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The Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor that is activated by phosphorylation at S102 whereby it induces the expression of growth promoting genes such as EGFR and HER-2. We recently illustrated by an in vitro kinase assay that a novel peptide to YB-1 was highly phosphorylated by the serine/threonine p90 S6 kinases RSK-1 and RSK-2, and to a lesser degree PKCα and AKT. Herein, we sought to develop this decoy cell permeable peptide (CPP) as a cancer therapeutic. This 9-mer was designed as an interference peptide that would prevent endogenous YB-1(S102) phosphorylation based on molecular docking. In cancer cells, the CPP blocked P-YB-1(S102) and down-regulated both HER-2 and EGFR transcript level and protein expression. Further, the CPP prevented YB-1 from binding to the EGFR promoter in a gel shift assay. Notably, the growth of breast (SUM149, MDA-MB-453, AU565) and prostate (PC3, LNCap) cancer cells was inhibited by ∼90% with the CPP. Further, treatment with this peptide enhanced sensitivity and overcame resistance to trastuzumab in cells expressing amplified HER-2. By contrast, the CPP had no inhibitory effect on the growth of normal immortalized breast epithelial (184htert) cells, primary breast epithelial cells, nor did it inhibit differentiation of hematopoietic progenitors. These data collectively suggest that the CPP is a novel approach to suppressing the growth of cancer cells while sparing normal cells and thereby establishes a proof-of-concept that blocking YB-1 activation is a new course of cancer therapeutics.

Here, we have studied the activity of a novel protein-tyrosine kinase inhibitor that is selective for the Src family of tyrosine kinases. We have focused our study on the effects of this compound on T cell receptor-induced T cell activation, a process dependent on the activity of the Src kinases Lck and FynT. This compound is a nanomolar inhibitor of Lck and FynT, inhibits anti-CD3-induced protein-tyrosine kinase activity in T cells, demonstrates selectivity for Lck and FynT over ZAP-70, and preferentially inhibits T cell receptor-dependent anti-CD3-induced T cell proliferation over non-T cell receptor-dependent phorbol 12-myristate 13-acetate/interleukin-2 (IL-2)-induced T cell proliferation. Interestingly, this compound selectively inhibits the induction of the IL-2 gene, but not the granulocyte-macrophage colony-stimulating factor or IL-2 receptor genes. This compound offers a useful new tool for examining the role of the Lck and FynT tyrosine kinases versus ZAP-70 in T cell activation as well as the role of other Src family kinases in receptor function.

Induced pluripotent stem (iPS) cells are generated from somatic cells by genetic manipulation. Reprogramming entails multiple transgene integrations and occurs apparently stochastically in rare cells over many days. Tissue stem cells may be subject to less-stringent epigenetic restrictions than other cells and might therefore be more amenable to deprogramming. We report that brain-derived neural stem (NS) cells acquire undifferentiated morphology rapidly and at high frequency after a single round of transduction with reprogramming factors. However, critical attributes of true pluripotency--including stable expression of endogenous Oct4 and Nanog, epigenetic erasure of X chromosome silencing in female cells, and ability to colonise chimaeras--were not attained. We therefore applied molecularly defined conditions for the derivation and propagation of authentic pluripotent stem cells from embryos. We combined dual inhibition (2i) of mitogen-activated protein kinase signalling and glycogen synthase kinase-3 (GSK3) with the self-renewal cytokine leukaemia inhibitory factor (LIF). The 2i/LIF condition induced stable up-regulation of Oct4 and Nanog, reactivation of the X chromosome, transgene silencing, and competence for somatic and germline chimaerism. Using 2i /LIF, NS cell reprogramming required only 1-2 integrations of each transgene. Furthermore, transduction with Sox2 and c-Myc is dispensable, and Oct4 and Klf4 are sufficient to convert NS cells into chimaera-forming iPS cells. These findings demonstrate that somatic cell state influences requirements for reprogramming and delineate two phases in the process. The ability to capture pre-pluripotent cells that can advance to ground state pluripotency simply and with high efficiency opens a door to molecular dissection of this remarkable phenomenon.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) eliminates many epigenetic modifications that characterize differentiated cells. In this study, we tested whether functional differences between chronic obstructive pulmonary disease (COPD) and non-COPD fibroblasts could be reduced utilizing this approach. Primary fibroblasts from non-COPD and COPD patients were reprogrammed to iPSCs. Reprogrammed iPSCs were positive for oct3/4, nanog, and sox2, formed embryoid bodies in vitro, and induced teratomas in nonobese diabetic/severe combined immunodeficient mice. Reprogrammed iPSCs were then differentiated into fibroblasts (non-COPD-i and COPD-i) and were assessed either functionally by chemotaxis and gel contraction or for gene expression by microarrays and compared with their corresponding primary fibroblasts. Primary COPD fibroblasts contracted three-dimensional collagen gels and migrated toward fibronectin less robustly than non-COPD fibroblasts. In contrast, redifferentiated fibroblasts from iPSCs derived from the non-COPD and COPD fibroblasts were similar in response in both functional assays. Microarray analysis identified 1,881 genes that were differentially expressed between primary COPD and non-COPD fibroblasts, with 605 genes differing by more than twofold. After redifferentiation, 112 genes were differentially expressed between COPD-i and non-COPD-i with only three genes by more than twofold. Similar findings were observed with microRNA (miRNA) expression: 56 miRNAs were differentially expressed between non-COPD and COPD primary cells; after redifferentiation, only 3 miRNAs were differentially expressed between non-COPD-i and COPD-i fibroblasts. Interestingly, of the 605 genes that were differentially expressed between COPD and non-COPD fibroblasts, 293 genes were changed toward control after redifferentiation. In conclusion, functional and epigenetic alterations of COPD fibroblasts can be reprogrammed through formation of iPSCs.

Background Proteoglycans are found on the cell surface and in the extracellular matrix, and serve as prime sites for interaction with signaling molecules. Proteoglycans help regulate pathways that control stem cell fate, and therefore represent an excellent tool to manipulate these pathways. Despite their importance, there is a dearth of data linking glycosaminoglycan structure within proteoglycans with stem cell differentiation. Methods Human embryonic stem cell line WA09 (H9) was differentiated into early mesoderm and endoderm lineages, and the glycosaminoglycanomic changes accompanying these transitions were studied using transcript analysis, immunoblotting, immunofluorescence and disaccharide analysis. Results Pluripotent H9 cell lumican had no glycosaminoglycan chains whereas in splanchnic mesoderm lumican was glycosaminoglycanated. H9 cells have primarily non-sulfated heparan sulfate chains. On differentiation towards splanchnic mesoderm and hepatic lineages N-sulfo group content increases. Differences in transcript expression of NDST1, HS6ST2 and HS6ST3, three heparan sulfate biosynthetic enzymes, within splanchnic mesoderm cells compared to H9 cells correlate to changes in glycosaminoglycan structure. Conclusions Differentiation of embryonic stem cells markedly changes the proteoglycanome. General significance The glycosaminoglycan biosynthetic pathway is complex and highly regulated, and therefore, understanding the details of this pathway should enable better control with the aim of directing stem cell differentiation. ?? 2014 Elsevier B.V. All rights reserved.

The mechanism of uptake of the thyroid hormones, 3,5,3'-triiodo-L-thyronine (L-T3) and L-thyroxine (L-T4), was studied in rat pituitary GH4C1 cells. The major portion (approximately 65{\%}) of L-T3 transport was stereospecific and saturable. Transport of L-T3 was 8-10 times more rapid than transport of D-T3. [125I]L-T3 transport was saturable at microM concentrations; a Lineweaver-Burk plot was linear with Km = 0.4 microM and Vmax = 4 pmol/min/10(6) cells. Unlabeled analogs competed with [125I]L-T3 uptake in the order L-T3 {\textgreater} or = L-T4 {\textgreater} 3,3',5'-triiodo-L-thyronine (reverse-T3), D-T3, D-T4, and L-thyronine. L-T3 and L-T4 also both effectively inhibited [125I]L-T4 transport. Uptake of [125I]L-T3 was inhibited 40-55{\%} by large neutral amino acids and 77{\%} by 80 microM beta-2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, an inhibitor selective for the L system of amino acid uptake. Conversely, L-T3 inhibited the transport of [3H]leucine by pituitary cells (IC50 = 2 microM), but D-T3 and 3,5,3'-triiodothyroacetic acid (Triac) did not. L-Leucine was transported much more efficiently (Vmax = 0.65 mumol/min/10(6) cells) than L-T3 by GH4C1 cells. The results show that L-T3 and L-T4 share the same stereospecific transport pathway in pituitary cells, that the transport mechanism is saturable at supraphysiological thyroid hormone concentrations, and that the L system is partially responsible for L-T3 transport.

Human embryonic stem cells (hESC) can differentiate to cardiomyocytes in vitro but with generally poor efficiency. Here, we describe a novel method for the efficient generation of cardiomyocytes from hESC in a scalable suspension culture process. Differentiation in serum-free medium conditioned by the cell line END2 (END2-CM) readily resulted in differentiated cell populations with more than 10% cardiomyocytes without further enrichment. By screening candidate molecules, we have identified SB203580, a specific p38 MAP kinase inhibitor, as a potent promoter of hESC-cardiogenesis. SB203580 at concentrations textless10 microM, induced more than 20% of differentiated cells to become cardiomyocytes and increased total cell numbers, so that the overall cardiomyocyte yield was approximately 2.5-fold higher than controls. Gene expression indicated that early mesoderm formation was favored in the presence of SB203580. Accordingly, transient addition of the inhibitor at the onset of differentiation only was sufficient to determine the hESC fate. Patch clamp electrophysiology showed that the distribution of cardiomyocyte phenotypes in the population was unchanged by the compound. Interestingly, cardiomyogenesis was strongly inhibited at SB203580 concentrations textgreater or =15 microM. Thus, modulation of the p38MAP kinase pathway, in combination with factors released by END2 cells, plays an essential role in early lineage determination in hESC and the efficiency of cardiomyogenesis. Our findings contribute to transforming human cardiomyocyte generation from hESC into a robust and scalable process.

Electrospun poly-l-lactic acid (PLLA) nanofiber mats carrying surface amine groups, previously introduced by nitrogen atmospheric pressure nonequilibrium plasma, are embedded into aqueous solutions of oligomeric acrylamide-end capped AGMA1, a biocompatible polyamidoamine with arg-gly-asp (RGD)-reminiscent repeating units. The resultant mixture is finally cured giving PLLA-AGMA1 hydrogel composites that absorb large amounts of water and, in the swollen state, are translucent, soft, and pliable, yet as strong as the parent PLLA mat. They do not split apart from each other when swollen in water and remain highly flexible and resistant, since the hydrogel portion is covalently grafted onto the PLLA nanofibers via the addition reaction of the surface amine groups to a part of the terminal acrylic double bonds of AGMA1 oligomers. Preliminary tested as scaffolds, the composites prove capable of maintaining short-term undifferentiated cultures of human pluripotent stem cells in feeder-free conditions.

Recent studies suggest that forced activation of AMP-activated protein kinase (AMPK) could inhibit melanoma cell proliferation. In this report, we evaluated the anti-melanoma cell activity by a novel small-molecular AMPK activator, GSK621. Treatment of GSK621 decreased survival and proliferation of human melanoma cells (A375, WM-115 and SK-Mel-2 lines), which was accompanied by activation of caspase-3/-9 and apoptosis. Reversely, caspase inhibitors attenuated GSK621-induced cytotoxicity against melanoma cells. Significantly, GSK621 was more potent than other AMPK activators (A769662, Compound 13 and AICAR) in inhibiting melanoma cells. Intriguingly, same GSK621 treatment was non-cytotoxic or pro-apoptotic against human melanocytes. Molecularly, we showed that activation of AMPK mediated GSK621's activity against melanoma cells. AMPK$\alpha$1 shRNA knockdown or dominant negative mutation (T172A) dramatically attenuated GSK621-induced melanoma cell lethality. Further studies revealed that MEK-ERK activation might be the primary resistance factor of GSK621. MEK-ERK inhibition, either genetically or pharmacologically, significantly sensitized melanoma cells to GSK-621. Remarkably, intraperitoneal (i.p.) injection of GSK621 inhibited A375 tumor growth in SCID mice. Co-administration of MEK-ERK inhibitor MEK162 further sensitized GSK621-induced anti-A375 tumor activity in vivo. Together, the results imply that targeted activation of AMPK by GSK621 inhibits melanoma cell survival and proliferation. MEK-ERK inhibition may further sensitize GSK621's anti-melanoma cell activity in vitro and in vivo.

There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB), used in the Aldeflour assay, has been considered a specific inhibitor for ALDH1A1 isoform. In this study, we explore the effects of human ALDH isoenzymes, ALDH1A2 and ALDH2, on drug resistance and proliferation, and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay, Western blot, RT-PCR, and Aldefluor assay. Both cell lines, with either ALDH1A2 or ALDH2, exhibited higher cell proliferation rates, higher clonal efficiency, and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors, DEAB and disulfiram, we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65-90%. Furthermore, our TLDA results revealed that ALDH1, ALDH7, ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition, other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells.

The immunosuppressive macrolide rapamycin shows marked structural similarity to FK-506, and like FK-506 inhibits the activation of cultured T and B lymphocytes at concentrations as low as 10(-10) M. However, rapamycin blocks T-lymphocyte proliferation at a much later stage than FK-506. It also inhibits human, porcine and murine T- and B-lymphocyte activation by all pathways tested, including pathways which are insensitive to FK-506, such as interleukin-2 (IL-2)-mediated proliferation of IL-2-dependent T-cell lines, activation of human peripheral blood T lymphocytes by phorbol ester and anti-CD28 and activation of murine B lymphocytes by bacterial lipopolysaccharide. Thus these two macrolides that bind competitively to the same major intracellular receptor protein inhibit T- and B-lymphocyte activation by quite distinct mechanisms. View Publication

In recent years, human embryonic stem (hES) cells have become a promising cell source for regenerative medicine. Although hES cells have the ability for unlimited self-renewal, potential adverse effects of long-term cell culture upon hES cells must be investigated before therapeutic applications of hES cells can be realized. Here we investigated changes in molecular profiles associated with young (textless60 passages) and old (textgreater120 passages) cells of the H9 hES cell line as well as young (textless85 passages) and old (textgreater120 passages) cells of the PKU1 hES cell line. Our results show that morphology, stem cell markers, and telomerase activity do not differ significantly between young and old passage cells. Cells from both age groups were also shown to differentiate into derivatives of all 3 germ layers upon spontaneous differentiation in vitro. Interestingly, mitochondrial dysfunction was found to occur with prolonged culture. Old passage cells of both the H9 and PKU1 lines were characterized by higher mitochondrial membrane potential, larger mitochondrial morphology, and higher reactive oxygen species content than their younger counterparts. Teratomas derived from higher passage cells were also found to have an uneven preference for differentiation compared with tumors derived from younger cells. These findings suggest that prolonged culture of hES cells may negatively impact mitochondrial function and possibly affect long-term pluripotency.