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In the present study, we report a new method for enrichment and analysis of fetal CD34+ stem cells after culture in order to determine whether it is feasible for noninvasive prenatal diagnosis. We also determined whether fetal CD34+ stem cells persist in maternal blood after delivery and assessed whether they have an impact on noninvasive prenatal diagnosis of genetic abnormalities. Peripheral blood samples were obtained from 35 pregnant women, 13 non-pregnant women who had given birth to male offsprings, 12 women who had never been pregnant, and eight pregnant women with male fetuses. CD34+ stem cells were enriched and either cultured for prenatal diagnosis or analyzed with fluorescence in situ hybridization (FISH)/polymerase chain reaction (PCR) to determine peristance in maternal blood. Fetal/maternal cells can be isolated and grown in vitro" to provide enough cells for a more accurate fetal sex or aneuploid prediction than is provided by unenriched and uncultured CD34+ stem cells. The presence of fetal cells in maternal blood samples from mothers who had given birth to male offspring was found in 3 of 13 blood samples. PCR was positive for Y chromosome in one woman who had never been pregnant. Analysis of cultured CD34+ stem cells from mothers with Y PCR positivity did not detect any male cells in any samples. Even if PCR positivity is due to persistence of fetal stem cells from previous pregnancies�

Jun N-terminal kinase (JNK) is a stress-activated protein kinase that can be induced by inflammatory cytokines, bacterial endotoxin, osmotic shock, UV radiation, and hypoxia. We report the identification of an anthrapyrazolone series with significant inhibition of JNK1, -2, and -3 (K(i) = 0.19 microM). SP600125 is a reversible ATP-competitive inhibitor with textgreater20-fold selectivity vs. a range of kinases and enzymes tested. In cells, SP600125 dose dependently inhibited the phosphorylation of c-Jun, the expression of inflammatory genes COX-2, IL-2, IFN-gamma, TNF-alpha, and prevented the activation and differentiation of primary human CD4 cell cultures. In animal studies, SP600125 blocked (bacterial) lipopolysaccharide-induced expression of tumor necrosis factor-alpha and inhibited anti-CD3-induced apoptosis of CD4(+) CD8(+) thymocytes. Our study supports targeting JNK as an important strategy in inflammatory disease, apoptotic cell death, and cancer.

Augmenting the host's natural immune response to viruses by the administration of exogenous cytokines such as interferon-alpha (IFN-alpha) is a strategy increasingly employed in antiviral therapeutics. Enhancing the release of endogenous cytokines is, however, an alternative approach. The imidazoquinolinamines imiquimod and resiquimod have demonstrated potency as inducers of IFN-alpha and other cytokines both in vitro and in vivo. Cytokine gene activation is mediated via the signal transducer and activator of transcription 1 (STAT-1) and involves the transcription factors NFkappaB and alpha4F1. Antiviral activity has been demonstrated against a variety of viruses, and clinical efficacy has been demonstrated against genital warts, herpes genitalis and molluscum contagiosum. Imiquimod is administered as a 5% cream (Aldara) and has been licensed for the treatment of anogenital warts in immunocompetent patients. Complete clearance of warts has been observed in up to half of treated patients with only local side effects reported. Resiquimod can be administered topically but also exists as an oral formulation. The range of potential infections for which these agents may have clinical utility includes chronic hepatitis C virus infection and Kaposi's sarcoma. In addition, the imidazoquinolinamines may find roles in the therapy of cancers and as vaccine adjuvants.

Patients with heart failure are predisposed to infections and anemia, possibly due to reduced hematopoiesis. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is increased in heart failure, and it inhibits normal hematopoiesis, partly due to apoptosis through the effector molecule Fas. We examined bone marrow progenitor cells of mice with heart failure induced by acute myocardial infarction. The fraction of progenitor cells in mice with heart failure was only approximately 40% of control. Measured with in vitro clonal assays, the proliferative capacity of the progenitor cells in mice with heart failure was reduced to approximately 50% of control. Flow cytometry with specific markers revealed a threefold increase in apoptosis among progenitor cells from mice with heart failure. In these mice, TNF-alpha/Fas expression was increased in bone marrow natural killer (NK) and T cells, and these lymphocytes showed increased cytolytic activity in vitro against progenitor cells. We conclude that the TNF-alpha/Fas pathway in lymphocytes is activated in the bone marrow during heart failure, which may play a pathogenic role in the observed decrease in hematopoiesis.

Histone deacetylases (HDACs) play important roles in transcriptional regulation and pathogenesis of cancer. Thus, HDAC inhibitors are candidate drugs for differentiation therapy of cancer. Here, we show that the well-tolerated antiepileptic drug valproic acid is a powerful HDAC inhibitor. Valproic acid relieves HDAC-dependent transcriptional repression and causes hyperacetylation of histones in cultured cells and in vivo. Valproic acid inhibits HDAC activity in vitro, most probably by binding to the catalytic center of HDACs. Most importantly, valproic acid induces differentiation of carcinoma cells, transformed hematopoietic progenitor cells and leukemic blasts from acute myeloid leukemia patients. More over, tumor growth and metastasis formation are significantly reduced in animal experiments. Therefore, valproic acid might serve as an effective drug for cancer therapy.

STI571 is a 2-phenylalaminopyrimidine derivative that inhibits c-abl, Bcr-Abl, and platelet-derived growth factor receptor tyrosine kinases. Recently, inhibition of stem cell factor (SCF)-induced c-kit phosphorylation and cell proliferation by STI571 was reported in the human myeloid cell line MO7e. Because approximately 70% of acute myelogenous leukemia (AML) cases are c-kit positive, we evaluated in vitro effects of STI571 on c-kit-positive cell lines and primary AML blast cells. At concentrations textgreater5 microM, the drug marginally inhibited SCF-independent proliferation of cell lines and most of AML blasts. Treatment of AML cells with cytarabine and STI571 showed synergistic effect at low concentrations. Western blotting analysis documented a distinct band of M(r) 145,000 specific for c-kit in cell lines and in AML samples. There was no correlation between the level of the c-kit expression evaluated by Western blotting and percentage of c-kit-positive blasts as measured by flow cytometry. Neither in cell lines nor in primary AML cells, c-kit autophosphorylation was detectable under standard growth conditions. SCF-induced phosphorylation of c-kit in MO7e cells was inhibited by STI571. In a c-kit-positive AML-4 cell line, as well as in AML samples, c-kit phosphorylation was not induced by SCF exposure, suggesting that in these cases, the receptor could not be functionally activated. In conclusion, with the exception of MO7e, SCF did not induce phosphorylation of c-kit, and cell proliferation was not modulated in the presence of STI571. We did not detect any SCF-independent c-kit phosphorylation in our experimental systems. Consequently, STI571 exerted only a limited inhibitory effect on the cell growth.

Sir2p is an NAD(+)-dependent histone deacetylase required for chromatin-dependent silencing in yeast. In a cell-based screen for inhibitors of Sir2p, we identified a compound, splitomicin, that creates a conditional phenocopy of a sir2 deletion mutant in Saccharomyces cerevisiae. Cells grown in the presence of the drug have silencing defects at telomeres, silent mating-type loci, and the ribosomal DNA. In addition, whole genome microarray experiments show that splitomicin selectively inhibits Sir2p. In vitro, splitomicin inhibits NAD(+)-dependent histone deacetylase activity (HDA) of the Sir2 protein. Mutations in SIR2 that confer resistance to the drug map to the likely acetylated histone tail binding domain of the protein. By using splitomicin as a chemical genetic probe, we demonstrate that continuous HDA of Sir2p is required for maintaining a silenced state in nondividing cells.

BACKGROUND: Ex vivo expansion of cord blood (CB) hematopoietic stem and progenitor cells increases cell dose and may reduce the severity and duration of neutropenia and thrombocytopenia after transplantation. This study's purpose was to establish a clinically applicable culture system by investigating the use of cytokines, serum-free media, and autologous plasma for the expansion of CB cells and the engraftment of expanded product in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. STUDY DESIGN AND METHODS: Enriched CB CD34+ cells were cultured in four media (Iscove's modified Dulbecco's medium with FCS, Gibco; X-Vivo-10, BioWhittaker; QBSF-60, Quality Biological; and StemSpan SFEM, Stem Cell Technologies) with four cytokine combinations (thrombopoietin [TPO], SCF, Flt-3 ligand [FL] with and without G-CSF, and/or IL-6). The effect of autologous CB plasma was also investigated. The read-out measures were evaluated on Days 8 and 12. After expansion at the optimized condition, cultured cells were transplanted into sublethally irradiated NOD/SCID mice. The engraftment of human CD45+ cells and subsets in the bone marrow, spleen, and peripheral blood was determined. RESULTS: QBSF-60 or StemSpan SFEM supported high yields of early progenitors (CD34+ cells, textlessor= 64.8-fold; CD34+CD38- cells, 330-fold; CFU-granulocyte erythroid macrophage megakaryocyte [GEMM], 248-fold) and CFUs of the myeloid (CFU-GM, 407-fold) and erythroid (BFU/CFU-E, 144-fold) lineages. The expansion of the megakaryocytic lineage was consistently higher in X-Vivo-10 (CFU-megakaryocyte, 684-fold). Autologous plasma promoted colony formation but reduced CD34+ cells and CFU-GEMM. The addition of G-CSF or IL-6 improved cell yields; G-CSF was more effective for committed progenitors. Expansion products from cultures in QBSF-60 with the cytokines engrafted and differentiated into the myeloid and lymphoid lineages in NOD/SCID mice. CONCLUSION: The data supported the strategy of expansion. The optimized condition may be applicable to clinical expansion for the abrogation or reduction of posttransplant cytopenia.

Regulation of the homeostasis of vascular endothelium is critical for the processes of vascular remodeling and angiogenesis under physiological and pathological conditions. Here we show that doxorubicin (Dox), a drug used in antitumor therapy, triggered a marked accumulation of p53 and induced CD95 gene expression and apoptosis in proliferating human umbilical vein endothelial cells (HUVECs). Transfection and site-directed mutagenesis experiments using the CD95 promoter fused to an intronic enhancer indicated the requirement for a p53 site for Dox-induced promoter activation. Furthermore, the p53 inhibitor pifithrin-alpha (PFT-alpha) blocked both promoter inducibility and protein up-regulation of CD95 in response to Dox. Up-regulated CD95 in Dox-treated cells was functional in eliciting apoptosis upon incubation of the cells with an agonistic CD95 antibody. However, Dox-mediated apoptosis was independent of CD95/CD95L interaction. The analysis of apoptosis in the presence of PFT-alpha and benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone revealed that both p53 and caspase activation are required for Dox-mediated apoptosis of HUVECs. Finally, Dox triggered Bcl-2 down-regulation, cytochrome c release from mitochondria, and the activation of caspases 9 and 3, suggesting the involvement of a mitochondrially operated pathway of apoptosis. These results highlight the role of p53 in the response of primary endothelial cells to genotoxic drugs and may reveal a novel mechanism underlying the antitumoral properties of Dox, related to its ability to induce apoptosis in proliferating endothelial cells.

A promising and increasingly exploited property of hematopoietic stem cells is their ability to efflux the fluorescent dye Hoechst 33342. The Hoechst-negative cells are isolated by fluorescence-activated cell sorting as a so-called side population" (SP) of bone marrow. This SP from bone marrow�

Characterization of molecules with tightly controlled expression patterns during differentiation represents an approach to understanding regulation of hematopoietic stem cell commitment. The multidrug resistance-1 (MDR1) gene product, P-glycoprotein, and the breast cancer resistance protein (BCRP) are expressed differentially during hematopoiesis, with the highest levels in primitive bone marrow stem cell populations that are CD34(low) and CD34(-), respectively. Roles for ATP-binding cassette (ABC) transporter superfamily members in conferring drug resistance have been extensively described. However, recent hematopoietic overexpression studies have begun to reveal previously unknown roles for ABC transporter function in normal and malignant hematopoiesis. Expression of MDR1 and BCRP transporters in the myeloid lineage has been reported in blasts from acute myeloid leukemia, but very low to undetectable in normal myelomonocytic cells. Retroviral-mediated dysregulated expression of the MDR1 transporter resulted in increased hematopoietic repopulating activity and myeloproliferative disease in mice. A distinct functional role for the BCRP transporter as a negative regulator of hematopoietic repopulating activity has recently been demonstrated using the same approach. Additionally, the presence of BCRP expression specifically on hematopoietic side-population stem cells and neural stem/progenitors, makes BCRP an attractive candidate marker for isolation of stem cells with the ability to respond to diverse environmental cues. Regulation of stem cell biology by ABC transporters has emerged as an important new field of investigation. In light of these findings, it will be critical to further characterize this family of proteins in hematopoietic lineage-restricted stem cells and in pluripotent stem cells capable of crossing lineage barriers.

The human ATP-binding cassette superfamily G (White) member 2 (ABCG2) gene and its murine homologue breast cancer resistance protein 1 (Bcrp1) are recently described ATP-binding cassette transporters associated with drug resistance in tumor cell lines, including the MCF-7 cell line, selected for its resistance to mitoxantrone (MCF-7/MitoR). Infection of MCF-7 cells with the retroviral vector containing ABCG2 cDNA (G1-ABCG2) resulted in cells (MCF-7/ABCG2) that were resistant to mitoxantrone at levels similar to those observed in MCF-7/MitoR cells. Previous studies have shown that pluripotent hematopoietic stem cells overexpress the multidrug-resistant transport (MDR1) gene and efflux rhodamine, a substrate for the MDR1 transporter. Other studies have identified a primitive hematopoietic stem cell population, or side population (SP) cells, which are identified by their efflux of the fluorescent dye, Hoechst 33342. In an attempt to identify the transport genes responsible for this phenotype, we examined the uptake of Hoechst 33342 into MCF-7, MCF-7/MitoR, and MCF-7 cells infected with a retroviral vector expressing the ABCG2 gene (MCF-7/ABCG2). MCF-7/MitoR cells as well as MCF-7/ABCG2 cells demonstrated lower levels of Hoechst 33342 uptake compared with the parental MCF-7 cells. We also examined the level of the mouse Bcrp1 RNA in SP cells and non-SP cells isolated from mouse hematopoietic cells. Mouse SP cells expressed relatively high levels of Bcrp1 mRNA relative to non-SP cells. These results suggest that Hoechst 33342 is a substrate for the ABCG2 transporter and that ABCG2/Bcrp1 expression may serve as a marker for hematopoietic stem cells in hematopoietic cells.