Biomedical subjects
T McGrath
Publications and source records attributed to T McGrath.
Mechanisms of multidrug resistance in HL60 cells. Analysis of resistance associated membrane proteins and levels of mdr gene expression.
HL60 cells isolated for resistance to Adriamycin do not contain P-glycoprotein, as determined with immunological probes. These cells, however, are multidrug resistant and defective in the cellular accumulation of drug. In view of these findings, we have examined in greater detail certain properties of the HL60/Adr cells and have compared these properties to an HL60 drug-resistant isolate (HL60/Vinc) which contains high levels of P-glycoprotein. The results of these studies demonstrated that verapamil induces a major increase in cellular drug accumulation in both HL60/Adr and HL60/Vinc isolates. An 125I-labeled photoaffinity analog of verapamil labeled P-glycoprotein contained in membranes of HL60/Vinc cells. In contrast, this agent did not label any protein selectively associated with drug resistance in membranes of the HL60/Adr isolate. The photoactive dihydropyridine calcium channel blocker [3H]azidopine and [125I]NASV, a photoaffinity analog of vinblastine, labelled P-glycoprotein in membranes from HL60/Vinc cells, whereas in experiments with the HL60/Adr isolate there was no detectable labeling of a drug resistance associated membrane protein. Additional studies have been carried out to analyze membrane proteins of HL60/Adr cells labeled with the photoaffinity agent 8-azido-alpha-[32P]ATP (AzATP32). The results demonstrate that this agent labeled a resistance associated membrane protein of 190 kilodaltons (P190). P190 is essentially absent in membranes of drug-sensitive cells. Labeling of P190 with AzATP32 in membranes of resistant cells was blocked completely when incubations were carried out in the presence of excess unlabeled ATP. Additional studies were carried out to analyze mdr gene amplification and expression in sensitive and resistant cells. Experiments carried out with human 5',mdr1 (1.1 kb) and mdr3 (1.0 kb) cDNAs demonstrate that both of these sequences were highly amplified in the HL60/Vinc isolate. Only the mrd1 gene sequence however, was overexpressed. In contrast, there was no detectable amplification or overexpression of mdr1 or mdr3 sequences in HL60/Adr cells. The results of this study thus identify a new nucleotide binding protein which is overexpressed in membranes of HL60 cells isolated for resistance to Adriamycin. P190, which exhibits properties distinct from P-glycoprotein, possibly functions in the energy-dependent drug efflux system contained in the HL60/Adr resistant isolate.
Multiple mechanisms of adriamycin resistance in the human leukemia cell line CCRF-CEM.
CEM cells exhibiting a 25-fold (C25X) or 80-fold (C80X) increase in resistance to adriamycin were isolated and characterized. C25X cells were cross-resistant to daunomycin and etoposide (VP-16) but not to vincristine or colchicine. These cells were not defective in the cellular accumulation of drug and did not contain detectable levels of P-glycoprotein. Continued exposure of C25X cells to adriamycin resulted in increased levels of resistance and additional phenotypic changes. These cells (C80X) now contained high levels of P-glycoprotein and were cross-resistant to a variety of agents including vincristine and colchicine. A fluorometric assay for DNA unwinding was used to measure levels of drug-induced DNA breaks in sensitive and C25X resistant cells. Studies carried out with VP-16, 4'9-acridinyl-aminomethanesulfon-m-anisidide (m-AMSA), adriamycin, or daunomycin showed that the level of drug-induced DNA strand breakage in resistant cells was considerably less than that occurring in drug-treated sensitive cells. These studies, therefore, show that treatment of CEM cells with adriamycin resulted in a nuclear alteration that contributed to drug resistance. They also demonstrate that prolonged treatment of cells with adriamycin resulted in membrane alterations that affect cellular drug accumulation. Adriamycin resistance in CEM cells can thus occur as a result of at least two distinct mechanisms.
Mechanisms of multidrug resistance in HL60 cells: evidence that a surface membrane protein distinct from P-glycoprotein contributes to reduced cellular accumulation of drug.
HL60 cells exhibiting a 140-fold increase in resistance to vincristine contain three surface membrane proteins with molecular weights of 210,000 (P210), 180,000 (P180), and 150,000 (P150) which are highly phosphorylated in vivo and in an in vitro system in the presence of Mn2+ and [gamma-32P]ATP. These phosphorylated proteins are either absent or present in very low levels in membranes of drug-sensitive cells. Growth of the vincristine-resistant isolate in the absence of drug results in a decrease in the level of resistance and a major reduction in the phosphorylation of P210 and P180. The phosphorylation of P150 is not altered in the revertant which still exhibits substantial levels of resistance. Further studies show that P210 and P180 are highly reactive with a monoclonal antibody against P-glycoprotein. These two proteins are present in only very low levels in revertant cells. The monoclonal antibody exhibits no reactivity with P150. In HL60 cells isolated for a 25-fold increase in vincristine resistance proteins reactive with P-glycoprotein monoclonal antibody are essentially absent. P150 is however highly phosphorylated in these cells. Additional experiments using lectin binding of 32P-labeled proteins demonstrates that P150 has properties distinct from P210 and P180. Analysis of drug uptake patterns in the vincristine-resistant isolates and the revertant shows that resistance is related to a reduced intracellular accumulation of drug. Reduced accumulation of vincristine is also found in HL60 cells isolated for resistance to Adriamycin. These cells are devoid of P-glycoprotein but contain phosphorylated P150. These results suggest that proteins P150, P180, and P210 may contribute to multidrug resistance in HL60 cells through a mechanism which involves reduced cellular accumulation of drug. P180 and P210 are structurally related whereas P150 is distinct from these two proteins.
Adriamycin resistance in HL60 cells in the absence of detectable P-glycoprotein.
Previous studies have shown that the development of multi-drug resistance in cell lines treated with chemotherapeutic agents is closely associated with the overexpression of a 170-180 kilodalton surface membrane glycoprotein (P-glycoprotein). In the present study a monoclonal antibody against the P-glycoprotein was used to determine if this protein is overexpressed in multi-drug resistant HL60 cells. Using either indirect immunofluorescent staining or immunoblot analysis P-glycoprotein could not be detected in HL60 cells isolated for resistance to adriamycin. In contrast HL60 cells isolated for resistance to vincristine contain the P-glycoprotein and the amount of this material increases with increasing levels of resistance. These studies thus demonstrate adriamycin resistance in P-glycoprotein negative HL60 cells. Furthermore adriamycin and vincristine are found to have distinct effects in inducing overexpression of P-glycoprotein in the HL60 cell line. This information could be useful in the development of therapeutic strategies for the treatment of certain forms of cancer.
A new Legionella species, Legionella feeleii species nova, causes Pontiac fever in an automobile plant.
From 15 to 21 August 1981, Pontiac fever affected 317 automobile assembly plant workers. Results of serologic tests were negative for Mycoplasma, Chlamydia, respiratory tract viruses, and previously described legionellae. A gram-negative, rod-shaped organism (WO-44C) that did not grow on blood agar, required L-cysteine for growth, and contained large amounts of branched-chain fatty acids was isolated from a water-based coolant. The organism did not react with antisera against other legionellae, and on DNA hybridization the organism was less than 10% related to other Legionella species. Geometric mean titers found by indirect fluorescent antibody testing to WO-44C were significantly higher in ill employees than in controls (p = 0.0001). Attack rates by department decreased linearly with the department's distance from the implicated coolant system. The etiologic agent apparently was a new Legionella species; we propose the name Legionella feeleii species nova (AATC 35072). This is the first outbreak of nonpneumonic legionellosis in which the etiologic agent is not L. pneumophila, serogroup 1.
Substrate stabilization of lysozyme to thermal and guanidine hydrochloride denaturation.
The thermal and guanidine hydrochloride (GdnHCl) denaturation of lysozyme has been investigated at various concentrations of tri-N-acetylglucosamine ((GlcNAc)3), a trisaccharide which binds specifically at the active site of native lysozyme. The presence of (GlcNAc)3 leads to a readily observable stabilization of the protein to thermal and GdnHCl denaturation. An analysis of guanidine hydrochloride denaturation curves shows that the stability of lysozyme is increased by 495 cal/mol by the presence of 3 x 10(-4) M (GlcNAc)3. The midpoint of the thermal denaturation curve, T 1/2, is increased 1.6 and 5.3 degrees C by 2.02 x 10(-4) M and 1.38 x 10(-3) M (GlcNAc)3, respectively. This corresponds to an increase in the stability of lysozyme of 385 and 1275 cal/mol. These results are in excellent agreement with predictions based on an equation derived by Schellman ((1975) Biopolymers 14, 999-1018) to take into account the effect of ligand binding on the melting temperature of a protein. delta T 1/2 = TT0R divided by delta HD ln (1 + KB[S]) where T and T0 are T1/2 values in the presence and absence of (GlcNAc)3, delta HD is the enthalpy of denaturation in the presence of (GlcNAc)3, KB in the equilibrium constant for the binding of (GlcNAc)3 to lysozyme, and [S] is the free concentration of (GlcNAc)3. Thus, the increased stability of an enzyme in the presence of its substrate, coenzyme, or any small molecule that it binds specifically results because binding to the native state shifts the unfolding equilibrium and decreases the concentration of unfolded states of the enzyme. It is suggested that this may be a more important factor than substrate-induced conformational changes in acccounting for the decreased rates of protein catabolism frequently observed in vivo at elevated substrate concentrations.
Effect of cardiac arrest on cerebral circulation. An experimental investigation.
The effect of 2 to 15 min of cardiac arrest on cerebral circulation was investigated in dogs and Rhesus monkeys. When circulatory arrest lasted longer than 5 minutes, angiographic changes of the no-reflow phenomenon were observed between 3 and 4 h after resuscitation in dogs but not in monkeys. These findings were (1) marked prolongation of the arterial phase in both intracranial and extracranial arteries; followed by (2) occasional evidence of dilatation of the proximal intracranial arteries with non-filling of the distal intracranial arteries; and (3) faint demonstration of the venous phase. Carbon black perfusion results were (1) diffuse lack of perfusion at the arteriolar-capillary level and (2) multifocal areas of filling defects. Regional cortical blood flow measurement in monkeys disclosed a significant decrease in flow starting from 3 h after re-establishment of circulation for those in which longer than 5 min of arrest was induced.
Total body irradiation and syngeneic marrow transplantation in a inbred rat model of acute myelogenous leukemia.
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An early stage in the comparison of methods of modification of a field strain of foot-and-mouth disease, type A (Turkey 19-64).
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Reaching poor populations with cancer prevention and early detection programs.
The American Cancer Society (ACS), Philadelphia Division, through its Nurses' Education Committee (NEC), accepted the challenge to provide cancer prevention and early detection programs to several culturally diverse populations to directly reach the underserved, the working poor, and healthcare professionals who care for the poor. Populations selected included (1) students enrolled in nursing-assistant programs, (2) committed members of the ACS's NEC, (3) registered nurses employed by the local department of public health, and (4) a group of female immigrants from Russia.