Introduction: Therapeutic agents like doxorubicin, an anthracycline antibiotic drug, are widely used in cancer chemotherapy. The use of doxorubicin is limited however by an increased risk of cardiac damage as a side effect, and an increased cancer cell drug resistance mediated by efflux drug transporters. Strategies are needed to protect the heart and still allow the benefits of drug treatment. “Basic” fibroblast growth factor-2 (FGF-2) is a multi-functional protein. It is angiogenic and cardioprotective against ischemia-reperfusion injury. FGF-2 can also regulate cancer cell drug resistance or sensitivity, however, so far, there is no evidence that FGF-2 protects against doxorubicin-induced cardiac damage through effects on efflux drug transporter levels or function. Aims: To investigate whether: (1) FGF-2 can increase resistance to doxorubicin-induced neonatal rat cardiac myocyte damage; and if so whether (2) an effect on efflux drug transporters might contribute to this cardioprotection by FGF-2. Methods: Neonatal rat cardiac myocyte cultures were treated with doxorubicin in the absence or presence of pre-treatment with FGF-2. To assess cell damage: (i) culture medium was tested for lactate dehydrogenase (LDH) activity as an indication of plasma membrane disruption; (ii) cells were stained with fluorescent apoptosis and necrosis biomarkers as well as (iii) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and acridine orange to assess DNA fragmentation or compaction. The role of FGF receptor (FGFR) or protein kinase C (PKC) was addressed through use of inhibitors including SU5402, or chelerythrine as well as bisindomaleimide. Multidrug resistance gene 1a and 1b (MDR1a, 1b), multidrug resistance gene 2 (MDR2) and multidrug resistance-related protein 1 (MRP1) gene expression, as well as the function of MDRs and MRPs protein products were assessed by real-time reverse transcriptase-polymerase chain reaction (qPCR), as well as retention/extrusion of (fluorescent) doxorubicin/calcein in cardiac myocytes, respectively. Efflux drug transporter inhibitors, including 20 µM cyclosporine A (CsA), 2 µM verapamil and 1 µM Tariquidar (XR9576) were used to asssess for a direct effect of FGF-2 on transporter function. Fluorescence-activated cell sorting (FACS) was used to measure fluorescent doxorubicin/calcein levels inside treated cardiac myocytes. Results: Doxorubicin increased the incidence of programmed cell death, DNA damage, and lysosome and LDH activity, while decreasing cell number at 24 hours. FGF-2 prevented the detrimental effects of doxorubicin. In turn, the protective effects of FGF-2 were blocked in the presence of FGFR or PKC inhibitors. FGF-2 treatment significantly increased MDR1a, MDR1b, MDR2, MRP1 RNA levels by qPCR, and protein levels as assessed by function, and specifically extrusion of doxorubicin/calcein, in the presence of doxorubicin when compared to doxorubicin treatment alone. Furthermore, inhibition of efflux drug transporters with CsA and Tariquidar (XR9576) significantly reduced the ability of FGF-2 to protect against doxorubicin-induced damage; the beneficial effect of FGF-2 was completely blocked by pretreatment with verapamil. Conclusion(s): These data indicate for the first time that exogenous FGF-2 can increase resistance to doxorubicin-induced neonatal rat cardiac myocyte damage, and implicate PKC and regulation of efflux transporter protein levels and/or function in the mechanism.

No description available.

Introduction: Therapeutic agents like doxorubicin, an anthracycline antibiotic drug, are widely used in cancer chemotherapy. The use of doxorubicin is limited however by an increased risk of cardiac damage as a side effect, and an increased cancer cell drug resistance mediated by efflux drug transporters. Strategies are needed to protect the heart and still allow the benefits of drug treatment. “Basic” fibroblast growth factor-2 (FGF-2) is a multi-functional protein. It is angiogenic and cardioprotective against ischemia-reperfusion injury. FGF-2 can also regulate cancer cell drug resistance or sensitivity, however, so far, there is no evidence that FGF-2 protects against doxorubicin-induced cardiac damage through effects on efflux drug transporter levels or function. Aims: To investigate whether: (1) FGF-2 can increase resistance to doxorubicin-induced neonatal rat cardiac myocyte damage; and if so whether (2) an effect on efflux drug transporters might contribute to this cardioprotection by FGF-2. Methods: Neonatal rat cardiac myocyte cultures were treated with doxorubicin in the absence or presence of pre-treatment with FGF-2. To assess cell damage: (i) culture medium was tested for lactate dehydrogenase (LDH) activity as an indication of plasma membrane disruption; (ii) cells were stained with fluorescent apoptosis and necrosis biomarkers as well as (iii) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and acridine orange to assess DNA fragmentation or compaction. The role of FGF receptor (FGFR) or protein kinase C (PKC) was addressed through use of inhibitors including SU5402, or chelerythrine as well as bisindomaleimide. Multidrug resistance gene 1a and 1b (MDR1a, 1b), multidrug resistance gene 2 (MDR2) and multidrug resistance-related protein 1 (MRP1) gene expression, as well as the function of MDRs and MRPs protein products were assessed by real-time reverse transcriptase-polymerase chain reaction (qPCR), as well as retention/extrusion of (fluorescent) doxorubicin/calcein in cardiac myocytes, respectively. Efflux drug transporter inhibitors, including 20 µM cyclosporine A (CsA), 2 µM verapamil and 1 µM Tariquidar (XR9576) were used to asssess for a direct effect of FGF-2 on transporter function. Fluorescence-activated cell sorting (FACS) was used to measure fluorescent doxorubicin/calcein levels inside treated cardiac myocytes. Results: Doxorubicin increased the incidence of programmed cell death, DNA damage, and lysosome and LDH activity, while decreasing cell number at 24 hours. FGF-2 prevented the detrimental effects of doxorubicin. In turn, the protective effects of FGF-2 were blocked in the presence of FGFR or PKC inhibitors. FGF-2 treatment significantly increased MDR1a, MDR1b, MDR2, MRP1 RNA levels by qPCR, and protein levels as assessed by function, and specifically extrusion of doxorubicin/calcein, in the presence of doxorubicin when compared to doxorubicin treatment alone. Furthermore, inhibition of efflux drug transporters with CsA and Tariquidar (XR9576) significantly reduced the ability of FGF-2 to protect against doxorubicin-induced damage; the beneficial effect of FGF-2 was completely blocked by pretreatment with verapamil. Conclusion(s): These data indicate for the first time that exogenous FGF-2 can increase resistance to doxorubicin-induced neonatal rat cardiac myocyte damage, and implicate PKC and regulation of efflux transporter protein levels and/or function in the mechanism.

Polymorphisms in organic cation transporters SLC22A4, SLC22A23 and IBD5 locus have been associated with pathogenesis of inflammatory bowel disease (IBD). We sought to investigate the association of polymorphisms in these genes to IBD risk in a Canadian population, subclone and express human SLC22A23 gene to determine the localization in the cell. DNA samples from 160 patients with Crohn´s disease (CD), 149 patients with ulcerative colitis (UC) and 142 healthy controls were genotyped by PCR-RFLP analysis or TaqMan system. Gateway® recombination technology was used to transform and express SLC22A23 gene in HEK 293 cell line. Polymorphisms in the IBD5 locus rs17622208-AA genotype and rs11739135-CC genotype increase the risk of CD. Moreover, carriers of SLC22A23 polymorphisms rs4959235-TT genotype and rs9503518-GG genotype increase dramatically the risk of UC. We confirm that SLC22A23 polymorphisms are important in the pathogenesis of IBD and they can ultimately be used as biomarkers of the disease risk.

Two studies were conducted to compare nitrogen (N) and glutamate metabolism in Polypay and percentage White Dorper lambs.First, a two-phase digestion/N metabolism trial was conducted with 18 wether lambs of three genetic types: Polypay (PP), 1/2 White Dorper 1/2 Polypay (1/2 D), and 3/4 White Dorper 1/4 Polypay (3/4 D). Six lambs of each genetic type were fed a high roughage diet (HR; Phase 1) or high concentrate diet (HC; Phase 2). DM and N digestion was higher for 1/2 D than PP or 3/4 D fed HC. N retention was highest for 1/2 D regardless of diet.The second study analyzed the effect of genetic type on glutamate transporter and metabolizing enzyme expression in liver, kidney, longissimus dorsi muscle (LD muscle), and subcutaneous fat (Sub Q Fat) tissue of 18 wether lambs of three genetic types: PP, 1/2 D, and 15 /16 White Dorper 1/16 Polypay (15 /16 D). Tissue samples were analyzed for protein and mRNA content of GS, GDH, ALT, EAAC1, and GLT-1. Glutamate transport and metabolism capacity was lowest for the heavier muscled 15 /16 D lambs.The results suggest genetic type has an effect on N metabolism due to differential expression of glutamate transporters and metabolizing enzymes.

Polymorphisms in organic cation transporters SLC22A4, SLC22A23 and IBD5 locus have been associated with pathogenesis of inflammatory bowel disease (IBD). We sought to investigate the association of polymorphisms in these genes to IBD risk in a Canadian population, subclone and express human SLC22A23 gene to determine the localization in the cell. DNA samples from 160 patients with Crohn´s disease (CD), 149 patients with ulcerative colitis (UC) and 142 healthy controls were genotyped by PCR-RFLP analysis or TaqMan system. Gateway® recombination technology was used to transform and express SLC22A23 gene in HEK 293 cell line. Polymorphisms in the IBD5 locus rs17622208-AA genotype and rs11739135-CC genotype increase the risk of CD. Moreover, carriers of SLC22A23 polymorphisms rs4959235-TT genotype and rs9503518-GG genotype increase dramatically the risk of UC. We confirm that SLC22A23 polymorphisms are important in the pathogenesis of IBD and they can ultimately be used as biomarkers of the disease risk.

The protein SiaA (Streptococcal iron acquisition) is involved in heme uptake in the bacterium Streptococcus pyogenes. It is difficult to obtain this protein in its fully holo form (completely loaded with heme). To increase the concentration of heme in the growing cell, we added ä-aminolevulinic acid (ALA) and ferrous sulfate (FeSO4), precursors of heme, to the growth media. Neither increasing the concentration of heme in vivo, nor growth at lower temperature for longer times, increased the production of holoprotein. The classical method of measuring the concentration of heme in a newly discovered heme protein is cumbersome. We have developed an improved method, which gives a solution that is more stable and has a cleaner spectrum. With further development, this new technique may replace the classical assay. Background information on S. pyogenes, SiaA, ABC transporters, heme biosynthesis, and the pyridine hemochrome assay are described.

Chemotherapy is used in the treatment of cancer. Unfortunately, drugs often fail due to multidrug resistance (MDR) caused by P-glycoprotein (P-gp1or ABCB1) and the multidrug resistance-associated protein (MRP1 or ABCC1). These proteins bind and transport drugs out of cancer cells, thereby conferring MDR. / The second chapter of this thesis addresses an unexplained phenomenon that accompanies P-gp1 expression, collaterally sensitive to verapamil. The collective results of this work demonstrated that treatment of cells that over-express P-gp1 with verapamil induces apoptosis. Furthermore, the findings show that the ATPase activity of P-gp1 was activated by verapamil. The degree of ATPase activation was proportional to the level of apoptosis and the increased demand for ATP resulted in the production of reactive oxygen species (ROS). Finally, the production of ROS led to cell death mediated by apoptosis in that experimental model system. / Chapters three and four are devoted to understanding the binding characteristics of MRP1 with two of its physiological substrates, glutathione (GSH) and leucotriene C4(LTC4). Photoreactive derivatives of these substrates were synthesised to address this objective, IAAGSH and IAALTC4. Photolabelling and transport studies showed that these derivatives have similar binding characteristics as the native compounds. In addition, photolabelling of MRP1 occurred with a high specificity with both compounds. IAAGSH and IAALTC 4 were also used to determine the locations of GSH and LTC4 binding sites. This was accomplished using MRP1-variants containing hemagglutinin (HA) epitopes at specific locations in the amino acid sequence. Through photoaffinity labelling, immunoprecipitation, and trypsin digestion, a map of binding sites for IAAGSH or IAALTC4 was obtained. Both LTC4 and GSH bound to transmembrane (TM) regions 10-11 and 16-17 which have been previously implicated in drug binding. Furthermore, novel binding sites for both substrates were discovered. IAALTC4 photolabelled a novel site within the first five TMs (TMD0) of MRP1, whereas IAAGSH labelled two cytoplasmic regions (L1 and L0). These may represent specific binding sites for LTC4 and GSH. / The work within this thesis explores some of the biochemical characteristics of Pgp1 and MRP1 that are not directly related to drug resistance and may lead to new strategies in cancer treatment.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p. 92-107.

Thesis (honors)--Georgia State University, 2007. / Title from file title page. Under the direction of Dabney White Dixon. Electronic text (88 p. : col. ill.) : digital, PDF file. Description based on contents viewed Sept. 30, 2008. Includes bibliographical references (p. 46-47).