Oxidative stress protection by novel telomerase activators in mesenchymal stem cells derived from healthy and diseased individuals.

Human Bone Marrow Mesenchymal Stem cells (hMSC) are a promising candidate for cytotherapy. However, the therapeutic potential is limited since the therapy requires ex-vivo cell culturing in which deterioration in cellular viability and aging is observed with time.Telomerase ribonucleoprotein complex re-elongates telomeres and therefore promotes genomic integrity, proliferation and lifespan. Recently we showed that increasing telomerase reverse transcriptase (TERT) expression by novel compound confers resistance from apoptosis induced by oxidative stress. Here we investigated the possibility that a controlled induction of human TERT (hTERT) levels by chemical compounds (AGS-499 and AGS-500) might improve the functionality of hMSC derived from healthy and neurodegenerative diseased individuals. We demonstrate that AGS treatments of hMSC increased telomerase activity and hTERT levels in a time and dose dependent manner. Prolonged treatments with the compounds increased the average telomeres length, without altering population doublings (PD) or inducing chromosomal aberrations. AGS treatments of hMSC protected the cells from apoptosis and DNA damages induced by H2O2, and from the toxicity induced by long term exposure to DMSO. These AGS effects were shown to be mediated by telomerase since they were not observed when TERT was depleted from hMSC or in mouse embryonic stem cells derived from TERT knockout mice. Furthermore, AGS compounds did not alter the functionality of hMSC as examined by their ability to differentiate into various lineages in the presence of the compounds. These results suggest that pharmaceutical increase of telomerase may confer a beneficial therapeutic advantage in regenerative medicine when hMSC therapy is applied.

Percutaneous transaortic occlusion of patent ductus arteriosus using a new versatile angiographic and delivery catheter.

We evaluated the usefulness of patent ductus arteriosus (PDA) occlusion using a new catheter that combines good angiographic properties, easy manipulation across the duct, documentation of aortic and pulmonary artery pressure, and coil delivery for percutaneous transaortic ductal occlusion. Thirty patients (13 females and 17 males) who met the clinical and echocardiographic criteria for the percutaneous closure of PDA were catheterized. The patients selected for this study had a small to moderate PDA with a conal or tubular shape, adequate aortic ampula, and normal or mildly elevated pulmonary artery pressure. The median age was 4.8 years (range, 10 months-20 years). Patients were catheterized by the transaortic approach using the new angiographic and coil delivery catheter specially designed for this procedure. A PDA was closed in every case. A single DuctOcclud pfm coil was delivered in 28 patients, and 2 patients had NitOcclud pfm coils for ductal occlusion. The screening time was measured and found to be 50% less than the measured screening time using other catheters in our laboratory. Only one catheter and one guidewire were used per procedure, with no need for exchange wires or the establishment of an arteriovenous loop. The procedure time was also reduced by 50%. A minimal residual shunt, demonstrated by color-flow Doppler mapping, was present in 2 patients. The new combined angiographic and coil delivery catheter is most suitable for transaortic PDA coil occlusion, significantly decreasing the procedure and screening time.

Cardiac manifestations in thiamine-responsive megaloblastic anemia syndrome.

Thiamine-responsive megaloblastic anemia (TRMA) syndrome is a rare autosomal recessive disorder defined by the occurrence of megaloblastic anemia, diabetes mellitus, and sensorineural deafness, responding in varying degrees to thiamine treatment. Other features of this syndrome gradually develop. We describe three TRMA patients with heart rhythm abnormalities and structural cardiac anomalies. Eight other reported TRMA patients also had cardiac anomalies. Recently, the TRMA gene, SLC19A2, was identified, encoding a functional thiamine transporter. Characterization of the metabolic defect of TRMA may shed light on the role of thiamine in common cardiac abnormalities.

Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow.

Wnt signalling has an important role in cell fate determination, tissue patterning and tumorigenesis. Secreted antagonists of Wnt include Frizzled (Fz)-related proteins (FRPs), Cerberus, Wnt inhibitory factor (WIF) and Dickkopf (Dkk). FRPs, Cerberus and WIF have all been shown to act by binding and sequestering Wnt. We report a novel mechanism of Wnt-signalling inhibition by human Dkk-1. Dkk-1 demonstrated no interaction with Wnt but bound a single cell surface site with high affinity (K(D) = 0.39 nM). Its receptor was detectable in a complex with a relative molecular mass of 240,000 (M(r) 240K) with [(125)I] Dkk-1 by covalent affinity cross-linking. Wnt signalling through beta-catenin is mediated by the Fz receptor and a recently identified low-density-lipoprotein-receptor-related co-receptor, LRP6/Arrow. Overproduction of the 200K LRP6 protein, but not of Fz, strikingly increased Dkk-1 binding as well as the amount of the 240K cross-linked complex, which was shown to be composed of Dkk-1 and LRP6. Moreover, Dkk-1 function was completely independent of Fz but LRP6 dramatically interfered with the Dkk-1 inhibition of Wnt signalling. Thus, unlike Wnt antagonists, which exert their effects by molecular mimicry of Fz or Wnt sequestration through other mechanisms, Dkk-1 specifically inhibits canonical Wnt signalling by binding to the LRP6 component of the receptor complex.

Potential (18)F-labeled biomarkers for epidermal growth factor receptor tyrosine kinase.

As PET candidate tracers for EGFr-TK, five 4-(anilino)quinazoline derivatives, each fluorinated in the aniline moiety, were prepared. Each was tested in vitro for inhibition of EGFr autophosphorylation in A431 cell line. The leading compounds were then radiolabeled with (18)F and cell binding experiments, biodistribution and PET studies in A431 tumor-bearing mice were performed. Metabolic studies were carried out in a mice control group. From our results, we concluded that while in vitro experiments indicates efficacy of 4-(anilino)quinazoline compounds, kinetic factors and rapid blood clearance make them unsuitable as tracers for nuclear medicine imaging of EGFr-TK.

Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells.

Constitutive activation of signal transducer and activator of transcription (STAT) proteins has been detected in a wide variety of human primary tumor specimens and tumor cell lines including blood malignancies, head and neck cancer, and breast cancer. We have previously demonstrated a high frequency of Stat3 DNA-binding activity that is constitutively-induced by an unknown mechanism in human breast cancer cell lines possessing elevated EGF receptor (EGF-R) and c-Src kinase activities. Using tyrosine kinase selective inhibitors, we show here that Src and JAK family tyrosine kinases cooperate to mediate constitutive Stat3 activation in the absence of EGF stimulation in model human breast cancer cell lines. Inhibition of Src or JAKs results in dose-dependent suppression of Stat3 DNA-binding activity, which is accompanied by growth inhibition and induction of programmed cell death. In addition, transfection of a dominant-negative form of Stat3 leads to growth inhibition involving apoptosis of breast cancer cells. These results indicate that the biological effects of the Src and JAK tyrosine kinase inhibitors are at least partially mediated by blocking Stat3 signaling. While EGF-R kinase activity is not required for constitutive Stat3 activation in breast cancer cells, EGF stimulation further increases STAT DNA-binding activity, consistent with an important role for EGF-R in STAT signaling and malignant progression. Analysis of primary breast tumor specimens from patients with advanced disease revealed that the majority exhibit elevated STAT DNA-binding activity compared to adjacent non-tumor tissues. Our findings, taken together, suggest that tyrosine kinases transduce signals through Stat3 protein that contribute to the growth and survival of human breast cancer cells in culture and potentially in vivo.

The cytoskeleton-associated TCR zeta chain is constitutively phosphorylated in the absence of an active p56(lck) form.

The TCR recognizes peptide-MHC complexes and transmits activation signals leading to cellular responses. We have previously characterized two TCR populations expressed on the T cell surface; one is linked to the cytoskeleton via a detergent-insoluble cytoskeleton-associated zeta (cska-zeta) chain, while the other is detergent soluble and not linked to the cytoskeleton. The cska-zeta form displays unique properties: it is constitutively phosphorylated, does not undergo hyperphosphorylation upon TCR stimulation as opposed to its non-cytoskeleton-associated counterpart (non-cska-zeta) and it maintains a molecular mass of 16 kDa. It is well established that p56(lck) and possibly p59(fyn) are responsible for the generation of the 21/23-kDa phosphorylated detergent-soluble zeta form. We now demonstrate that the phosphorylation of cska-zeta does not require the activity of p56(lck). We also show that although Lck does not phosphorylate cska-zeta in vivo, it retains the capacity to phosphorylate cska-zeta in vitro. Moreover, differences in zeta-associated kinase activity were detected for non-cska-zeta and cska-zeta. Our results indicating that different kinases phosphorylate the two zeta forms are consistent with a growing consensus that each TCR form may regulate distinct cellular functions.

Inhibition of murine AIDS (MAIDS) development in C57BL/6J mice by tyrphostin AG-1387.

We previously showed that certain tyrphostin derivatives, known as protein tyrosine kinase inhibitors, also act as topoisomerase I-specific antagonists and inhibit Moloney murine leukemia virus replication in vitro in acutely and chronically infected cells. However, an accurate portrayal of retroviral-induced disease cannot rely exclusively on extrapolations from in vitro data. Therefore, experiments with animal models are essential for evaluating the efficacy of a specific drug in vivo. In this study, we examined the effect of tyrphostin AG-1387 on murine AIDS (MAIDS) development in C57BL/6J mice injected with the LP-BM5 virus mixture. A single dose of tyrphostin, administered together with or 24 h post virus inoculation, decreased the development of MAIDS symptoms as measured by spleen and lymph node weight, the T-cell response to concanavalin A (con A), and spleen architecture. Furthermore, weekly treatment with tyrphostins totally abolished MAIDS symptoms and prevented the viral infection of the spleen cells as measured by the absence of viral RNA and the restoration of T-cell function in these spleens. These results implicate that prolonged treatment with tyrphostins is needed for the prevention of MAIDS development in infected mice and suggest that it may be applied as a legitimate remedy for the treatment of retroviral-induced diseases.

Substrate competitive inhibitors of IGF-1 receptor kinase.

IGF-1 and its receptor play a pivotal role in many cancers, and therefore, IGF-1R is an attractive target for the design of inhibitors. In this communication, we report on a number of lead compounds for inhibitors of the isolated IGF-1R kinase. The search for these compounds utilized two novel in vitro assays and was aided by the knowledge of the three-dimensional structure of the insulin receptor kinase domain, which is 84% homologous to the IGF-1R kinase domain. The most potent inhibitor found in these assays was tyrphostin AG 538, with an IC(50) = 400 nM. In computer modeling, AG 538 was placed in the kinase domain of the insulin receptor and was able to sit in place of tyrosines 1158 and 1162, which undergo autophosphorylation. Experimentally it is indeed found that AG 538 does not compete with ATP but competes with the IGF-1R substrate. We prepared I-OMe AG 538, which is more hydrophobic and less sensitive to oxidation than AG 538. Both AG 538 and I-OMe AG 538 inhibit IGR-1R autophosphorylation in intact cells in a dose-dependent manner but I-OMe-AG 538 is superior, probably because of its enhanced hydrophobic nature. Both compounds inhibit the activation of the downstream targets PKB and Erk2. These findings suggest that AG 538 and I-OMe-AG 538 can serve as a lead compound for the development of substrate competitive inhibitors of the IGF-1R. The possible advantage of substrate competitive inhibitors vis-à-vis ATP competitive inhibitors is discussed.

Deregulated expression of homeobox-containing genes, HOXB6, B8, C8, C9, and Cdx-1, in human colon cancer cell lines.

Previously we have demonstrated a reciprocal deregulation of various homeobox genes (HOXB6, B8, C8 and C9 vs Cdx-1) in human colorectal cancer (CRC). In the present study, using RT-PCR, we have investigated the expression pattern of these homeobox genes in various human colon cell lines, representing various stages of colon cancer progression and differentiation. Thus, we have tested polyposis coli Pc/AA adenoma cells, Caco-2, HT-29 and LS174T adenocarcinoma cell lines. All cell lines, except LS174T, demonstrated a pattern of deregulated homeobox gene expression which resembled that of CRC. In contrast, the pattern of expression of these genes in the highly oncogenic LS174T cells, as well as in Caco-2 cells transfected with activated Ha-ras or Polyoma middle T oncogene, resembled that of the normal mucosa. The reciprocal deregulation of HOX and Cdx-1 genes in CRC and in CRC-derived cell lines suggests a possible role in human CRC development.

Suboptimal splice sites of equine infectious anaemia virus control Rev responsiveness.

The Rev protein of equine infectious anaemia virus (EIAV) was shown previously to stimulate the expression of a heterologous CAT reporter gene when the 3' half of the EIAV genome was present downstream in cis. However, computer analysis could not reveal the existence of a stable RNA secondary structure that could be analogous to the Rev-responsive element of other lentiviruses. In the present study, the inhibitory RNA element designated the cis-acting repressing sequence (CRS) has been localized to the centre of the EIAV genome. The inhibition exerted by this element could be overcome by supplying Rev in trans. The ability of the EIAV CRS to function in a heterologous context suggests that it does not require interactions with other viral proteins. Site-directed mutagenesis showed that the various centrally located suboptimal splice sites of the EIAV genome function as CRS and confer Rev-dependence on the CRS-containing transcripts. In addition, the data suggest that in canine Cf2Th cells, which are highly permissive for EIAV replication, CRS prevents nuclear export of CRS-containing transcripts and the supply of Rev relieves this suppression.

Disruption of microtubules in living cells by tyrphostin AG-1714.

Tyrphostin AG-1714 and several related molecules with the general structure of nitro-benzene malononitrile (BMN) disrupt microtubules in a large variety of cultured cells. This process can be inhibited by the stabilization of microtubules with taxol or by pretreatment of the cells with pervanadate, which inhibits tyrosine phosphatases and increases the overall levels of phosphotyrosine in cells. Unlike other microtubule-disrupting drugs such as nocodazole or colchicine, tyrphostin AG-1714 does not interfere with microtubule polymerization or stability in vitro, suggesting that the effect of this tyrphostin on microtubules is indirect. These results imply an involvement of protein tyrosine phosphorylation in the regulation of overall microtubule dynamics. Tyrphostins of AG-1714 type could thus be powerful tools for the identification of such microtubule regulatory pathways.

Local delivery of platelet-derived growth factor receptor-specific tyrphostin inhibits neointimal formation in rats.

Signal transduction through the platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR) system is involved in the process of postangioplasty restenosis. Tyrphostins are low molecular weight inhibitors of protein tyrosine kinases. We assessed the antiproliferative effects of PDGFRbeta-specific tyrphostin AG-1295 in vitro and in vivo. AG-1295 significantly inhibited rat smooth muscle cell growth stimulated by PDGF-BB or FCS. This antiproliferative effect was paralleled by reversible reduction of the total phosphotyrosine level and the degree of PDGFRbeta phosphorylation by the drug in vitro. Local sustained delivery of the drug from perivascularly implanted polymeric matrices resulted in focal AG-1295 levels of 711 and 29.1 ng/mg of dry arterial tissue 1 and 14 days after implantation in rats. AG-1295 delivered from polymeric matrices resulted in a 35% reduction of neointimal formation on day 14 after balloon injury in the rat carotid model. Tyrosine phosphorylation of certain transduction proteins in arterial tissue extracts was significantly upregulated by balloon injury on day 3 but was essentially returned to or below basal levels 14 days after injury. Tyrphostin treatment decreased tyrosine phosphorylation at both time points below the basal levels. Moreover, the enhancement of PDGFRbeta expression 3 and 14 days after arterial injury was strongly inhibited by AG-1295 treatment. It can be concluded that AG-1295 reduces neointimal formation by inhibiting PDGFbeta-triggered tyrosine phosphorylation.

Growth inhibition of psoriatic keratinocytes by quinazoline tyrosine kinase inhibitors.

Psoriasis is characterized by hyperproliferation of keratinocytes associated with an inflammatory infiltrate in the epidermis. Among factors which may be related to hyperplasia of psoriatic keratinocytes is the persistent autocrine stimulation of the epidermal growth factor receptor (EGFR) by transforming growth factor-alpha. Owing to the pivotal role of the EGFR in driving the growth of human psoriatic keratinocytes, we examined two selective inhibitors of EGFR kinase activity: 4-(3-bromophenylamino)-6, 7-dimethoxyquinazoline (AG1517/SU5271) and 4-(3-chlorophenylamino)-6, 7-dimethoxyquinazoline (AG1478) on psoriatic keratinocytes. SU5271 potently inhibits ligand-induced autophosphorylation of EGFR, and downstream signal transduction events, including DNA replication and cell cycle progression. SU5271, at micromolar concentrations, inhibited the proliferation of keratinocytes isolated from psoriatic lesions in excellent correlation with its EGFR kinase inhibitory activity in these cells. Biologically active concentrations of SU5271 penetrated human cadaver skin, suggesting that this compound is a strong candidate as an antipsoriatic agent.

Human frizzled 1 interacts with transforming Wnts to transduce a TCF dependent transcriptional response.

The human homologue of fz1 (Hfz1) was cloned from a cDNA library. Hfz1 was shown to couple to Wnt signal transduction pathways by its ability to enhance Wnt induced TCF dependent transcription in both autocrine and paracrine modes. Enhanced TCF dependent signaling was dose dependent with respect to both Wnt-3A and Hfz1. Moreover, Hfz1 deletion mutants with truncated carboxy termini showed markedly reduced capacity to enhance Wnt signal transduction. Specificity was demonstrated with respect to signal transduction by different Wnts. While Wnt-3a, -3, -1 and to a lesser extent Wnt-2 cooperated with Hfz1 in the paracrine assay for TCF dependent signaling, neither Wnt-4, -5a, -5b, -6, -7a nor -7b did so, despite similar levels of expression. However, coimmunoprecipitation of Hfz1 with both Wnt-3a and Wnt-5a indicated that TCF dependent signaling in response to Wnts is not determined solely by their ability to bind the receptor. All of these findings provide strong evidence that Hfz1 is a functional partner for certain Wnts in inducing TCF dependent transcription.

Alpha-cyanocinnamide derivatives: a new family of non-peptide, non-sulfhydryl inhibitors of Ras farnesylation.

Farnesylation of Ras and other proteins is required for their membrane attachment and normal function. Here we report on the synthesis of alpha-cyanocinnamide derivatives, a new family of farnesyltransferase inhibitors. These compounds are nonpeptidic and do not contain sulfhydryl groups. The most potent compound is a pure competitive inhibitor with respect to the Ras protein and mixed competitive with respect to farnesyl diphosphate. Selectivity studies against geranylgeranyltransferase and biological activities of selected compounds are described.

Tyrphostins that suppress the growth of human papilloma virus 16-immortalized human keratinocytes.

Human papilloma virus 16 (HPV16) is considered to be the causative agent for cervical cancer, which ranks second to breast cancer in women's malignancies. In an attempt to develop drugs that inhibit the malignant transformation of HPV16-immortalized epithelial cells, we examined the effect of tyrphostins on such cells. We examined the effect of tyrphostins from four different families on the growth of HPV16-immortalized human keratinocytes (HF-1) cells. We found that they alter their cell cycle distribution, their morphology, and induce cell death by apoptosis. The effects of tyrphostins on HF-1 cells are different from their effects on normal keratinocytes. Growth suppression by AG555 and AG1478 is accompanied by 30% apoptosis in HF-1 cells, but this is not observed in normal keratinocytes. Tyrphostin treatment produces distinctive morphological changes in HF-1 cells and in normal keratinocytes; however, the culture organization of normal keratinocytes is less disrupted. These differential effects of the tyrphostins on HPV16-immortalized keratinocytes compared with their effects on normal keratinocytes suggests that these compounds are suitable candidates for the treatment of papilloma. Previous and present results indicate that group 1 tyrphostins, which inhibit Cdk2 activation, and group 2 tyrphostins, represented by AG1478, a potent epidermal growth factor receptor kinase inhibitor, induce cell cycle arrest; and, in the case of HF-1 cells, apoptosis and differentiation. Cells accumulate in the G(1) phase of the cell cycle at the expense of S and G(2) + M. These compounds block the growth of normal keratinocytes without inducing apoptosis or differentiation, causing them to accumulate in G(1). AG17, which belongs to group 4, exerts its antiproliferative effect mainly by increasing the fractions of cells in G(1) with a concomitant decrease in the fraction of cells in S and G(2) + M.

A dual inhibitor of platelet-derived growth factor beta-receptor and Src kinase activity potently interferes with motogenic and mitogenic responses to PDGF in vascular smooth muscle cells. A novel candidate for prevention of vascular remodeling.

PP1 has previously been described as an inhibitor of the Src-family kinases p56(Lck) and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed, and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60(c-src) in vitro and Src-dependent tyrosine phosphorylation in p60(c-srcF572)-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs, as determined in the modified Boyden chamber, as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly, in addition to inhibition of Src kinase, PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells, including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR, PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases, the activities of the epidermal growth factor receptor, fibroblast growth factor receptor-1, and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872, whereas PDGF alpha-receptor was susceptible, albeit to a lesser extent than PDGF beta-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the Src family of tyrosine kinases. It is also a potent inhibitor of the PDGF beta-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events, creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity, such as vascular remodeling and restenosis.