Abstract: Heparanase, endo-beta-D-glucuronidase, degrades heparan sulfate glycosaminoglycans - the principal polysaccharide of the basement membrane and extracellular matrix. Heparanase activity plays a decisive role in biological processes associated with remodeling of the extracellular matrix, such as cancer metastasis, angiogenesis and inflammation. In the hematopoietic system, heparanase is thought to be associated with normal differentiation and function of myeloid cells and platelets. We investigated heparanase polymorphisms in patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), Hodgkin's disease (HD) and multiple myeloma (MM). Significant correlation was found between rs11099592 and rs6535455 heparanase gene (HPSE) single nucleotide polymorphisms (SNPs) and ALL (chi(2)(1d.f.)=4.96, P=0.026). Genotype frequency comparisons revealed a significant association with rs4693602 (chi(2)(2d.f.)=7.276, P=0.026) in MM patients and rs4364254 (chi(2)(2d.f.)=6.226, P=0.044) in AML patients. Examination of HPSE gene mRNA expression by real-time RT-PCR indicated a significant low HPSE gene expression level in ALL patients and a high expression level in MM and AML patients, compared to healthy controls. Moreover, statistically significant correlation was found between heparanase mRNA expression level and three HPSE gene SNPs (rs4693608, rs11099592 and rs4364254) among healthy individuals. These data suggest that certain HPSE gene SNPs may contribute to basal heparanase gene expression and that alterations in this gene are an important determinant in the pathogenesis of ALL, AML and MM.Leukemia advance online publication, 5 July 2007; doi:10.1038/sj.leu.2404821.
Abstract: Heparanase is a mammalian endoglucuronidase responsible for heparan sulfate (HS) degradation. HS is a major constituent of the extracellular matrix (ECM) and HS-degrading activity plays a decisive role in fundamental biological processes associated with remodeling of the ECM, such as cancer metastasis, angiogenesis and inflammation. There is great interest in the prospect of genome-wide association studies to identify genetic factors underlying complex diseases. It is important to establish a detailed description of the heparanase (HPSE) gene single nucleotide polymorphisms (SNPs). In this study, four Israeli Jewish populations (Ashkenazi, North African, Mediterranean and Near Eastern) were examined for 7 HPSE gene SNPs. Four out of 7 SNPs (rs4693608, db11099592, rs4364254, db6856901) were found to be polymorphic. Population comparisons revealed significant differences in SNPs allele frequency between Near Eastern and each of the other three populations. Genotype and allele frequencies in Jewish populations were different from non-Jewish populations, except for a certain similarity to Caucasians. Although the distance between SNPs is relatively small, the db11099592 SNP was in linkage disequilibrium (LD) only with the proximal SNP rs4693608. LD between distal SNPs rs4364254 and db6856901 was found only in Mediterraneans and North Africans. The current study provides a characterization of the normally occurring HPSE gene SNPs in different populations. This information is obligatory for further studies on the linkage between these SNPs and heparanase expression and function in various pathological processes, primarily cancer progression.
Abstract: The aim of the study was to isolate and characterize a population of neuronal progenitors in the human umbilical cord blood (HUCB) mononuclear cell
(MNC) fraction, for in vitro manipulation towards neuronal differentiation. Selection of the HUCB neuronal progenitors (HUCBNPs) was based on the neuronal prerequisite for adherence to collagen. Populations of collagen-adherent, nestin-positive (94.8±2.9%) progenitors expressing α1/2 integrin receptors, as revealed by Western blot and adhesion
assay using selective antagonists, were isolated and
survived for more than 14 days. In vitro differentiation
of the HUCBNPs was achieved by treatment with 10%
human SH-SY5Y neuroblastoma cell-conditioning media (CM) supplemented with 10 ng/ml nerve growth factor (NGF). Some 83±8.2% of the surviving progenitors acquired a neuronal-like morphology, expressed by cellular outgrowths of different lengths. About 35±6% of the HUCBNPs had long outgrowths with a length/cell diameter ratio greater than 2, typical of developing neurons. The majority of these progenitors, analyzed by immunocytochemistry
and/or RT-PCR, expressed common neuronal
markers such as microtubule-associated protein 2 (MAP-2; 98.5±2%), neurotrophin receptor (TrkA; 98.5±0.06%), neurofillament-160 (NF-160; 94.2±1%), beta-tubulin III (89.8±4.2%) and neuron specific enolase (NSE). Combined CM and NGF treatment induced constitutive activation of the mitogen-activated protein kinases ERK2 (36-fold vs control), p38α (nine-fold vs control) and p38beta (23-fold vs
control), most likely related to survival and/or differentiation. The results point to operationally defined conditions for activating neuronal differentiation of HUCBNPs ex vivo and emphasize the crucial role of neuronal CM and NGF in this process.
Abstract: The use of adult stem cells for myocardial tissue repair might be limited in elderly and sick people because their cells are depleted and exhausted. The present study was conducted to explore the potential of human umbilical cord blood (UCB) CD133+ progenitor cells for myocardial tissue repair in a model of extensive myocardial infarction (MI). CD133+ progenitor cells were isolated from newborn UCB. Cells (1.2-2 x 10(6)) or saline (control) was infused intravenously 7 days after permanent coronary artery ligation in athymic nude rats. Left ventricular (LV) function was assessed before and 1 month after infusion by echocardiography. Tracking of human cells was performed by fluorescent in situ hybridization for human X and Y chromosomes or by immunostaining for HLA-DR or HLA-ABC. One month after delivery, LV fractional shortening improved by 42 +/- 17% in cell-treated hearts and decreased by 39 +/- 10% in controls (p = .001). Anterior wall thickness decreased significantly in controls but not in treated hearts. Microscopic examination revealed that the UCB cells were able to migrate, colonize, and survive in the infarcted myocardium. Human cells were identified near vessel walls and LV cavity and were occasionally incorporated into endothelial cells in six of nine cell-treated animals but not in controls. Scar tissue from cell-treated animals was significantly populated with autologous myofibroblasts as indicated by colocalization of HLA-DR and alpha-smooth muscle actin staining. In conclusion, the present work suggests that, after MI, intravenous delivery of human UCB-derived CD133+ cells can produce functional recovery by preventing scar thinning and LV systolic dilatation.
Abstract: Multidrug resistance (MDR) due to over-expression of the MDR1 (ABCB1) gene and its P-glycoprotein (Pgp) product is an obstacle in the treatment of hematological malignancies. In this study, we have evaluated the potency of KT-5720 to reverse Pgp-dependent MDR in vitro and in vivo. KT-5720 (but not its close derivatives, K252a and K252b) reversed multidrug resistance of LM1/MDR cell line at non-toxic concentrations and increased accumulation of rhodamine 123 (Rh123). KT-5720 significantly reversed MDR1-dependent resistance of primary malignant cells from patients with chronic myelogenous leukemia in blast crisis (CML-BC) and advanced multiple myeloma (MM). Moreover, KT-5720 (at 5 mg/kg) sensitized the bone marrow of MDR1 transgenic mice model towards daunorubicin (at 8 mg/kg) without general toxic effects. Therefore, KT-5720 can be considered as candidate for combination therapy in various hematological malignancies where Pgp activity is a major impediment for cure.
Abstract: Multidrug resistance (MDR) due to the expression of the MDR1 gene and its P-glycoprotein (Pgp) product is a major factor in the prognosis and clinical outcome of patients with refractory lymphomas and other malignancies. The aim of our study was to establish a lymphoma, cellular system where a de novo acquisition of multidrug resistance is specifically related to overexpression of a transgenic, human MDR1. A multidrug sensitive lymphoma cell line (LM1) was established from a sporadic T-cell lymphoma of BALB/c mouse and was transduced by a retroviral vector containing the human MDR1 cDNA. The resultant cell variant (LM1/MDR) was characterized in comparison to the parental LM1 cells. The LM1/MDR cell variant is cross-resistant to DOX, COL, ACT D and VBL. This cell variant expresses the human MDR1 and exhibits de novo functional Pgp activity that can be blocked by the Pgp-modulators VRP and KT-5720. The acquired MDR of LM1/MDR is not accompanied with gene amplification, alternative splicing or up-regulation of the murine endogenous mdr1a, mdr1b, mrp1, mrp2 and mrp3 transporter-genes. Therefore, the acquired MDR is, specifically, human MDR1-dependent as it has been found in malignant cells of most lymphoma patients. Moreover, this system can be used as a model to study MDR and the efficacy of drugs and modulators on malignant cells where human Pgp is a major factor of multidrug resistance.
Abstract: The human multidrug-resistant gene (MDR1) encodes for P-glycoprotein (P-gp), which is a membrane-bound efflux-transporter conferring resistance to a number of natural cytotoxic drugs and potentially toxic xenobiotics. The wobble C3435T polymorphism at exon 26 was associated with different expression levels of the MDR1 gene and substrate uptake. Differences in allele frequencies of the C3435T polymorphism have previously been demonstrated between racial groups. In this study, 500 individuals from 5 Jewish populations of Israel (Ashkenazi, Yemenite, North African, Mediterranean, Near-Eastern) were examined for C3435T polymorphism using a PCR-RFLP-based technique to calculate genotype and allele frequencies. Frequencies of the C allele were quite similar among the Ashkenazi (0.65), Yemenite (0.645), and North-African (0.615) Jewish populations. However, the frequency of this allele was slightly lower among Mediterranean Jews (0.58) and significantly lower among Near-Eastern Jews (0.445). The frequency of the C allele among Near-Eastern Jews is, therefore, significantly different from those of all other tested Jewish populations. In comparison to previously studied non-Jewish populations, the frequency of this allele among Near-Eastern Jews is different from that in West Africans (0.91) but is similar to that in whites (0.497). However, the C allele frequencies among the other 4 Jewish populations are significantly lower than that found among West Africans and significantly higher than among non-Jewish whites. These data may have important therapeutic and prognostic implication for P-gp-related drug dosage recommendation in Jewish populations.
Abstract: T-25-Adh cells, cell variants derived from S49 mouse lymphoma, were transduced with a retrovirus containing the human MDR1 cDNA. The resultant cells (HU-1) are cross-resistant to colchicine, doxorubicin, vinblastine and actinomycin D, and their resistance to colchicine is reversed by verapamil. HU-1 cells were used to screen several protein kinase modulators for their ability to reverse multidrug resistance. Among the tested indole carbazole (K-252a) family of protein kinase inhibitors, only the antibiotic alkaloid KT-5720 (9-n-hexyl derivative of K-252a) could overcome the multidrug resistance of HU-1 cells and KB-V1 human carcinoma cells. Since other protein kinase A, C and G modulators did not reverse multidrug resistance in the tested multidrug-resistant cells, the chemosensitising activity of KT-5720 on these cells is apparently independent of its kinase inhibitory effects. Since KT-5720 fully reversed multidrug resistance at non-toxic concentrations, it might be a candidate for clinical chemosensitisation in combination chemotherapy.
Abstract: Many human cancers that are initially responsive to chemotherapy eventually fail to respond to treatment. For some drugs, dose escalation that may be required for a cure cannot be achieved because sensitive tissues such as bone marrow (BM) limit cytotoxic therapy. Approaches to prevent or circumvent BM toxicity are therefore a high priority of research on dose escalation protocols. In this study, we have transplanted BM cells from transgenic mice that constitutively express physiologic amounts of a functional human multidrug resistance (MDR1) cDNA to lethally irradiated C57BL/6 x SJL F1 mice (n = 36). From 6 weeks to 10 months after the transplant, all animals contained MDR1 DNA in spleen and BM specimens as indicated by Southern blot analysis, and expressed MDR1 messenger RNA in BM samples as detected by slot blot analysis. In addition, these animals were resistant to the myelosuppressive effect of doxorubicin, daunomycin, taxol, vinblastine, vincristine, etoposide, and actinomycin D, whereas control animals that were reconstituted with normal BM were drug sensitive. Finally, the chemoprotection afforded by the MDR1 gene could readily be reversed by adding chemosensitizers such as cyclosporin A and R-verapamil to chemotherapy. Hence, it appears that BM cells expressing the human MDR1 gene maintain this function after transplantation to host animals for a minimum of 10 months, and confer multidrug resistance to these BM recipients. This selective advantage conferred by expression of the MDR1 cDNA suggests a strategy for the use of MDR1 gene therapy in cancer chemotherapy and for the introduction of otherwise nonselectable genes into BM.
Abstract: The development of preclinical models for the rapid testing of agents that circumvent multidrug resistance in cancer is a high priority of research on drug resistance. A common form of multidrug resistance in human cancer results from expression of the MDR1 gene, which encodes a Mr 170,000 glycoprotein that functions as a plasma membrane energy-dependent multidrug efflux pump. We have engineered transgenic mice that express this multidrug transporter in their bone marrow and demonstrated that these animals are resistant to leukopenia by a panel of anticancer drugs including anthracyclines, vinca alkaloids, etoposide, taxol, and actinomycin D. Differential leukocyte counts indicate that both neutrophils and lymphocytes are protected. Drugs such as cisplatin, methotrexate, and 5-fluorouracil, which are not handled by the multidrug transporter, produce bone marrow suppression in both normal and transgenic mice. The resistance conferred by the MDR1 gene can be circumvented in a dose-dependent manner by simultaneous administration of agents previously shown to be inhibitors of the multidrug transporter in vitro, including verapamil isomers, quinidine, and quinine. Verapamil and quinine, both at levels suitable for human trials that produced only partial sensitization of the MDR1-transgenic mice, were fully sensitizing when used in combination. We conclude that MDR1-transgenic mice provide a rapid and reliable system to determine the bioactivity of agents that reverse multidrug resistance in animals.
Abstract: Many cancers have been cured by chemotherapeutic agents. However, other cancers are intrinsically drug resistant, and some acquire resistance following chemotherapy. Cloning of the cDNA for the human MDR1 gene (also known as PGY1), which encodes the multidrug efflux protein P-glycoprotein, has made it possible to measure levels of MDR1 RNA in human cancers. We report the levels of MDR1 RNA in greater than 400 human cancers. MDR1 RNA levels were usually elevated in untreated, intrinsically drug-resistant tumors, including those derived from the colon, kidney, adrenal gland, liver, and pancreas, as well as in carcinoid tumors, chronic myelogenous leukemia in blast crisis, and cell lines of non-small cell carcinoma of the lung (NSCLC) with neuroendocrine properties. MDR1 RNA levels were occasionally elevated in other untreated cancers, including neuroblastoma, acute lymphocytic leukemia (ALL) in adults, acute nonlymphocytic leukemia (ANLL) in adults, and indolent non-Hodgkin's lymphoma. MDR1 RNA levels were also increased in some cancers at relapse after chemotherapy, including ALL, ANLL, breast cancer, neuroblastoma, pheochromocytoma, and nodular, poorly differentiated lymphoma. Many types of drug-sensitive and drug-resistant tumors, including NSCLC and melanoma, contained undetectable or low levels of MDR1 RNA. The consistent association of MDR1 expression with several intrinsically resistant cancers and the increased expression of the MDR1 gene in certain cancers with acquired drug resistance indicate that the MDR1 gene contributes to multidrug resistance in many human cancers. Thus, evaluation of MDR1 gene expression may prove to be a valuable tool in the identification of individuals whose cancers are resistant to specific agents. The information may be useful in designing or altering chemotherapeutic protocols in these patients.
Abstract: The human multidrug resistance gene (MDR1) encodes a drug efflux pump glycoprotein (P-glycoprotein) responsible for resistance to multiple cytotoxic drugs. A plasmid carrying a human MDR1 cDNA under the control of a chicken beta-actin promoter was used to generate transgenic mice in which the transgene was mainly expressed in bone marrow and spleen. Immunofluorescence localization studies showed that P-glycoprotein was present on bone marrow cells. Furthermore, leukocyte counts of the transgenic mice treated with daunomycin did not fall, indicating that their bone marrow was resistant to the cytotoxic effect of the drug. Since bone marrow suppression is a major limitation to chemotherapy, these transgenic mice should serve as a model to determine whether higher doses of drugs can cure previously unresponsive cancers.
Abstract: Free mRNPs isolated from human term placental tissue were examined for protein kinase and phosphoprotein-phosphatase activities. Free mRNPs incubated with [gamma-32P]ATP in a protein kinase standard buffer show self-phosphorylation in the absence of exogenous substrates. Treatment of phosphorylated products with alkali showed a significant phosphorylation of tyrosine residues within the mRNP-proteins. An alkaline-phosphatase activity was found to be tightly associated with the mRNPs. Both heat stable and heat labile alkaline phosphatase activities were found in the mRNPs. Heat labile alkaline phosphatase is the major isoenzyme form of the mRNPs. The existence of both protein kinase(s) and alkaline phosphatase activities in placental free cytoplasmic mRNPs might suggest that a balance between phosphorylation, specifically on tyrosine residues, and dephosphorylation states of some of the mRNP-proteins is relevant for their physiological functions, and may therefore play a role in the regulation of mRNPs' metabolism and, consequently, in mRNA translation.
Abstract: The [32P]phosphoamino acids in proteins of first-trimester and term-cultured human placentas have been separated and their relative amounts have been measured. Significant phosphorylation of tyrosine residues could be detected in the cultured placental tissue at different stages of gestation. The phosphotyrosine accounts for 2-4% of the total acid-stable phosphate in the phosphoamino acids after partial acid hydrolysis. The difference in the extent of [32P]tyrosine in various placentas seems to be a function of biological variation of the individual placentas, rather than a function of placental age and stage of gestation. In contrast, a significant difference in the phosphorylation ratio of serine and threonine could be measured between first-trimester and term placentas. As more evidence is accumulating that protein phosphorylation of tyrosine is involved in the processes of cellular growth and proliferation, our findings of the relatively high tyrosine phosphorylation in human placenta strongly suggest that this type of protein phosphorylation may play an important role in the placental growth and development. Furthermore, these findings may correlate with the existence of the endogenous RNA virus-like particles found in normal human placenta.
Abstract: The [32P]phosphoamino acids in proteins of first trimester and term-cultured human placentas have been separated and their relative amounts were measured. A significant phosphorylation of tyrosine residues could be detected in the cultured placental tissue at different stages of gestation. The phosphotyrosine accounts for 2-4% of the total acid-stable phosphate in the phosphoamino acids after partial acid hydrolysis. The difference in the extent of [32P]tyrosine in various placentas seems to be a function of biological variation of the individual placentas, rather than a function of placental age and stage of gestation. In contrast, a significant difference in the phosphorylation ratio of serine and threonine could be measured between first trimester and term placentas. As more evidence is accumulating that protein phosphorylation of tyrosine is involved in the processes of cellular growth and proliferation, our findings of the relatively high tyrosine phosphorylation in human placenta strongly suggest that this type of protein phosphorylation may play an important role in the placental growth and development. Furthermore, these findings may correlate with the existence of the endogenous RNA virus-like particles found in normal human placenta.
Abstract: The in vitro synthesis of RNA in the human placental tissue, incubated in organ culture, was investigated. We followed the synthesis of the poly A(-) and poly A(+) RNA fractions, and investigated the distribution of the newly synthesized RNA among the subcellular fractions isolated from first and third trimester placentas. The poly A(-) RNA was the major fraction of the RNA synthesized in vitro. The incorporation of [3H]uridine into the poly A(+) RNA fraction was very low. As protein synthesis occurred during the entire incubation period, we suggest the presence of a pool of mRNA molecules in the form of mRNP particles.
Abstract: We studied the in vitro synthesis and secretion of alkaline phosphatase by human first trimester decidual tissue incubated in organ culture. Decidua synthesizes two different alkaline phosphatase isoenzymes; heat stable and heat labile. decidual tissue maintains a constant pool of alkaline phosphatases. The synthesis of alkaline phosphatase may be the driving force for its secretion. The de novo synthesis and secretion rate of alkaline phosphatases were also investigated. The higher specific radioactivities of the secreted enzymes than those found in the tissue may suggest that newly synthesized alkaline phosphatase is preferentially released. The intracellular distribution of the alkaline phosphatase isoenzymes was compared as well. The characteristics of the two isoenzymes are different in human decidua of maternal origin from those previously reported in the human placenta originating from embryonic stem cells.
Abstract: We studied the in vitro synthesis and secretion of hPL by human term placental tissue incubated in organ culture. Placental tissue maintains a constant pool of hPL. The synthesis of hPL may be the driving force for its secretion. The de novo synthesis and secretion rates of hPL were also investigated. The higher specific radioactivities of the secreted hPL than those found in the tissue may suggest that newly synthesized hPL is preferentially released. The intracellular distribution of hPL was also compared.
Abstract: The synthesis and secretion of alkaline phosphatases in vitro by human placental tissue incubated in organ culture were studied. First-trimester placenta synthesizes and secretes two different alkaline phosphatase isoenzymes (heat-labile and heat-stable), whereas in term placenta nearly all the alkaline phosphatase synthesized and secreted is heat-stable. The specific activities of alkaline phosphatases in first-trimester and term placental tissue remain constant throughout the time course of incubation. In the media, specific activities increase with time. Hence, alkaline phosphatase synthesis seems to be the driving force for its own secretion. The rates of synthesis de novo and of alkaline phosphatases were measured. The specific radioactivities of the secreted alkaline phosphatases were higher than the corresponding specific radioactivities in the tissue throughout the entire incubation period. The intracellular distribution of the alkaline phosphatase isoenzymes was compared.
