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

Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1,a cell surface glycoprotein expressed on MM cells. In preclinical models,elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein,we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1-CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary,human MM cells. Taken together,these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone. View Publication

Under standard culture conditions,tumor cells are exposed to 20% O(2),whereas the mean tumor oxygen levels within the tumor are much lower. We demonstrate,using low-passaged human tumor cell cultures established from glioma,that a reduction in the oxygen level in these cell cultures dramatically increases the percentage of CD133 expressing cells. View Publication

Mature mammary epithelial cells are generated from undifferentiated precursors through a hierarchical process,but the molecular mechanisms involved,particularly in the human mammary gland,are poorly understood. To address this issue,we isolated highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue and compared their transcriptomes obtained using three different methods. Elements unique to each subset of mammary cells were identified,and changes that accompany their differentiation in vivo were shown to be recapitulated in vitro. These include a stage-specific change in NOTCH pathway gene expression during the commitment of bipotent progenitors to the luminal lineage. Functional studies further showed NOTCH3 signaling to be critical for this differentiation event to occur in vitro. Taken together,these findings provide an initial foundation for future delineation of mechanisms that perturb primitive human mammary cell growth and differentiation. View Publication

As a critical tumor suppressor,p53 is inactivated in human cancer cells by somatic gene mutation or disruption of pathways required for its activation. Therefore,it is critical to elucidate the mechanism underlying p53 activation after genotoxic and cellular stresses. Accumulating evidence has indicated the importance of posttranslational modifications such as acetylation in regulating p53 stability and activity. However,the physiological roles of the eight identified acetylation events in regulating p53 responses remain to be fully understood. By employing homologous recombination,we introduced various combinations of missense mutations (lysine to arginine) into eight acetylation sites of the endogenous p53 gene in human embryonic stem cells (hESCs). By determining the p53 responses to DNA damage in the p53 knock-in mutant hESCs and their derivatives,we demonstrate physiological importance of the acetylation events within the core domain (K120 and K164) and at the C-terminus (K370/372/373/381/382/386) in regulating human p53 responses to DNA damage. View Publication

An in vivo transplantation system has been used to evaluate the developmental capacities of specific mouse mammary epithelial cell populations. Specifically,mouse mammary epithelial cells with distinctly limited developmental potentials have been identified using this procedure. Two distinct epithelial cell progenitors have been identified by experiments designed to determine whether basal lobular and ductal phenotypes could develop independently under conditions imposed by a limiting dilution. The prediction that these separate epithelial progenitors must exist was based upon the results from transplantation experiments carried out in epithelium-divested mammary fat pads of syngeneic mice with mammary epithelium from two different transgenic mouse models. The results presented here demonstrate the following points: 1) lobular,i.e. secretory,progenitor cells are present as distinct entities among the mammary epithelial cells found in immature virgin female mice; 2) similarly,ductal epithelial progenitors are present within the same population; 3) lobular progenitors are present in greater numbers,although both cell populations are extremely small; 4) as expected,some inocula produce outgrowths with simultaneous development of both lobular and ductal phenotypes--it is not known whether this indicates cooperative interaction between the two epithelial progenitors or signals the presence of a third progenitor type capable of producing both ductular and lobular committed daughters; 5) these findings have important consequences in the design of experiments aimed at testing the effects of known and putative mammary oncogenes and tumor suppressor genes,using techniques which include cellular transformation in vitro followed by in vivo cultivation and evaluation. View Publication

Although cancer is a disease with genetic and epigenetic origins,the possible effects of reprogramming by defined factors remain to be fully understood. We studied the effects of the induction or inhibition of cancer-related genes and immature status-related genes whose alterations have been reported in gastrointestinal cancer cells. Retroviral-mediated introduction of induced pluripotent stem (iPS) cell genes was necessary for inducing the expression of immature status-related proteins,including Nanog,Ssea4,Tra-1-60,and Tra-1-80 in esophageal,stomach,colorectal,liver,pancreatic,and cholangiocellular cancer cells. Induced cells,but not parental cells,possessed the potential to express morphological patterns of ectoderm,mesoderm,and endoderm,which was supported by epigenetic studies,indicating methylation of DNA strands and the histone H3 protein at lysine 4 in promoter regions of pluripotency-associated genes such as NANOG. In in vitro analysis induced cells showed slow proliferation and were sensitized to differentiation-inducing treatment,and in vivo tumorigenesis was reduced in NOD/SCID mice. This study demonstrated that pluripotency was manifested in induced cells,and that the induced pluripotent cancer (iPC) cells were distinct from natural cancer cells with regard to their sensitivity to differentiation-inducing treatment. Retroviral-mediated introduction of iPC cells confers higher sensitivity to chemotherapeutic agents and differentiation-inducing treatment. View Publication

Chronic myeloid leukaemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL,a constitutively active tyrosine kinase. It is widely believed that BCR-ABL activates Akt signalling that suppresses the forkhead O transcription factors (FOXO),supporting the proliferation or inhibiting the apoptosis of CML cells. Although the use of the tyrosine kinase inhibitor imatinib is a breakthrough for CML therapy,imatinib does not deplete the leukaemia-initiating cells (LICs) that drive the recurrence of CML. Here,using a syngeneic transplantation system and a CML-like myeloproliferative disease mouse model,we show that Foxo3a has an essential role in the maintenance of CML LICs. We find that cells with nuclear localization of Foxo3a and decreased Akt phosphorylation are enriched in the LIC population. Serial transplantation of LICs generated from Foxo3a(+/+) and Foxo3a(-/-) mice shows that the ability of LICs to cause disease is significantly decreased by Foxo3a deficiency. Furthermore,we find that TGF-beta is a critical regulator of Akt activation in LICs and controls Foxo3a localization. A combination of TGF-beta inhibition,Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo. Furthermore,the treatment of human CML LICs with a TGF-beta inhibitor impaired their colony-forming ability in vitro. Our results demonstrate a critical role for the TGF-beta-FOXO pathway in the maintenance of LICs,and strengthen our understanding of the mechanisms that specifically maintain CML LICs in vivo. View Publication

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and Akt are important regulators of the phosphatidylinositol 3-kinase (PI3K) pathway and thus are important to the regulation of a wide spectrum of tumor-related biological processes. Akt regulates several critical cellular functions,including cell cycle progression; cell migration,invasion,and survival; and angiogenesis. Decreased expression of PTEN and overexpression of the Akt proto-oncogene,which is located downstream of PI3K,have been shown in a variety of cancers,including glioblastoma. Novel small-molecule inhibitors of receptors and signaling pathways,including inhibitors of the PI3K pathway,have shown antitumor activity,but inhibitors of Akt have not been examined. In this study,we tested our hypothesis that the pharmacologic inhibition of Akt has an antiproliferative effect on gliomas. We showed that two newly developed Akt inhibitors,KP-372-1 and KP-372-2 (herein called KP-1 and KP-2),effectively inhibited the PI3K/Akt signaling cascade. KP-1 and KP-2 blocked both the basal and epidermal growth factor-induced phosphorylation of Akt Ser473 at 125 and 250 nmol/L,which,in turn,reduced the activation of intracellular downstream targets of Akt,including GSK-3beta and p70s6k. Furthermore,the treatment of U87 and U251 glioma cells with 125 to 250 nmol/L KP-1 and KP2 for 48 hours inhibited cell growth by approximately 50%. This decrease in cell growth stemmed from the induction of apoptosis. Collectively,these results provide a strong rationale for the pharmacologic targeting of Akt for the treatment of gliomas. View Publication

Although Hodgkin and Reed-Sternberg (HRS) cells are B lymphoid cells,they are unlike any normal cells of that lineage. Moreover,the limited proliferative potential of HRS cells belies the clinical aggressiveness of Hodgkin lymphoma (HL). More than 20 years ago,the L428 HL cell line was reported to contain a small population of phenotypic B cells that appeared responsible for the continued generation of HRS cells. This observation,however,has never been corroborated,and such clonotypic B cells have never been documented in HL patients. We found that both the L428 and KM-H2 HL cell lines contained rare B-cell subpopulations responsible for the generation and maintenance of the predominant HRS cell population. The B cells within the HL cell lines expressed immunoglobulin light chain,the memory B-cell antigen CD27,and the stem cell marker aldehyde dehydrogenase (ALDH). Clonal CD27(+)ALDH(high) B cells,sharing immunoglobulin gene rearrangements with lymph node HRS cells,were also detected in the blood of most newly diagnosed HL patients regardless of stage. Although the clinical significance of circulating clonotypic B cells in HL remains unclear,these data suggest they may be the initiating cells for HL. 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

The human blood group i and I antigens are determined by linear and branched poly-N-acetyllactosamine structures,respectively. In erythrocytes,the fetal i antigen is converted to the adult I antigen by I-branching beta-1,6-N-acetylglucosaminyltransferase (IGnT) during development. Dysfunction of the I-branching enzyme may result in the adult i phenotype in erythrocytes. However,the I gene responsible for blood group I antigen has not been fully confirmed. We report here a novel human I-branching enzyme,designated IGnT3. The genes for IGnT1 (reported in 1993),IGnT2 (also presented in this study),and IGnT3 consist of 3 exons and share the second and third exons. Bone marrow cells preferentially expressed IGnT3 transcript. During erythroid differentiation using CD34(+) cells,IGnT3 was markedly up-regulated with concomitant decrease in IGnT1/2. Moreover,reticulocytes expressed the IGnT3 transcript,but IGnT1/2 was below detectable levels. By molecular genetic analyses of an adult i pedigree,individuals with the adult i phenotype were revealed to have heterozygous alleles with mutations in exon 2 (1006GtextgreaterA; Gly336Arg) and exon 3 (1049GtextgreaterA; Gly350Glu),respectively,of the IGnT3 gene. Chinese hamster ovary (CHO) cells transfected with each mutated IGnT3 cDNA failed to express I antigen. These findings indicate that the expression of the blood group I antigen in erythrocytes is determined by a novel IGnT3,not by IGnT1 or IGnT2. 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