Toxicity of clopidogrel and ticlopidine on human myeloid progenitor cells: importance of metabolites

Ticlopidine and clopidogrel are thienopyridine derivatives used for inhibition of platelet aggregation. Not only hepatotoxicity, but also bone marrow toxicity may limit their use. Aims of the study were to find out whether non-metabolized drug and/or metabolites are responsible for myelotoxicity and whether the inactive clopidogrel metabolite clopidogrel carboxylate contributes to myelotoxicity. We used myeloid progenitor cells isolated from human umbilical cord blood in a colony-forming unit assay to assess cytotoxicity. Degradation of clopidogrel, clopidogrel carboxylate or ticlopidine (studied at 10 and 100 μM) was monitored using LC/MS. Clopidogrel and ticlopidine were both dose-dependently cytotoxic starting at 10 μM. This was not the case for the major clopidogrel metabolite clopidogrel carboxylate. Pre-incubation with recombinant human CYP3A4 not only caused degradation of clopidogrel and ticlopidine, but also increased cytotoxicity. In contrast, clopidogrel carboxylate was not metabolized by recombinant human CYP3A4. Pre-incubation with freshly isolated human granulocytes was not only associated with a myeloperoxidase-dependent degradation of clopidogrel, clopidogrel carboxylate and ticlopidine, but also with dose-dependent cytotoxicity of these compounds starting at 10 μM. In conclusion, both non-metabolized clopidogrel and ticlopidine as well as metabolites of these compounds are toxic towards myeloid progenitor cells. Taking exposure data in humans into account, the myelotoxic element of clopidogrel therapy is likely to be secondary to the formation of metabolites from clopidogrel carboxylate by myeloperoxidase. Concerning ticlopidine, both the parent compound and metabolites formed by myeloperoxidase may be myelotoxic in vivo. The molecular mechanisms of cytotoxicity have to be investigated in further studies.

Increased cytotoxicity of cisplatin in SK-MEL 28 melanoma cells upon down-regulation of melanoma inhibitor of apoptosis protein

BACKGROUND: Malignant melanoma is a highly metastatic cutaneous cancer and typically refractory to chemotherapy. Deregulated apoptosis has been identified as a major cause of cancer drug resistance, and upregulated expression of the inhibitor of apoptosis protein melanom, an inhibitor of apoptosis (ML-IAP) is frequent in melanoma. METHODS: Based on the conclusion that ML-IAP expression contributes to a malignant phenotype, we down-regulated the ML-IAP mRNA using sequence optimized antisense oligonucleotides. RESULTS: As measured by real-time PCR, oligonucleotides M706 and M711 inhibited ML-IAP mRNA expression by 47% and 52%, respectively in the highly metastatic and drug resistant SK-MEL28 cell line. Oligonucleotide M706, which was previously evaluated in G361 cells as the most efficient inhibitor of ML-IAP expression, was chosen to compare cell viability and drug sensitivity of these two melanoma cell lines with different p53 functionality. Protein expression was reduced by oligonucleotide M706 to 49% of the normal level and resulted in a dose-dependent specific reduction of cell viability with a maximum of 39% at 600 nM. Typical morphological changes showed that loss of viability was mainly due to cell death. In combination experiments, the use of oligonucleotide M706 resulted in a two-fold increase of cisplatin cytotoxicity at different concentrations of oligonucleotide and cisplatin (P<0.05). This is in line with our previous findings in G361 melanoma cell line, in which oligonucleotide M706 caused a 3-fold increase in cisplatin cytotoxicity. CONCLUSION: Our data suggest the use of ML-IAP antisense oligonucleotides to overcome drug resistance in metastatic melanoma, in spite of its p53 status.

Studies on the adhesion properties of the liver-metastatic murine RAW117 large-cell lymphoma cells: Roles of $\beta\sb3$ integrin

Metastasis is the major cause of death in cancer patients. Since many cancers show organ-preference of metastasis, elucidation of the underlying mechanisms of metastasis will benefit diagnosis or treatment of metastatic diseases. Adhesion mechanisms are thought to be involved in organ-preference of metastasis, because metastatic cells show organ preference in adhering to organ-derived microvascular endothelial cells. The adhesion molecules in this process remain largely unidentified. I have examined a series of murine RAW117 large-cell lymphoma cells variants selected in vivo for liver-colonizing properties ($\rm{H10{>>}L17>P}$). The highly liver-metastatic H10 cells were found to differentially express much higher levels of integrin $\alpha\rm\sb{v}\beta\sb3$ than L17 or P cells. H10 cells also adhered at higher rates to vitronectin and fibronectin than to fibrinogen, fibrin, laminin and type I collagen, and adhered at significantly higher rates to (GRGDS)$\sb4$ than to monomeric RGD-peptides. In contrast, P and L17 cells did not adhere well to the above substrates. H10 cells also spread well on vitronectin and migrated toward vitronectin concentration gradients. Pretreament of H10 cells with anti-$\beta\sb3$ monoclonal antibodies resulted in significant decreases in adhesion of H10 cells to vitronectin and immobilized (GRGDS)$\sb4$, and reduced the formation of experimental liver metastases in syngeneic Balb/c mice.^ Adhesion of RAW117 cells under hydrodynamic shear stresses was also studied because tumor cell adhesion occurs under fluid shear stresses in target organ microvessels. Similar to their properties found with static adhesion assays, H10 cells stabilized their hydrodynamic adhesion to vitronectin, fibronectin and (GRGDS)$\sb4$ much more quickly than P or L17 cells. Unlike their static adhesion properties, RAW117 cells showed differential adhesion stabilization to liver-sinusoidal endothelial cell-derived extracellular matrix ($\rm{H10{>>}L17>P}$). Although not supporting static adhesion of RAW117 cells, monomeric RGD-peptides mediated adhesion stabilization of H10 cells but not L17 or P cells. Integrin $\rm\alpha\sb{v}\beta\sb3$ was found to be involved in stabilizing H10 cell adhesion to vitronectin, (GRGDS)$\sb4$, monomeric RGD-peptide R1, and liver sinusoidal endothelial cell-derived extracellular matrix.^ This study is the first to provide evidence that integrin $\rm\alpha\sb{v}\beta\sb3$ is differentially expressed in liver-metastatic lymphoma cells and involved in differential adhesion of these cells. The results indicate that strong static adhesion and especially the unique hydrodynamic adhesion of RAW117 cells to the RGD-containing substrates correlate with liver-metastatic potentials. Thus, integrin $\rm\alpha\sb{v}\beta\sb3$ may play an important role in liver-preferential metastasis of RAW117 large-cell lymphoma cells. ^

Comparative dendritic cell biology of veterinary mammals.

Dendritic cells (DC) have a main function in innate immunity in that they sense infections and environmental antigens at the skin and mucosal surfaces and thereby critically influence decisions about immune activation or tolerance. As professional antigen-presenting cells, they are essential for induction of adaptive immune responses. Consequently, knowledge on this cell type is required to understand the immune systems of veterinary mammals, including cattle, sheep, pigs, dogs, cats, and horses. Recent ontogenic studies define bona fide DC as an independent lineage of hematopoietic cells originating from a common precursor. Distinct transcription factors control the development into the two subsets of classical DC and plasmacytoid DC. These DC subsets express a distinguishable transcriptome, which differs from that of monocyte-derived DC. Using a comparative approach based on phenotype and function, this review attempts to classify DC of veterinary mammals and to describe important knowledge gaps.

IDENTIFICATION AND CHARACTERIZATION OF DISTINCT POPULATIONS OF CLONOGENIC BONE MARROW STROMAL CELLS CAPABLE OF TRANSFERRING THE HEMATOPOIETIC MICROENVIRONMENT IN VIVO AND SUPPORTING LT-HSCs IN VITRO

Bone marrow (BM) stromal cells are ascribed two key functions, 1) stem cells for non-hematopoietic tissues (MSC) and 2) as components of the hematopoietic stem cell niche. Current approaches studying the stromal cell system in the mouse are complicated by the low yield of clonogenic progenitors (CFU-F). Given the perivascular location of MSC in BM, we developed an alternative methodology to isolate MSC from mBM. An intact ‘plug’ of bone marrow is expelled from bones and enzymatically disaggregated to yield a single cell suspension. The recovery of CFU-F (1917.95+199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32+1.9) by at least 2 orders of magnitude (P<0.001; N = 8) with an accompanying 196-fold enrichment of CFU-F frequency. Purified BM stromal and vascular endothelial cell populations are readily obtained by FACS. A detailed immunophenotypic analysis of lineage depleted BM identified PDGFRαβPOS stromal cell subpopulations distinguished by their expression of CD105. Both subpopulations retained their original phenotype of CD105 expression in culture and demonstrate MSC properties of multi-lineage differentiation and the ability to transfer the hematopoietic microenvironment in vivo. To determine the capacity of either subpopulation to support long-term multi-lineage reconstituting HSCs, we fractionated BM stromal cells into either the LinNEGPDGFRαβPOSCD105POS and LINNEGPDGFRαβPOSCD105LOW/- populations and tested their capacity to support LT-HSC by co-culturing each population with either 1 or 10 HSCs for 10 days. Following the 10 day co-culture period, both populations supported transplantable HSCs from 10 cells/well co-cultures demonstrating high levels of donor repopulation with an average of 65+23.6% chimerism from CD105POS co-cultures and 49.3+19.5% chimerism from the CD105NEG co-cultures. However, we observed a significant difference when mice were transplanted with the progeny of a single co-cultured HSC. In these experiments, CD105POS co-cultures (100%) demonstrated long-term multi- lineage reconstitution, while only 4 of 8 mice (50%) from CD105NEG -single HSC co-cultures demonstrated long-term reconstitution, suggesting a more limited expansion of functional stem cells. Taken together, these results demonstrate that the PDGFRαβCD105POS stromal cell subpopulation is distinguished by a unique capacity to support the expansion of long-term reconstituting HSCs in vitro.

Mechanical stretch induces vascular permeability factor in human mesangial cells: Mechanisms of signal transduction

Hemodynamic abnormalities have been implicated in the pathogenesis of the increased glomerular permeability to protein of diabetic and other glomerulopathies. Vascular permeability factor (VPF) is one of the most powerful promoters of vascular permeability. We studied the effect of stretch on VPF production by human mesangial cells and the intracellular signaling pathways involved. The application of mechanical stretch (elongation 10%) for 6 h induced a 2.4-fold increase over control in the VPF mRNA level (P < 0.05). There was a corresponding 3-fold increase in VPF protein level by 12 h (P < 0.001), returning to the baseline by 24 h. Stretch-induced VPF secretion was partially prevented both by the protein kinase C (PKC) inhibitor H7 (50 μM: 72% inhibition, P < 0.05) and by pretreatment with phorbol ester (phorbol-12-myristate-13 acetate 10−7 M: 77% inhibition, P < 0.05). A variety of protein tyrosine kinase (PTK) inhibitors, genistein (20 μg/ml), herbimycin A (3.4 μM), and a specific pp60src peptide inhibitor (21 μM) also significantly reduced, but did not entirely prevent, stretch-induced VPF protein secretion (respectively 63%, 80%, and 75% inhibition; P < 0.05 for all). The combination of both PKC and PTK inhibition completely abolished the VPF response to mechanical stretch (100% inhibition, P < 0.05). Stretch induces VPF gene expression and protein secretion in human mesangial cells via PKC- and PTK-dependent mechanisms.

Dendritic cell ontogeny: A human dendritic cell lineage of myeloid origin

Dendritic cells (DC) have been thought to represent a family of closely related cells with similar functions and developmental pathways. The best-characterized precursors are the epidermal Langerhans cells, which migrate to lymphoid organs and become activated DC in response to inflammatory stimuli. Here, we demonstrate that a large subset of DC in the T cell-dependent areas of human lymphoid organs are nonactivated cells and belong to a separate lineage that can be identified by high levels of the interleukin 3 receptor α chain (IL-3Rαhi). The CD34+IL-3Rαhi DC progenitors are of myeloid origin and are distinct from those that give rise to Langerhans cells in vitro. The IL-3Rαhi DC furthermore appear to migrate to lymphoid organs independently of inflammatory stimuli or foreign antigens. Thus, DC are heterogeneous with regard to function and ontogeny.

Activation of RhoA by Lysophosphatidic Acid and Gα12/13 Subunits in Neuronal Cells: Induction of Neurite Retraction

Neuronal cells undergo rapid growth cone collapse, neurite retraction, and cell rounding in response to certain G protein–coupled receptor agonists such as lysophosphatidic acid (LPA). These shape changes are driven by Rho-mediated contraction of the actomyosin-based cytoskeleton. To date, however, detection of Rho activation has been hampered by the lack of a suitable assay. Furthermore, the nature of the G protein(s) mediating LPA-induced neurite retraction remains unknown. We have developed a Rho activation assay that is based on the specific binding of active RhoA to its downstream effector Rho-kinase (ROK). A fusion protein of GST and the Rho-binding domain of ROK pulls down activated but not inactive RhoA from cell lysates. Using GST-ROK, we show that in N1E-115 neuronal cells LPA activates endogenous RhoA within 30 s, concomitant with growth cone collapse. Maximal activation occurs after 3 min when neurite retraction is complete and the actin cytoskeleton is fully contracted. LPA-induced RhoA activation is completely inhibited by tyrosine kinase inhibitors (tyrphostin 47 and genistein). Activated Gα12 and Gα13 subunits mimic LPA both in activating RhoA and in inducing RhoA-mediated cytoskeletal contraction, thereby preventing neurite outgrowth. We conclude that in neuronal cells, LPA activates RhoA to induce growth cone collapse and neurite retraction through a G12/13-initiated pathway that involves protein-tyrosine kinase activity.

Regulation of the Actin Cytoskeleton by Thrombin in Human Endothelial Cells: Role of Rho Proteins in Endothelial Barrier Function

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.

Vasopressin mRNA localization in nerve cells: Characterization of cis-acting elements and trans-acting factors

mRNA localization is a complex pathway. Besides mRNA sorting per se, this process includes aspects of regulated translation. It requires protein factors that interact with defined sequences (or sequence motifs) of the transcript, and the protein/RNA complexes are finally guided along the cytoskeleton to their ultimate destinations. The mRNA encoding the vasopressin (VP) precursor protein is localized to the nerve cell processes in vivo and in primary cultured nerve cells. Sorting of VP transcripts to dendrites is mediated by the last 395 nucleotides of the mRNA, the dendritic localizer sequence, and it depends on intact microtubules. In vitro interaction studies with cytosolic extracts demonstrated specific binding of a protein, enriched in nerve cell tissues, to the radiolabeled dendritic localizer sequence probe. Biochemical purification revealed that this protein is the multifunctional poly(A)-binding protein (PABP). It is well known for its ability to bind with high affinity to poly(A) tails of mRNAs, prerequisite for mRNA stabilization and stimulation of translational initiation, respectively. With lower affinities, PABP can also associate with non-poly(A) sequences. The physiological consequences of these PABP/RNA interactions are far from clear but may include functions such as translational silencing. Presumably, the translational state of mRNAs subject to dendritic sorting is influenced by external stimuli. PABP thus could be a component required to regulate local synthesis of the VP precursor and possibly of other proteins.

c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1.

Two genetic events contribute to the development of endemic Burkitt lymphoma (BL) infection of B lymphocytes with Epstein-Barr virus (EBV) and the activation of the protooncogene c-myc through chromosomal translocation. The viral genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for transformation of primary human B cells by EBV in vitro; however, these genes are not expressed in BL cells in vivo. To address the question whether c-myc activation might abrogate the requirement of the EBNA2 and LMP1 function, we have introduced an activated c-myc gene into an EBV-transformed cell line in which EBNA2 was rendered estrogen-dependent through fusion with the hormone binding domain of the estrogen receptor. The c-myc gene was placed under the control of regulatory elements of the immunoglobulin kappa locus composed a matrix attachment region, the intron enhancer, and the 3' enhancer. We show here that transfection of a c-myc expression plasmid followed by selection for high MYC expression is capable of inducing continuous proliferation of these cells in the absence of functional EBNA2 and LMP1. c-myc-induced hormone-independent proliferation was associated with a dramatic change in the growth behavior as well as cell surface marker expression of these cells. The typical lymphoblastoid morphology and phenotype of EBV-transformed cells completely changed into that of BL cells in vivo. We conclude that the phenotype of BL cells reflects the expression pattern of viral and cellular genes rather than its germinal center origin.

Disaccharide uptake and priming in animal cells: inhibition of sialyl Lewis X by acetylated Gal beta 1-->4GlcNAc beta-O-naphthalenemethanol.

Inhibitors of glycosylation provide a tool for studying the biology of glycoconjugates. One class of inhibitors consists of glycosides that block glycoconjugate synthesis by acting as primers of free oligosaccharide chains. A typical primer contains one sugar linked to a hydrophobic aglycone. In this report, we describe a way to use disaccharides as primers. Chinese hamster ovary cells readily take up glycosides containing a pentose linked to naphthol, but they take up hexosides less efficiently and disaccharides not at all. Linking phenanthrol to a hexose improves its uptake dramatically but has no effect on disaccharides. To circumvent this problem, analogs of Xyl beta 1-->6Gal beta-O-2-naphthol were tested as primers of glycosaminoglycan chains. The unmodified disaccharide did not prime, but methylated derivatives had activity in the order Xyl beta 1-->6Gal(Me)3-beta-O-2-naphthol > Xyl beta 1-->6Gal (Me)2 beta-O-2-naphthol >> Xyl beta 1-->6Gal(Me)beta-O-2-naphthol. Acetylated Xyl beta 1-->6Gal beta-O-2-naphthol also primed glycosaminoglycans efficiently, suggesting that the terminal xylose residue was exposed by removing the acetyl groups. The general utility of using acetyl groups to create disaccharide primers was shown by the priming of oligosaccharides on peracetylated Gal beta 1-->4GlcNAc beta-O-naphthalenemethanol. This disaccharide inhibited sialyl Lewis X expression on HL-60 cells.

Epstein-Barr virus-mediated protection against etoposide-induced apoptosis in BJA-B B cell lymphoma cells: role of Bcl-2 and caspase proteins

Epstein-Barr virus (EBV)-infected B cell lymphomas are resistant to apoptosis during cancer development and treatment with therapies. The molecular controls that determine why EBV infection causes apoptosis resistance need further definition. EBV-positive and EBV-negative BJA-B B cell lymphoma cell lines were used to compare the expression of selected apoptosis-regulating Bcl-2 and caspase proteins in EBV-related apoptosis resistance, after 8 hr or 18-24 hr etoposide treatment (80 muM). Apoptosis was quantified using morphology and verified with Hoechst 33258 nuclear stain and electron microscopy. Fluorescence activated cell sorting (FACS) was used to analyse effects on cell cycle of the EBV infection as well as etoposide treatment. Anti-apoptotic Bcl-2 and Bcl-XL, pro-apoptotic Bax, caspase-3 and caspase-9 expression and activation were analysed using Western immunoblots and densitometry. EBV-positive cultures had significantly lower levels of apoptosis in untreated and etoposide-treated cultures in comparison with EBV-negative cultures (p &lt; 0.05). FACS analysis indicated a strong G2/M block in both cell sublines after etoposide treatment. Endogenous Bcl-2 was minimal in the EBV-negative cells in comparison with strong expression in EBV-positive cells. These levels did not alter with etoposide treatment. Bcl-XL was expressed endogenously in both cell lines and had reduced expression in EBV-negative cells after etoposide treatment. Bax showed no etoposide-induced alterations in expression. Pro-caspase-9 and -3 were seen in both EBV-positive and -negative cells. Etoposide induced cleavage of caspase-9 in both cell lines, with the EBV-positive cells having proportionally less cleavage product, in agreement with their lower levels of apoptosis. Caspase-3 cleavage occurred in the EBV-negative etoposide-treated cells but not in the EBV-positive cells. The results indicate that apoptosis resistance in EBV-infected B cell lymphomas is promoted by an inactive caspase-3 pathway and elevated expression of Bcl-2 that is not altered by etoposide drug treatment.

Thyroid hormone export from cells: contribution of P-glycoprotein

Verapamil inhibits tri-iodothyronine (T-3) efflux from several cell types, suggesting the involvement of multidrug resistance-associated (MDR) proteins in T-3 transport. The direct involvement of P-glycoprotein (P-gp) has not, however, been investigated. We compared the transport of I-125-T-3 in MDCKII cells that had been transfected with mdr1 cDNA (MDCKII-MDR) versus wild-type MDCKII cells (MDCKII), and examined the effect of conventional (verapamil and nitrendipine) and specific MDR inhibitors (VX 853 and VX 710) on I-125-T-3 efflux. We confirmed by Western blotting the enhanced expression of P-gp in MDCKII-MDR cells. The calculated rate of I-125-T-3 efflux from MDCKII-MDR cells (around 0.30/min) was increased twofold compared with MDCKII cells (around 0.15/min). Overall, cellular accumulation of I-125-T-3 was reduced by 26% in MDCKII-MDR cells compared with MDCKII cells, probably reflecting enhanced export of T-3 from MDCKII-MDR cells rather than reduced cellular uptake, as P-gp typically exports substances from cells. Verapamil lowered the rate of I-125-T-3 efflux from both MDCKII and MDCKII-MDR cells by 42% and 66% respectively, while nitrendipine reduced I-125-T-3 efflux rate by 36% and 48% respectively, suggesting that both substances inhibited other cellular T-3 transporters in addition to P-gp. The specific MDR inhibitors VX 853 and VX 710 had no effect of I-125-T-3 efflux rate from wild-type MDCKII cells but reduced I-125-T-3 export in MDCKII-MDR cells by 50% and 53% respectively. These results have provided the first direct evidence that P-gp exports thyroid hormone from cells.