In a model of normal human breast,we have shown that progesterone increased incorporation of 5-bromo-2'-deoxyuridine and increased cell numbers by activation of pathways involved in DNA replication licensing,including E2F transcription factors,chromatin licensing and DNA replication factor 1 (Cdt1),and the minichromosome maintenance proteins and by increased expression of proteins involved in kinetochore formation including Ras-related nuclear protein (Ran) and regulation of chromosome condensation 1 (RCC1). Progenitor cells competent to give rise to both myoepithelial and luminal epithelial cells were increased by progesterone,showing that progesterone influences epithelial cell lineage differentiation. Therefore,we have demonstrated that progesterone augments proliferation of normal human breast cells by both activating DNA replication licensing and kinetochore formation and increasing bipotent progenitor numbers. View Publication

Retinoids triggers differentiation of acute promyelocytic leukemia (APL) blasts by transcriptional regulation of myeloid regulatory genes. Using a microarray approach,we have identified a novel retinoid-responsive gene (CXXC5) encoding a nuclear factor,retinoid-inducible nuclear factor (RINF),that contains a CXXC-type zinc-finger motif. RINF expression correlates with retinoid-induced differentiation of leukemic cells and with cytokine-induced myelopoiesis of normal CD34(+) progenitors. Furthermore,short hairpin RNA (shRNA) interference suggests for this gene a regulatory function in both normal and tumoral myelopoiesis. Interestingly,RINF localizes to 5q31.3,a small region often deleted in myeloid leukemia (acute myeloid leukemia [AML]/myelodysplasia [MDS]) and suspected to harbor one or several tumor suppressor gene. View Publication

T-cell expression of programmed death receptor-1 (PD-1) down-regulates the immune response against malignancy by interacting with cognate ligands (eg,PD-L1) on tumor cells; however,little is known regarding PD-1 and natural killer (NK) cells. NK cells exert cytotoxicity against multiple myeloma (MM),an effect enhanced through novel therapies. We show that NK cells from MM patients express PD-1 whereas normal NK cells do not and confirm PD-L1 on primary MM cells. Engagement of PD-1 with PD-L1 should down-modulate the NK-cell versus MM effect. We demonstrate that CT-011,a novel anti-PD-1 antibody,enhances human NK-cell function against autologous,primary MM cells,seemingly through effects on NK-cell trafficking,immune complex formation with MM cells,and cytotoxicity specifically toward PD-L1(+) MM tumor cells but not normal cells. We show that lenalidomide down-regulates PD-L1 on primary MM cells and may augment CT-011's enhancement of NK-cell function against MM. We demonstrate a role for the PD-1/PD-L1 signaling axis in the NK-cell immune response against MM and a role for CT-011 in enhancing the NK-cell versus MM effect. A phase 2 clinical trial of CT-011 in combination with lenalidomide for patients with MM should be considered. View Publication

The notion that gliomas could originate from mutated glial precursor cells highlights the possibility of modulating the proliferative and migratory behaviour of glioma cells by acting on the molecular mechanisms operative during the development of the Central Nervous System (CNS),but absent in the normal adult brain. We show that the GL15 glioblastoma derived human cell line displays a high expression of nestin which,combined with the previously demonstrated high expression of vimentin,constitutes a characteristic of astrocyte restricted precursors. We also show that,in analogy with some leukaemia cells,GL15 cells display the constitutively phosphorylated form of Janus kinase 2 (JAK2),a tyrosine kinase expressed during CNS development but undetectable in the normal adult brain. The constitutive activation of JAK2 does not result from chromosomal aberrations involving the JAK2 gene,but most probably from abnormally activated transduction systems operative in glioblastoma cells. We then investigated the effects of tyrphostin AG490,an inhibitor of JAK2 autophosphorylation,on GL15 cell growth. In the absence of exogenous growth factors and cytokines,10 microM tyrphostin AG490 induces an S phase arrest,combined with a partial impairment of the G2 phase of the cell cycle. The abnormally activated JAK2 could then potentially represent a target for a selective pharmacological approach in glioblastoma cells in which a combination of glial precursor characteristics and genetic alterations occurs. View Publication

Both innate and adaptive immune systems are considered important for cancer prevention,immunosurveillance,and control of cancer progression. It is known that,although both systems initially eliminate emerging tumor cells efficiently,tumors eventually escape immune attack by a variety of mechanisms,including differentiation and recruitment of immunosuppressive CD11b(+)Gr-1(+) myeloid suppressor cells into the tumor microenvironment. However,we show that CD11b(+)Gr-1(+) cells found in ascites of epithelial ovarian cancer-bearing mice at advanced stages of disease are immunostimulatory rather than being immunosuppressive. These cells consist of a homogenous population of cells that morphologically resemble neutrophils. Moreover,like dendritic cells,immunostimulatory CD11b(+)Gr-1(+) cells can strongly cross-prime,augmenting the proliferation of functional CTLs via signaling through the expression of costimulatory molecule CD80. Adoptive transfer of these immunostimulatory CD11b(+)Gr-1(+) cells from ascites of ovarian cancer-bearing mice results in the significant regression of s.c. tumors even without being pulsed with exogenous tumor Ag prior to adoptive transfer. We now show for the first time that adaptive immune responses against cancer can be augmented by these cancer-induced granulocyte-like immunostimulatory myeloid (CD11b(+)Gr-1(+)) cells,thereby mediating highly effective antitumor immunity in an adoptive transfer model of immunity. View Publication

BACKGROUND Although studies have shown that Oct4,Sox2,Klf4,and c-Myc (OKSM)-mediated induced pluripotent stem cell (iPSC) technology sensitizes cancer cells to drugs,the potential risk of inserting c-Myc and random insertions of exogenous sequences into the genome persists. Several authors,including us,have presented microRNA (miRNA)-mediated reprogramming as an alternative approach. Herein,we evaluated the efficacy of miRNA-mediated reprogramming on hepatocellular carcinoma (HCC) cells. METHODS Among three miRNAs (miR-200c,miR-302s,and miR-369s) that were previously presented for miRNA-mediated reprogramming,miR-302 was expressed at low levels in HCC cells. After transfecting three times with miR-302,the cells were incubated in ES medium for 3 weeks and then characterized. RESULTS iPSC-like spheres were obtained after the 3-week incubation. Spheres presented high NANOG and OCT4 expression,low proliferation,high apoptosis,low epithelial-mesenchymal transition marker expression (N-cadherin,TGFBR2),and sensitization to drugs. Several miRNAs were changed (e.g.,low oncomiR miR-21,high miR-29b). cMyc was decreased,and methylation was elevated on histone 3 at lysine 4 (H3K4). Differentiated cells expressed markers of each germ layer (GFAP,FABP4,and ALB). AOF2 (also known as LSD1 or KDM1),one of the targets for miR-302,was repressed in iPSC-like-spheres. Silencing of AOF2 resulted in similar features of iPSC-like-spheres,including cMyc down-regulation and H3K4 methylation. In drug-resistant cells,sensitization was achieved through miR-302-mediated reprogramming. CONCLUSIONS miR-302-mediated iPSC technology reprogrammed HCC cells and improved drug sensitivity through AOF2 down-regulation,which caused H3K4 methylation and c-Myc repression. View Publication