Publications (18)66.85 Total impact
-
Article: Energetic domains and conformational analysis of human serum albumin upon co-incubation with sodium benzoate and glucose.
[show abstract] [hide abstract]
ABSTRACT: Sodium benzoate (SB), a powerful inhibitor of microbial growth, is one of the most commonly used food preservative. Here, we determined the effects of SB on human serum albumin (HSA) structure in the presence or absence of glucose after 35 days of incubation under physiological conditions. The biochemical, biophysical, and molecular approaches including free amine content assay (TNBSA assay), fluorescence, and circular dichroism spectroscopy (CD), differential scanning calorimetry (DSC), and molecular docking and LIGPLOT studies were utilized for structural studies. The TNBSA results indicated that SB has the ability to bind Lys residues in HSA through covalent bonds. The docking and LIGPLOT studies also determined another specific site via hydrophobic interactions. The CD results showed more structural helicity for HSA incubated with SB, while HSA incubated with glucose had the least, and HSA incubated with glucose + SB had medium helicity. Fluorescence spectrophotometry results demonstrated partial unfolding of HSA incubated with SB in the presence or absence of glucose, while maximum partial unfolding was observed in HSA incubated with glucose. These results were confirmed by DSC and its deconvoluted thermograms. The DSC results also showed significant changes in HSA energetic structural domains due to HSA incubation with SB in the presence or absence of glucose. Together, our studies showed the formation of three different intermediates and indicate that biomolecular investigation are effective in providing new insight into safety determinations especially in health-related conditions including diabetes.Journal of biomolecular structure & dynamics 04/2013; · 4.99 Impact Factor -
Article: Acidic residue modifications restore chaperone activity of β-casein interacting with lysozyme.
[show abstract] [hide abstract]
ABSTRACT: An important factor in medicine and related industries is the use of chaperones to reduce protein aggregation. Here we show that chaperone ability is induced in β-casein by modification of its acidic residues using Woodward's Reagent K (WRK). Lysozyme at pH 7.2 was used as a target protein to study β-casein chaperone activities. The mechanism for chaperone activity of the modified β-casein was determined using UV-vis absorbencies, fluorescence spectroscopy, differential scanning calorimetry and theoretical calculations. Our results indicated that the β-casein destabilizes the lysozyme and increases its aggregation rate. However, WRK-ring sulfonate anion modifications enhanced the hydrophobicity of β-casein resulting in its altered net negative charge upon interactions with lysozyme. The reversible stability of lysozyme increased in the presence of WRK-modified β-casein, and hence its aggregation rate decreased. These results demonstrate the enhanced chaperone activity of modified β-casein and its protective effects on lysozyme refolding.International journal of biological macromolecules 07/2011; 49(4):616-21. · 2.37 Impact Factor -
Article: A distinct intermediate of RNase A is induced by sodium dodecyl sulfate at its pK(a).
[show abstract] [hide abstract]
ABSTRACT: The chemical denaturation of RNase A was found to be mediated by sodium dodecyl sulfate (SDS) at various pH. The characterization of the unfolding pathway was investigated by spectrophotometry and differential scanning calorimetry (DSC), and was analyzed by multivariate curve resolution (MCR) as a chemometric method. The spectrophotometric titration curve of RNase A upon interaction with SDS indicated a distinct complex intermediate in glycine buffer at pH 3.3. This was accompanied with the catalytic activation of the enzyme and was concurrent with maximum population of the intermediate, determined by MCR. This was confirmed by the DSC profile of RNase A in the presence of SDS, indicated by two transitions in thermal unfolding. The kinetic data on the unfolding process of RNase A upon addition of SDS showed a two-phase pathway under the same conditions. The intermediate appeared at low pH especially at the pK(a) of SDS (pH 3.3). These results provide strong evidence of the influence of low pH (around the pK(a) of SDS) on the existence of an intermediate upon interaction of RNase A with SDS.Colloids and Surfaces B Biointerfaces 08/2005; 43(3-4):150-7. · 3.46 Impact Factor -
Article: Electrochemical evidence for the molten globule states of cytochrome c induced by N-alkyl sulfates at low concentrations.
[show abstract] [hide abstract]
ABSTRACT: The molten globule state (MG) of cytochrome c is the major intermediate of protein folding. The formation of MG state of cytochrome c is induced by n-alkyl sulfates such as sodium octyl sulfate (SOS), sodium dodecyl sulfate (SDS), and sodium tetradecyl sulfate (STS). The folding state of cytochrome c was monitored using circular dichroism (CD), isothermal titration calorimetry (ITC) and partial specific volumes. To explore a new approach for characterizing the MG conformation, cyclic voltametric studies of n-alkyl sulfates induced transition at acidic pH of cytochrome c (unfolded state, U) was carried out. Here, we have used a cystein-modified gold electrode, which is effective for direct rapid electron transfer to cytochrome c even in acid solutions, to directly observe electrochemistry in native (N) cytochrome c. Our results show that the extent of electron transfer is increased for U --> MG, and also the easiness of electron transferring occurred from MG --> N transition. Thus we demonstrate that the MG state of cytochrome c, induced by n-alkyl sulfates as salts with hydrophobic chains (hydrophobic salts), with different compactness reaches to near identical amount of electron transferring as N state.Journal of Protein Chemistry 01/2003; 22(1):23-30. -
Article: Thermodynamic analysis of human serum albumin interactions with glucose: insights into the diabetic range of glucose concentration.
[show abstract] [hide abstract]
ABSTRACT: The interaction of proteins with glucose results in their non-enzymatic glycation and influences their structural and functional properties. Human serum albumin (HSA) interacts with glucose forming glycated HSA. However, the glucose binding sites and the thermodynamic characteristics of the glycated HSA require further delineation. Here, the binding properties of HSA and glucose were studied utilizing fluorescent techniques. HSA was incubated with glucose in the 0-300mM range at 27 or 37 degrees C. The interaction of HSA with glucose showed two sets of binding sites. The first set consists of two sites with positive cooperativity and the second set consists of nine identical non-cooperative sites. The percentage of glycated HSA (gly%) and the moles of glucose bound to moles of HSA (r) were utilized to obtain binding constants and thermodynamic parameters based on the Wyman binding potential. The enthalpy of binding, obtained by van't Hoff relation, presented exothermicity up to 7mM glucose (126mg/dl, normal range) and endothermic propensity at higher glucose concentrations (>7mM, diabetic range). The start of endothermic propensity was consistent with the diabetic range of glucose concentration and indicates unfolding of HSA. The Gibbs free energy and entropy of binding further supports the unfolding of HSA. Therefore, glucose interacts with multiple sites on HSA affecting its biochemical and biophysical properties. This may interfere with HSA normal function contributing to diabetic complications.The International Journal of Biochemistry & Cell Biology 10/2002; 34(9):1115-24. · 4.63 Impact Factor -
Article: Macrophage receptors responsible for distinct recognition of low density lipoprotein containing pyrrole or pyridinium adducts: models of oxidized low density lipoprotein.
[show abstract] [hide abstract]
ABSTRACT: Oxidation of low density lipoproteins (LDL) induced by incubation with Cu(2+) ions results in the formation of a heterogeneous group of aldehydic adducts on lysyl residues (Lys) of apolipoprotein B (apoB) that are thought to be responsible for the uptake of oxidized LDL (oxLDL) by macrophages. To define the structural and chemical criteria governing such cell recognition, we induced two modifications of lysines in LDL that mimic prototypic adducts present in oxLDL; namely, epsilon-amino charge-neutralizing pyrrolation by treatment with 2,5-hexanedione (hdLDL), and epsilon-amino charge-retaining pyridinium formation via treatment with 2,4,6-trimethylpyrylium (tmpLDL). Both modifications led to recognition by receptors on mouse peritoneal macrophages (MPM). To assess whether the murine scavenger receptor class A-I (mSR-A) was responsible for recognition of hdLDL or tmpLDL in MPM, we measured binding at 4 degrees C and degradation at 37 degrees C of these modified forms of (125)I-labeled LDL by mSR-A-transfected CHO cells. Although uptake and degradation of hdLDL by mSR-A-transfected CHO cells was quantitatively similar to that of the positive control, acLDL, tmpLDL was not recognized by these cells. However, both tmpLDL and hdLDL were recognized by 293 cells that had been transfected with CD36. In the human monocytic cell line THP-1 that had been activated with PMA, uptake of tmpLDL was significantly inhibited by blocking monoclonal antibodies to CD36, further suggesting recognition of tmpLDL by this receptor. Macrophage uptake and degradation of LDL oxidized by brief exposure to Cu(2+) was inhibited more effectively by excess tmpLDL and hdLDL than was more extensively oxidized LDL, consistent with the recognition of the former by CD36 and the latter primarily by SR-A.Collectively, these studies suggest that formation of specific pyrrole adducts on LDL leads to recognition by both the mSR-A and mouse homolog of CD36 expressed on MPM, while formation of specific pyridinium adducts on LDL leads to recognition by the mouse homolog of CD 36 but not by mSR-A. As such, these two modifications of LDL may represent useful models for dissecting the relative contributions of specific modifications on LDL produced during oxidation, to the cellular uptake of this heterogeneous ligand.The Journal of Lipid Research 10/2000; 41(9):1455-63. · 5.56 Impact Factor -
Article: Differential modulation of cadherin-mediated cell-cell adhesion by platelet endothelial cell adhesion molecule-1 isoforms through activation of extracellular regulated kinases.
[show abstract] [hide abstract]
ABSTRACT: The role of platelet endothelial cell adhesion molecule-1 (PECAM-1) in endothelial cell-cell interactions and its contribution to cadherin-mediated cell adhesion are poorly understood. Such studies have been difficult because all known endothelial cells express PECAM-1. We have used Madin-Darby canine kidney (MDCK) cells as a model system in which to evaluate the role of PECAM-1 isoforms that differ in their cytoplasmic domains in cell-cell interactions. MDCK cells lack endogenous PECAM-1 but form cell-cell junctions similar to those of endothelial cells, in which PECAM-1 is concentrated. MDCK cells were transfected with two isoforms of murine PECAM-1, Delta15 and Delta14&15, the predominant isoforms expressed in vivo. Expression of the Delta15 isoform resulted in apparent dedifferentiation of MDCK cells concomitant with the loss of adherens junctions, down-regulation of E-cadherin, alpha- and beta-catenin expression, and sustained activation of extracellular regulated kinases. The Delta15 isoform was not concentrated at cell-cell contacts. In contrast, the Delta14&15 isoform localized to sites of cell-cell contact and had no effect on MDCK cell morphology, cadherin/catenin expression, or extracellular regulated kinase activity. Thus, the presence of exon 14 in the cytoplasmic domain of PECAM-1 has dramatic effects on the ability of cells to maintain adherens junctions and an epithelial phenotype. Therefore, changes in the expression of exon 14 containing PECAM-1 isoforms, which we have observed during development, may have profound functional consequences.Molecular Biology of the Cell 08/2000; 11(8):2793-802. · 4.94 Impact Factor -
Article: Focal adhesion kinase, paxillin, and bcl-2: analysis of expression, phosphorylation, and association during morphogenesis.
[show abstract] [hide abstract]
ABSTRACT: Cell adhesive mechanisms which determine tissue architecture during morphogenesis are tightly regulated and have an impact on apoptosis, cell migration, proliferation, and differentiation. Bcl-2 is a death repressor that protects cells from apoptosis initiated by a variety of stimuli including loss of cell adhesion. Utilizing the kidney as a model of an organ that undergoes three-dimensional development we demonstrate that bcl-2 directly associates with paxillin. Focal adhesion kinase (FAK)(p125) and paxillin(p68) were highly expressed and tyrosine phosphorylated during development but declined to low levels following renal maturation (postnatal day 20) in normal mice. The decline in the expression of p125 FAK and p68 paxillin occurred together with an increase in specific cleavage products of lower molecular weights. Mice deficient in bcl-2 are born with renal hypoplasia and succumb to renal failure as a result of renal multicystic disease. In kidneys from postnatal day 20 bcl-2 -/- mice, tyrosine phosphorylation of p125 FAK and p68 paxillin was not down-regulated. However, the level of expression was similar to that of normal mice. These results demonstrate that the developmentally regulated expression and phosphorylation of FAK and paxillin, in the presence of bcl-2, is necessary for normal morphogenesis. The interaction of paxillin with bcl-2 during nephrogenesis may provide an alternative to integrin(s) signaling through paxillin/FAK thus bypassing the need for adhesion-mediated survival during three dimensional morphogenesis. Dev Dyn 1999;215:371-382.Developmental Dynamics 09/1999; 215(4):371-82. · 2.54 Impact Factor -
Article: Prokaryotic gene fusion expression systems and their use in structural and functional studies of proteins.
[show abstract] [hide abstract]
ABSTRACT: The ability to express and purify large quantity of proteins in bacteria has greatly impacted many aspects of biological research. These include their use as a source of reagent for biochemical and biophysical studies as well as a source of antigen for antibody production. Currently many different expression systems are available and new ones are being developed. These systems allow inducible expression of a desired protein as a fusion with an affinity tag for simple purification. The affinity tags can generally be removed by specific proteases which recognize cleavage sites engineered between the affinity tag and the desired protein. Presence of tags that encode epitopes of specific antibodies provide additional means for identification of recombinant proteins. This review provides an overview of some of the most commonly utilized expression systems and examples of the use of these proteins in biochemical and biophysical studies. I will also describe other available systems which may provide suitable alternative for expression of recombinant proteins.Preparative Biochemistry & Biotechnology 03/1999; 29(1):77-90. · 0.47 Impact Factor -
Article: Thrombospondin-1, PECAM-1, and regulation of angiogenesis.
[show abstract] [hide abstract]
ABSTRACT: Thrombospondin-1 (TSP1) is a multidomain glycoprotein expressed by many cell types. It is a multifunctional protein with important roles in regulation of vascular cell functions. Mutation or loss of tumor suppressor genes results in down regulation of TSP1 expression during malignant transformation. Thus, suggesting that down regulation of TSP1 may contribute to development of the tumor angiogenic phenotype and perhaps tumor metastasis. TSP1 was demonstrated to be a natural inhibitor of angiogenesis. Peptides from procollagen-like domain and type 1 repeats of TSP1, like whole TSP1, inhibit the angiogenic response to a variety of angiogenic stimuli in vivo and endothelial cell (EC) migration in vitro by directly acting on ECs. The molecular mechanisms which mediate these inhibitory effects of TSP1 and its peptides are not understood. TSP1 expression is down regulated in the Polyoma middle T transformed mouse brain ECs (bEND.3). This may remove the TSP1 inhibitory effects allowing ECs to rapidly proliferate in culture and form hemangiomas in vivo. Re-expression of TSP1 in bEND.3 cells restores a normal phenotype and suppresses their ability to form hemangiomas. This is mediated by modulating expression of several genes in concert favoring a differentiated state of endothelium. TSP1 transfected bEND.3 cells down regulate expression of PECAM-1, a multifunctional endothelial cell adhesion molecule with essential roles in angiogenesis. A similar phenotype to that of TSP1 transfected cells was observed when endogenous PECAM-1 levels were down regulated by anti-sense transfection of bEND.3 cells. The anti-sense PECAM-1 transfected cells turn on expression of endogenous TSP1 and its angioinhibitory receptor, CD36. Expression of other genes with potential roles in regulation of EC phenotype were also affected in patterns very similar to those observed in TSP1 transfected bEND.3 cells. Therefore, it appears that a reciprocal relationship exists between TSP1 and PECAM-1 such that they are constituents of a "switch" that regulates in concert many components of the angiogenic and differentiated phenotype of ECs.Histology and histopathology 02/1999; 14(1):285-94. · 2.48 Impact Factor -
Article: Tissue specific expression of alternatively spliced murine PECAM-1 isoforms.
[show abstract] [hide abstract]
ABSTRACT: PECAM-1 (CD31) is a cell adhesion molecule that is highly expressed at the sites of endothelial cell-cell contact and at lower levels on the surface of platelets and leukocytes. It is a member of the immunoglobulin gene superfamily and undergoes alternative splicing to generate several isoforms that differ only in their cytoplasmic domains. The tissue distribution of the expression of different PECAM-1 isoforms has not been previously defined. We have examined PECAM-1 expression in various mouse tissues and endothelial cells. PECAM-1 mRNA was highly expressed in lung, heart, and kidney, and to a lower extent in brain and liver. Most endothelial cells in culture expressed high levels of PECAM-1 mRNA; however, normal mouse brain endothelial cells rapidly lost PECAM-1 expression in culture. To examine the tissue distribution of PECAM-1 isoform expression, RT/PCR was performed on the RNA isolated from various mouse tissues and mouse endothelial cells. Cloning and sequencing of the cDNA products indicated that most tissues and endothelial cells expressed several PECAM-1 isoforms at different frequencies. The PECAM-1 isoform that lacks exons 14 and 15 was most frequently detected in all cases. A novel PECAM-1 isoform that lacks exons 12 and 14 was detected in brain. An antibody to the extracellular domain of PECAM-1 reacted with two major bands, at 130 kDa and 110-120 kDa, in lysates prepared from endothelial cells or kidneys at different stages of development. An antibody prepared against PECAM-1 exon 14, which reacts only with cytoplasmic domain of PECAM-1 isoforms that contain exon 14, failed to react with the major lower molecular weight form of PECAM-1 in these lysates. Therefore, PECAM-1 isoforms that lack exon 14 are expressed in endothelial cells and tissues in developmentally regulated fashion. These results illustrate that multiple PECAM-1 isoforms are expressed in various mouse tissues and endothelial cells. Understanding the distribution of PECAM-1 isoforms, and the identity of intracellular proteins with which they may interact, will help to elucidate the role of PECAM-1 in endothelial cell-cell interactions and morphogenesis.Developmental Dynamics 02/1999; 214(1):44-54. · 2.54 Impact Factor -
Article: Direct use of synthetic peptides for antiserum production.
BioTechniques 08/1998; 25(1):28-30, 32. · 2.67 Impact Factor -
Article: Down-regulation of platelet endothelial cell adhesion molecule-1 results in thrombospondin-1 expression and concerted regulation of endothelial cell phenotype.
[show abstract] [hide abstract]
ABSTRACT: bEND.3 cells are polyoma middle T-transformed mouse brain endothelial cells that express very little or no thrombospondin-1, a natural inhibitor of angiogenesis, but express high levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) that localizes to sites of cell-cell contact. Here, we have examined the role of PECAM-1 in regulation of bEND.3 cell proliferation, migration, morphogenesis, and hemangioma formation. We show that down-regulating PECAM-1 expression by antisense transfection of bEND. 3 cells has a dramatic effect on their morphology, proliferation, and morphogenesis on Matrigel. There is an optimal level for PECAM-1 expression such that high levels of PECAM-1 inhibit, whereas moderate levels of PECAM-1 stimulate, endothelial cell morphogenesis. The down-regulation of PECAM-1 in bEND.3 cells resulted in reexpression of endogenous thrombospondin-1 and its antiangiogenic receptor CD36. The expression of the vascular endothelial growth factor receptors flk-1 and flt-1, as well as integrins and metalloproteinases (which are involved in angiogenesis), were also affected. These observations are consistent with the changes observed in proliferation, migration, and adhesion characteristics of the antisense-transfected bEND.3 cells as well as with their lack of ability to form hemangiomas in mice. Thus, a reciprocal relationship exists between thrombospondin-1 and PECAM-1 expression, such that these two molecules appear to be constituents of a "switch" that regulates in concert many components of the angiogenic and differentiated phenotypes of endothelial cells.Molecular Biology of the Cell 04/1998; 9(4):701-13. · 4.94 Impact Factor -
Article: Miniprep DNA isolation for automated sequencing of multiple samples.
Analytical Biochemistry 08/1997; 250(1):117-9. · 3.00 Impact Factor -
Article: Thrombospondin-1, a natural inhibitor of angiogenesis, regulates platelet-endothelial cell adhesion molecule-1 expression and endothelial cell morphogenesis.
[show abstract] [hide abstract]
ABSTRACT: Expression of thrombospondin-1 (TS1) in polyoma middle-sized T (tumor)-transformed mouse brain endothelial cells (bEND.3) restores a normal phenotype and suppresses their ability to form hemangiomas in mice. We show that TS1 expression results in complete suppression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) expression and altered cell-cell interactions in bEND.3 cells. To further investigate the role of PECAM-1 in regulation of endothelial cell-cell interactions and morphogenesis, we expressed human (full length) or murine (delta 15) PECAM-1 isoforms in TS1-transfected bEND.3 (bEND/TS) cells. Expression of either human or murine PECAM-1 resulted in an enhanced ability to organize and form networks of cords on Matrigel, an effect that was specifically blocked by antibodies to PECAM-1. Anti-PECAM-1 antibodies also inhibited tube formation in Matrigel by normal human umbilical vein endothelial cells. However, PECAM-1-transfected bEND/TS cells did not regain the ability to form hemangiomas in mice and the expressed PECAM-1, unlike the endogenous PECAM-1 expressed in bEND.3 cells, failed to localize to sites of cell-cell contact. This may be, in part, attributed to the different isoforms of PECAM-1 expressed in bEND.3 cells. Using reverse transcription-polymerase chain reaction, we determined that bEND.3 cells express mRNA encoding six different PECAM-1 isoforms, the isoform lacking both exons 14 and 15 (delta 14&15) being most abundant. Expression of the murine delta 14&15 PECAM-1 isoform in bEND/TS cells resulted in a similar phenotype to that described for the full-length human or murine delta 15 PECAM-1 isoform. The delta 14&15 isoform, despite the lack of exon 14, failed to localize to sites of cell-cell contact even in clones that expressed it at very high levels. Thus, contrary to recent reports, lack of exon 14 is not sufficient to result in junctional localization of PECAM-1 isoforms in bEND/TS cells.Molecular Biology of the Cell 07/1997; 8(7):1329-41. · 4.94 Impact Factor -
Article: Thrombospondin 1 expression in transformed endothelial cells restores a normal phenotype and suppresses their tumorigenesis.
[show abstract] [hide abstract]
ABSTRACT: Murine endothelial cells are readily transformed in a single step by the polyomavirus oncogene encoding middle-sized tumor antigen. These cells (bEND.3) form tumors (hemangiomas) in mice which are lethal in newborn animals. The bEND.3 cells rapidly proliferate in culture and express little or no thrombospondin 1 (TS1). To determine the role of TS1 in regulation of endothelial cell phenotype, we stably transfected bEND.3 cells with a human TS1 expression vector. The cells expressing human TS1 were readily identified by their altered morphology and exhibited a slower growth rate and lower saturation density than the parental bEND.3 cells. The TS1-expressing cells also formed aligned cords of cells instead of clumps or cysts in Matrigel. Moreover, while the bEND.3 cells formed large tumors in nude mice within 48 hr, the TS1-expressing cells failed to form tumors even after 1 month. The TS1-transfected cells expressed transforming growth factor beta mRNA and bioactivity at levels similar to those of the parental or vector-transfected bEND.3 cells, indicating that the effects of TS1 expression are not due to the activation of transforming growth factor beta by TS1. TS1 expression resulted in a > 100-fold decrease in net fibrinolytic (urokinase-type plasminogen activator, uPA) activity due to more plasminogen-activator inhibitor 1 and less uPA secretion. TS1 thus appears to be an important regulator of endothelial cell phenotype required for maintaining the quiescent, differentiated state.Proceedings of the National Academy of Sciences 08/1995; 92(15):6788-92. · 9.68 Impact Factor -
Article: Analysis of various mRNA potentially involved in cisplatin resistance of murine leukemia L1210 cells.
[show abstract] [hide abstract]
ABSTRACT: Enhanced DNA repair has been identified as a major mechanism of resistance to the anticancer drug cisplatin in murine leukemia L1210 cells. Studies of other cells have implicated the elevation of a variety of RNA transcripts in cisplatin resistance. This study investigated potential changes in transcription of these genes as well as genes involved in DNA repair. No elevation in any of the following transcripts was observed: thymidylate synthase, dihydrofolate reductase, DNA polymerase alpha, DNA polymerase beta, topoisomerase II, Ha-ras, beta-tubulin, metallothionein and the DNA repair genes ERCC1 and ERCC2. Thymidine kinase was increased no more than 2-fold. None of these RNA were induced by incubation with cisplatin. High levels of cisplatin produced selective decreases in certain RNA. These results demonstrate that the previous observations of elevated RNA can not be universally applied to all cisplatin-resistant cells.Cancer Letters 08/1990; 52(3):179-85. · 4.24 Impact Factor -
Article: DNA repair in cells sensitive and resistant to cis-diamminedichloroplatinum(II): host cell reactivation of damaged plasmid DNA.
[show abstract] [hide abstract]
ABSTRACT: cis-Diamminedichloroplatinum(II) (cis-DDP) has a broad clinical application as an effective anticancer drug. However, development of resistance to the cytotoxic effects is a limiting factor. In an attempt to understand the mechanism of resistance, we have employed a host cell reactivation assay of DNA repair using a cis-DDP-damaged plasmid vector. The efficiency of DNA repair was assayed by measuring the activity of an enzyme coded for by the plasmid vector. The plasmid expression vector pRSVcat contains the bacterial gene coding for chloramphenicol acetyltransferase (CAT) in a configuration which permits expression in mammalian cells. The plasmid was transfected into repair-proficient and -deficient Chinese hamster ovary cells, and CAT activity was subsequently measured in cell lysates. In the repair-deficient cells, one cis-DDP adduct per cat gene was sufficient to eliminate expression. An equivalent inhibition of CAT expression in the repair-proficient cells did not occur until about 8 times the amount of damage was introduced into the plasmid. These results implicate DNA intrastrand cross-links as the lesions responsible for the inhibition of CAT expression. This assay was used to investigate the potential role of DNA repair in mediating cis-DDP resistance in murine leukemia L1210 cells. The parent cell line L1210/0 resembled repair-deficient cells in that about one adduct per cat gene eliminated expression. In three resistant L1210 cell lines, 3-6-fold higher levels of damage were required to produce an equivalent inhibition. This did not correlate with the degree of resistance as these cells varied from 10- to 100-fold resistant.(ABSTRACT TRUNCATED AT 250 WORDS)Biochemistry 05/1989; 28(7):3120-4. · 3.42 Impact Factor
Top co-authors
Top Journals
Institutions
-
2002–2011
-
University of Tehran
- Institute of Biochemistry and Biophysics
Tehrān, Ostan-e Tehran, Iran
-
-
1995–2000
-
University of Washington Seattle
- Department of Biochemistry
Seattle, WA, USA
-
-
1990
-
Dartmouth Medical School
- Department of Pharmacology and Toxicology
Hanover, NH, USA
-
-
1989
-
University of Nebraska at Omaha
- Eppley Institute for Research in Cancer and Allied Diseases
Omaha, NE, USA
-
