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

BACKGROUND Acute myeloid leukemia (AML) is initiated and maintained by a subset of self-renewing leukemia stem cells (LSCs),which contribute to the progression,recurrence and therapeutic resistance of leukemia. However,the mechanisms underlying the maintenance of LSCs drug resistance have not been fully defined. In this study,we attempted to elucidate the mechanisms of LSCs drug resistance. METHODS We performed reverse phase protein arrays to analyze the expression of anti-apoptotic proteins in the LSC-enriched leukemia cell line KG-1a. Immuno-blotting,cell viability and clinical AML samples were evaluated to verify the micro-assay results. The characteristics and transcriptional regulation of survivin were analyzed with the relative luciferase reporter assay,mutant constructs,chromatin immuno-precipitation (ChIP),quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR),and western blotting. The levels of Sp1,c-Myc,phospho-extracellular signal-regulated kinase (p-ERK),phospho-mitogen and stress-activated protein kinase (p-MSK) were investigated in paired CD34+ and CD34- AML patient samples. RESULTS Survivin was highly over-expressed in CD34 + CD38- KG-1a cells and paired CD34+ AML patients compared with their differentiated counterparts. Functionally,survivin contributes to the drug resistance of LSCs,and Sp1 and c-Myc concurrently regulate levels of survivin transcription. Clinically,Sp1 and c-Myc were significantly up-regulated and positively correlated with survivin in CD34+ AML patients. Moreover,Sp1 and c-Myc were further activated by the ERK/MSK mitogen-activated protein kinase (MAPK) signaling pathway,modulating survivin levels. CONCLUSION Our findings demonstrated that ERK/MSK/Sp1/c-Myc axis functioned as a critical regulator of survivin expression in LSCs,offering a potential new therapeutic strategy for LSCs therapy. View Publication

Inflammatory breast cancer (IBC) is an angioinvasive and most aggressive type of advanced breast cancer characterized by rapid proliferation,chemoresistance,early metastatic development and poor prognosis. IBC tumors display a triple-negative breast cancer (TNBC) phenotype characterized by centrosome amplification,high grade of chromosomal instability (CIN) and low levels of expression of estrogen receptor α (ERα),progesterone receptor (PR) and HER-2 tyrosine kinase receptor. Since the TNBC cells lack these receptors necessary to promote tumor growth,common treatments such as endocrine therapy and molecular targeting of HER-2 receptor are ineffective for this subtype of breast cancer. To date,not a single targeted therapy has been approved for non-inflammatory and inflammatory TNBC tumors and combination of conventional cytotoxic chemotherapeutic agents remains the standard therapy. IBC tumors generally display activation of epithelial to mesenchymal transition (EMT) that is functionally linked to a CD44+/CD24-/Low stem-like phenotype. Development of EMT and consequent activation of stemness programming is responsible for invasion,tumor self-renewal and drug resistance leading to breast cancer progression,distant metastases and poor prognosis. In this study,we employed the luminal ER+ MCF-7 and the IBC SUM149PT breast cancer cell lines to establish the extent to which high grade of CIN and chemoresistance were mechanistically linked to the enrichment of CD44+/CD24low/- CSCs. Here,we demonstrate that SUM149PT cells displayed higher CIN than MCF-7 cells characterized by higher percentage of structural and numerical chromosomal aberrations. Moreover,centrosome amplification,cyclin E overexpression and phosphorylation of retinoblastoma (Rb) were restricted to the stem-like CD44+/CD24-/Low subpopulation isolated from SUM149PT cells. Significantly,CD44+/CD24-/Low CSCs displayed resistance to conventional chemotherapy but higher sensitivity to SU9516,a specific cyclin-dependent kinase 2 (Cdk2) inhibitor,demonstrating that aberrant activation of cyclin E/Cdk2 oncogenic signaling is essential for the maintenance and expansion of CD44+/CD24-/Low CSC subpopulation in IBC. In conclusion,our findings propose a novel therapeutic approach to restore chemosensitivity and delay recurrence of IBC tumors based on the combination of conventional chemotherapy with small molecule inhibitors of the Cdk2 cell cycle kinase. View Publication

BACKGROUND: Previous studies have shown that patients with Bcr-Abl-positive acute lymphoblastic leukemia (ALL) either have primary disease that is refractory to imatinib mesylate or develop disease recurrence after an initial response. METHODS: The authors investigated the effects of a newly designed Bcr-Abl inhibitor,AMN107,by comparing its in vitro inhibitory potency on p190 Bcr-Abl ALL cell lines with that of imatinib. RESULTS: In two Philadelphia (Ph)-positive ALL cell lines,AMN107 was found to be 30-40 times more potent than imatinib in inhibiting cellular proliferation. AMN107 was also more effective than imatinib in inhibiting phosphorylation of p190 Bcr-Abl tyrosine kinase in cell lines and primary ALL cells. The inhibition of cellular proliferation was associated with the induction of apoptosis in only one of the cell lines. No activity was observed in cell lines lacking the BCR-ABL genotype. CONCLUSIONS: The results of the current study suggest the superior potency of AMN107 compared with imatinib in Ph-positive ALL and support clinical trials of AMN107 in patients with Ph-positive ALL. View Publication

Loss or mutation of the TP53 tumor suppressor gene is not commonly observed in acute myeloid leukemia (AML),suggesting that there is an alternate route for cell transformation. We investigated the hypothesis that previously observed Bcl-2 family member overexpression suppresses wild-type p53 activity in AML. We demonstrate that wild-type p53 protein is expressed in primary leukemic blasts from patients with de novo AML using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and phospho-specific flow cytometry. We found that p53 was heterogeneously expressed and phosphorylated in AML patient samples and could accumulate following DNA damage. Overexpression of antiapoptosis protein Bcl-2 in AML cells was directly correlated with p53 expression and phosphorylation on serine residues 15,46,and 392. Within those patients with the highest levels of Bcl-2 expression,we identified a mutation in FLT3 that duplicated phosphorylation site Y591. The presence of this mutation correlated with greater than normal Bcl-2 expression and with previously observed profiles of potentiated STAT and MAPK signaling. These results support the hypothesis that Flt3-mediated signaling in AML enables accumulation of Bcl-2 and maintains a downstream block to p53 pathway apoptosis. Bcl-2 inhibition might therefore improve the efficacy of existing AML therapies by inactivating this suppression of wild-type p53 activity. View Publication

In somatic cells,eroded telomeres can induce DNA double-strand break signaling,leading to a form of replicative senescence or apoptosis,both of which are barriers to tumorigenesis. However,cancer cells might display telomere dysfunctions which in conjunction with defects in DNA repair and apoptosis,enables them to circumvent these pathways. Chronic lymphocytic leukemia (CLL) cells exhibit telomere dysfunction,and a subset of these cells are resistant to DNA damage-induced apoptosis and display short telomeres. We show here that these cells exhibit significant resection of their protective telomeric 3' single-stranded overhangs and an increased number of telomere-induced foci containing gammaH2AX and 53BP1. Chromatin immunoprecipitation and immunofluorescence experiments demonstrated increased levels of telomeric Ku70 and phospho-S2056-DNA-PKcs,2 essential components of the mammalian nonhomologous end-joining DNA repair system. Notably,these CLL cells display deletions of telomeric signals on one or 2 chromatids in parallel with 11q22 deletions,or with 13q14 deletions associated with another chromosomal aberration or with a complex karyotype. Taken together,our results indicate that a subset of CLL cells from patients with an unfavorable clinical outcome harbor a novel type of chromosomal aberration resulting from telomere dysfunction. View Publication

Current treatment options for advanced and hormone refractory prostate cancer are limited and responses to commonly used androgen pathway inhibitors are often unsatisfactory. Our recent results indicated that sodium ionophore monensin is one of the most potent and cancer-specific inhibitors in a systematic sensitivity testing of most known drugs and drug-like molecules in a panel of prostate cancer cell models. Because monensin has been extensively used in veterinary applications to build muscle mass in cattle,the link to prostate cancer and androgen signaling was particularly interesting. Here,we showed that monensin effects at nanomolar concentrations are linked to induction of apoptosis and potent reduction of androgen receptor mRNA and protein in prostate cancer cells. Monensin also elevated intracellular oxidative stress in prostate cancer cells as evidenced by increased generation of intracellular reactive oxygen species and by induction of a transcriptional profile characteristic of an oxidative stress response. Importantly,the antiproliferative effects of monensin were potentiated by combinatorial treatment with the antiandrogens and antagonized by antioxidant vitamin C. Taken together,our results suggest monensin as a potential well-tolerated,in vivo compatible drug with strong proapoptotic effects in prostate cancer cells,and synergistic effects with antiandrogens. Moreover,our data suggest a general strategy by which the effects of antiandrogens could be enhanced by combinatorial administration with agents that increase oxidative stress in prostate cancer cells. View Publication

Adult T-cell leukemia-lymphoma (ATL) is an aggressive disease,incurable by standard chemotherapy. NK314,a new anticancer agent possessing inhibitory activity specific for topoisomerase IIα (Top2α),inhibited the growth of various ATL cell lines (50% inhibitory concentration: 23-70nM) with more potent activity than that of etoposide. In addition to the induction of DNA double-strand breaks by inhibition of Top2α,NK314 induced degradation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs),resulting in impaired DNA double-strand break repair. The contribution of DNA-PK to inhibition of cell growth was affirmed by the following results: NK314 inhibited cell growth of M059J (a DNA-PKcs-deficient cell line) and M059K (a cell line with DNA-PKcs present) with the same potency,whereas etoposide exhibited weak inhibition of cell growth with M059K cells. A DNA-PK specific inhibitor,NU7026,enhanced inhibitory activity of etoposide on M059K as well as on ATL cells. These results suggest that NK314 is a dual inhibitor of Top2α and DNA-PK. Because ATL cells express a high amount of DNA-PKcs,NK314 as a dual molecular targeting anticancer agent is a potential therapeutic tool for treatment of ATL. View Publication

OBJECTIVE: Identification of a clinical grade method for the ex vivo generation of donor-derived T cells cytotoxic against both myeloid and lymphoblastic cells still remains elusive. We investigated rapid generation and expansion of donor derived-allogeneic T-cell lines cytotoxic against patient leukemic cells. MATERIALS AND METHODS: Acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts were cultured 5 days in Stem Span,granulocyte macrophage colony-stimulating factor,interleukin-4,and calcium ionophore. All B-precursor ALL (N22) and AML (N13),but not T-cell ALL (N3),differentiated into mature leukemia-derived antigen-presenting cells (LD-APC). All but one LD-APC generated cytotoxic T lymphocyte (CTL) from adult human leukocyte antigen (HLA)-identical (N8) or unrelated donors (N2). RESULTS: Upon in vitro culture,donor-derived CTL acquired a memory T phenotype,showing concomitant high CD45RA,CD45RO,CD62L expression. CD8(+) cells,but not CD4(+) cells,were granzyme,perforine,and interferon-gamma-positive. Pooled CD4(+) and CD8(+) cells were cytotoxic against leukemic blasts (32%,30:1 E:T ratio),but not against autologous or patient-derived phytohemagglutinin blasts. LD-APC from five ALL patients were used to generate CTL from cord blood. A mixed population of CD4(+) and CD8(+) cells was documented in 54% of wells. T cells acquired classical effector memory phenotype and showed a higher cytotoxicity against leukemia blasts (47%,1:1 E:T ratio). Adult and cord blood CTL showed a skewing from a complete T-cell receptor repertoire to an oligo-clonal/clonal pattern. CONCLUSIONS: Availability of these cells should allow clinical trials for salvage treatment of leukemia patients relapsing after allogeneic stem cell transplantation. View Publication

Aurora kinases are key regulators of cell mitosis and have been implicated in the process of tumorigenesis. In recent years,the Aurora kinases have attracted much interest as promising targets for cancer treatment. Here we report on the roles of Aurora A and Aurora B kinases in clear cell renal cell carcinoma (ccRCC). Using genomewide expression array analysis of 174 patient samples of ccRCC,we found that expression levels of Aurora A and B were significantly elevated in ccRCC compared to normal kidney samples. High expression levels of Aurora A and Aurora B were significantly associated with advanced tumor stage and poor patient survival. Inhibition of Aurora kinase activity with the drug VX680 (also referred to as MK-0457) inhibited ccRCC cell growth in vitro and led to ccRCC cell accumulation in the G2/M phase and apoptosis. Growth of ccRCC xenograft tumors was also inhibited by VX680 treatment,accompanied by a reduction of tumor microvessel density. Analysis of endothelial cell lines demonstrated that VX680 inhibits endothelial cell growth with effects similar to that seen in ccRCC cells. Our findings suggest that VX680 inhibits the growth of ccRCC tumors by targeting the proliferation of both ccRCC tumor cells and tumor-associated endothelial cells. Aurora kinases and their downstream cell cycle proteins have an important role in ccRCC and may be potent prognostic markers and therapy targets for this disease. View Publication

Regulatory T cells (T(reg)) that prevent autoimmune diseases by suppression of self-reactive T cells may also suppress the immune response against cancer. In mice,depletion of T(reg) by Ab therapy leads to more efficient tumor rejection. T(reg)-mediated suppression of antitumor immune responses may partly explain the poor clinical response to vaccine-based immunotherapy for human cancer. In this study,we measured the prevalence of T(reg) that coexpress CD4 and CD25 in the PBLs,tumor-infiltrating lymphocytes,and regional lymph node lymphocytes from 65 patients with either pancreas or breast cancer. In breast cancer patients (n = 35),pancreas cancer patients (n = 30),and normal donors (n = 35),the prevalence of T(reg) were 16.6% (SE 1.22),13.2% (SE 1.13),and 8.6% (SE 0.71) of the total CD4(+) cells,respectively. The prevalence of T(reg) were significantly higher in breast cancer patients (p textless 0.01) and pancreas cancer patients (p textless 0.01) when compared with normal donors. In tumor-infiltrating lymphocytes and lymph node lymphocytes,the T(reg) prevalence were 20.2% (SE 3.93) and 20.1% (SE 4.3),respectively. T(reg) constitutively coexpressed CTLA-4 and CD45RO markers,and secreted TGF-beta and IL-10 but did not secrete IFN-gamma. When cocultured with activated CD8(+) cells or CD4(+)25(-) cells,T(reg) potently suppressed their proliferation and secretion of IFN-gamma. We conclude that the prevalence of T(reg) is increased in the peripheral blood as well as in the tumor microenvironment of patients with invasive breast or pancreas cancers. These T(reg) may mitigate the immune response against cancer,and may partly explain the poor immune response against tumor Ags. View Publication

Mechanisms of lethality of the three-substituted indolinone and putatively selective cyclin-dependent kinase (CDK)2 inhibitor 3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516) were examined in human leukemia cells. Exposure of U937 and other leukemia cells to SU9516 concentrations textgreater or =5 microM rapidly (i.e.,within 4 h) induced cytochrome c release,Bax mitochondrial translocation,and apoptosis in association with pronounced down-regulation of the antiapoptotic protein Mcl-1. These effects were associated with inhibition of phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase (Pol) II on serine 2 but not serine 5. Reverse transcription-polymerase chain reaction analysis revealed pronounced down-regulation of Mcl-1 mRNA levels in SU9516-treated cells. Similar results were obtained in Jurkat and HL-60 leukemia cells. Furthermore,cotreatment with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) blocked SU9516-mediated Mcl-1 down-regulation,implicating proteasomal degradation in diminished expression of this protein. Ectopic expression of Mcl-1 largely blocked SU9516-induced cytochrome c release,Bax translocation,and apoptosis,whereas knockdown of Mcl-1 by small interfering RNA potentiated SU9516 lethality,confirming the functional contribution of Mcl-1 down-regulation to SU9516-induced cell death. It is noteworthy that SU9516 treatment resulted in a marked increase in reactive oxygen species production,which was diminished,along with cell death,by the free radical scavenger N-acetylcysteine (NAC). We were surprised to find that NAC blocked SU9516-mediated inhibition of RNA Pol II CTD phosphorylation on serine 2,reductions in Mcl-1 mRNA levels,and Mcl-1 down-regulation. Together,these findings suggest that SU9516 kills leukemic cells through inhibition of RNA Pol II CTD phosphorylation in association with oxidative damage and down-regulation of Mcl-1 at the transcriptional level,culminating in mitochondrial injury and cell death. View Publication

Adult neural stem and progenitor cells (NSPCs) are usually defined retrospectively by their ability to proliferate in vivo (bromodeoxyuridine uptake) or to form neurospheres and to differentiate into neurons,astrocytes and oligodendrocytes in vitro. Additional strategies to identify and to isolate NSPCs are of great importance for the investigation of cell differentiation and fate specification. Using the cell surface molecules Prominin-1 and Lewis X and a metabolic marker,the aldehyde dehydrogenase activity,we isolated and characterized five main populations of NSPCs in the neurogenic subventricular zone (SVZ) and the non-neurogenic spinal cord (SC). We used clonal analysis to assess neurosphere formation and multipotency,BrdU retention to investigate in vivo proliferation activity and quantified the expression of NSPC associated genes. Surprisingly,we found many similarities in NSPC subpopulations derived from the SVZ and SC suggesting that subtypes with similar intrinsic potential exist in both regions. The marker defined classification of NSPCs will help to distinguish subpopulations of NSPCs and allows their prospective isolation using fluorescence activated cell sorting. View Publication