Biomedical subjects

R A Davey

Publications and source records attributed to R A Davey.

At least 37 records · Page 2Linked to original sources

PubMed1997

In vitro binding of purified murine ecotropic retrovirus envelope surface protein to its receptor, MCAT-1.

An amino-terminal portion of the Friend murine leukemia virus (MLV) envelope surface protein [SU, residues 1 to 236 [SU:(1-236)]] and its receptor, MCAT-1, were each purified from insect cells after expression by using recombinant baculoviruses. Friend SU:(1-236) bound specifically to Xenopus oocytes that expressed MCAT-1 with an affinity (Kd, 55 nM) similar to that of viral SU binding to permissive cells. Direct binding of Friend SU:(1-236) to purified MCAT-1 was observed in detergent and after reconstitution into liposomes. Analysis of binding demonstrated that MCAT-1 and Friend SU:(1-236) interact with a stoichiometry of near 1:1. These findings demonstrate that the amino-terminal domain from the SU of ecotropic murine retroviruses contains an MCAT-1 binding domain.

Animals↗

PubMed1996

Biochemical evidence that patched is the Hedgehog receptor.

The protein Sonic hedgehog (Shh) is essential for a variety of patterning events during development. It is the signal from the notochord that induces ventral cell fate in the neural tube and somites, and is the polarizing signal for patterning of the anterior-posterior axis of the developing limb bud. Because of these and other inductive functions of Shh, it is important to understand how the Hedgehog (Hh) signal is received by the target cells. Here we describe binding studies using labelled Shh that strongly suggest that the Hh receptor is encoded by patched (ptc), a gene first identified in genetic screens in Drosophila.

Animals↗

PubMed1996

Comparison of drug accumulation in P-glycoprotein-expressing and MRP-expressing human leukaemia cells.

P-glycoprotein- and multidrug resistance-associated protein (MRP)-mediated multidrug resistance is associated with decreased drug accumulation. The P-glycoprotein-expressing CCRF-CEM/VLB100 subline and the MRP-expressing CCRF-CEM/E1000 subline are both 50-fold resistant to daunorubicin. However, accumulation of daunorubicin and rhodamine 123 was > 85% reduced in the P-glycoprotein-expressing subline compared to 40-50% in the MRP-expressing subline. Further, the CCRF-CEM/E1000 cells were 30-fold resistant to idarubicin, without reduced accumulation. Verapamil and SDZ PSC 833 restored daunorubicin and rhodamine 123 accumulation, while buthionine sulphoximine affected only the CCRF-CEM/ E1000 subline. We conclude that the verapamil associated change in rhodamine 123 accumulation provides a sensitive functional assay for both P-glycoprotein- and MRP-mediated MDR.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

The anthracycline resistance-associated (ara) gene, a novel gene associated with multidrug resistance in a human leukaemia cell line.

Multidrug resistance (MDR) in cancer cells is a major contributor to the failure of chemotherapy treatment. This paper describes a novel protein named the anthracycline resistance associated (ARA) protein. The ara gene is amplified in the MDR leukaemia line CCRF-CEM/E1000 and its mRNA is overexpressed. ARA belongs to the ATP binding cassette (ABC) family of proteins. Another ABC protein, the multidrug resistance-associated protein (MRP), has previously been reported to be overexpressed in the CEM/E1000 subline. The primary amino acid sequence of ARA indicates that it is 49.5 kDa without glycosylation, and that it has one potential glycosylation site. ARA has one ATP binding site and associated transmembrane regions. This is in contrast to MRP (190 kDa, 172 kDa deglycosylated) and most other higher eukaryote ABC proteins, which consist of two similar halves, each having one ATP binding site. In addition to ARA being coexpressed with MRP, comparison of amino acid sequences showed that, among known proteins, ARA is most similar to the C-terminal half of MRP.

ATP-Binding Cassette Transporters↗

PubMed1996

Extended multidrug resistance in haemopoietic cells.

The development of drug resistance was studied in a series of haemopoietic cells to determine its relationship to cell lineage. Treatment of the U937 monocytic cell line with epirubicin (15 ng/ml) or vinblastine (8 ng/ml) induced drug-resistant sublines with cross-resistance to epirubicin (8- and 16-fold respectively), vinblastine (5- and 20-fold), paclitaxel (15- and 42-fold) and etoposide (19- and 13-fold). However, sublines were also 3-5-fold resistant to the alkylating agent chlorambucil, cis-platinum and methotrexate, demonstrating an extended multidrug resistance (MDR) phenotype. These cells over-expressed P-glycoprotein, but decreased drug accumulation was not restored in the presence of verapamil, suggesting that the P-glycoprotein was not functional. Similar drug treatment of the HL60 promyelocytic cell line also produced sublines exhibiting an extended MDR phenotype. The KG1a and the HEL cell lines expressed functional P-glycoprotein and were resistant to the drug concentrations used for treatment. Multidrug resistance as mediated by P-glycoprotein cannot explain the resistance of CML patients to chemotherapy, especially in blast crisis. The induction of an extended MDR phenotype specifically in myeloid cells in response to drug treatment may explain the resistance observed in the treatment of CMI.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Paclitaxel sensitizes multidrug resistant cells to radiation.

The unique action of paclitaxel, to stabilize microtubules and block cells at the radiosensitive G2M phase of the cell cycle, suggests, it may sensitize tumors to radiotherapy. We have investigated the potential of this interaction to overcome multidrug resistance in vitro using the HL60 cell line and its P-glycoprotein expressing, multidrug resistant H/E8 subline. HL60 cells showed a modest 1.4-fold (p < 0.01) increase in sensitivity to 2 Gy radiation given 24 h after a 1 h treatment with paclitaxel. The H/E8 subline, which has increased radiation resistance and expresses an extended multidrug resistance phenotype, showed significant sensitization to radiation (up to 2.3-fold sensitization; p < 0.01) even with doses of paclitaxel which had no effect on cell viability or were associated with any G2/M block in the cell cycle. In the presence of verapamil, an inhibitor of P-glycoprotein mediated efflux, drug resistant cells could be sensitized to 2 Gy radiation by similar paclitaxel doses as the parental cell (> or = 30 nM; p < 0.01). These results indicate a therapeutic advantage may be possible in the treatment of resistant tumors by the combined use of paclitaxel with radiation.

Antineoplastic Agents, Phytogenic↗

PubMed1995

Expression of multidrug resistance in response to differentiation in the K562 human leukaemia cell line.

With the increasing use of inducers of cellular differentiation in the treatment of leukaemia, it is essential to understand the relationship between differentiation and the expression of the multidrug resistance. Using the K562 human leukaemia cell line and its multidrug resistant subline K562/E15B, differentiation was examined along two different pathways, megakaryocyte in response to treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), and erythroid in response to treatment with sodium butyrate, in the same cell line. P-glycoprotein expression was increased in the multidrug resistant K562/E15B subline, but not induced in the parental K562 cell line. However, both treatments conferred a different phenotype on the drug resistant subline. TPA treatment caused an increase in P-glycoprotein, increased drug resistance and decreased rhodamine-123 accumulation which was verapamil sensitive, demonstrating that TPA induced a fully functional P-glycoprotein. However, sodium butyrate treatment caused an increase in P-glycoprotein without increased drug resistance or without decreased rhodamine-123 accumulation suggesting that the P-glycoprotein induced by sodium butyrate was nonfunctional. These results stress the importance of examining not only the expression of P-glycoprotein in cells, but also the function of the P-glycoprotein induced.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Drug resistance mechanisms and MRP expression in response to epirubicin treatment in a human leukaemia cell line.

A drug resistant series of sublines were developed by treating the human leukaemia CCRF-CEM cell line with 16-1000 ng/ml of the anthracycline, epirubicin. The sublines developed resistance in two stages, neither involving detectable levels of P-glycoprotein. Treatment with up to 50 ng/ml epirubicin produced sublines with cross resistance limited to the anthracyclines and etoposide. Treatment with 100-1000 ng/ml epirubicin produced sublines with increased expression of the mrp gene, increased resistance to the anthracyclines and etoposide, additional cross resistance to vincristine and colchicine, decreased drug accumulation and reversal of resistance by verapamil and by buthionine sulphoximine (BSO; an inhibitor of glutathione synthesis). Our results indicate an interaction between MRP and glutathione metabolism as a mechanism for multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Development of drug resistance is reduced with idarubicin relative to other anthracyclines.

Multidrug resistance (MDR) is associated with poor prognosis in leukemia, and anthracyclines, which are used in the treatment of leukemia, are associated with the expression of P-glycoprotein and the development of MDR. We report here that idarubicin, a new anthracycline approved for use in the treatment of acute myelogenous leukemia (AML), did not induce P-glycoprotein expression in the K562 human leukemia cell line under conditions where daunorubicin, doxorubicin and epirubicin did induce expression of P-glycoprotein. The P-glycoprotein expressing, multidrug resistant sublines developed to daunorubicin (K/DNR), doxorubicin (K/DOX) and epirubicin (K/EPR) were cross-resistant to the other anthracyclines and to vinblastine, taxol, colchicine and actinomycin D, but were not resistant to idarubicin or etoposide. The idarubicin treated subline, K/IDA, was only resistant to taxol but was 12-fold sensitized to etoposide, suggesting that idarubicin had affected topoisomerase II in this subline.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Development of extended multidrug resistance in HL60 promyelocytic leukaemia cells.

In an attempt to mimic clinical conditions for the treatment of leukaemia, the HL60 promyelocytic cell line was treated for 18 h with low, clinically relevant, levels of the anthracycline epirubicin and the Vinca alkaloid vinblastine. The resulting drug-resistant sublines not only expressed P-glycoprotein and the MDR phenotype but were also cross-resistant to chlorambucil, methotrexate and cisplatinum, and had increased resistance to radiation. Development of resistance was associated with an aberrant differentiation phenotype with decreased expression of myeloid antigens and expression of glycophorin A, an antigen normally associated with erythroid differentiation. The ability of HL60 cells to terminally differentiate in response to all-trans-retinoic acid (vitamin A acid) was lost in the sublines. These results suggest that either a single novel mechanism is responsible for multiple drug resistance or the initial response to drug treatment is the co-induction of multiple mechanisms. These cells and the method by which they were generated therefore provide a clinically relevant model for the study of the initial events in the development of not only multidrug resistance but also the extended multiple drug resistance usually encountered in the treatment of leukaemia.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Differentiation and multidrug resistance in response to drug treatment in the K562 human leukaemia cell line.

The relationship between differentiation and P-glycoprotein expression in response to chemotherapeutic drugs was studied in the K562 human leukaemia cell line by treatment with low, but clinically achievable levels of vinblastine and epirubicin. Resistant sublines were easily generated with the multidrug resistant phenotype being expressed in response to drug treatment as low as 1 ng/ml vinblastine and 10 ng/ml epirubicin. These sublines showed stable but heterogeneous expression of P-glycoprotein as revealed by immunocytochemistry, and confirmed by cloning. This heterogeneity was maintained over 18 months with intermittent drug treatment. While selection for resistance induced erythroid and myeloid differentiation, expression of P-glycoprotein was not correlated with the stem cell antigen CD34 or with specific markers of erythroid or myeloid differentiation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Identification of a broad-specificity nucleoside transporter with affinity for the sugar moiety in Giardia intestinalis trophozoites.

A broad-specificity nucleoside transporter has been identified in Giardia intestinalis trophozoites, using a rapid sampling assay to measure influx of [3H]deoxycytidine, [3H]adenosine and [3H]guanosine at 0 degrees C. The influx of each labelled nucleoside was inhibited strongly by all common, naturally-occurring nucleosides but only poorly or not at all by nucleobases, indicating that the transporter recognizes structural features on the furanosyl moiety of ribo- and 2'-deoxyribonucleosides. Both 2'- and 5'-deoxyadenosine were potent inhibitors of influx (greater than 95% inhibition at 2 mM), whereas 3'-deoxyadenosine was significantly less effective (approx. 70% inhibition), and 2',3'-dideoxycytidine and cytosine arabinoside were virtually inactive (0-20% inhibition). The data reveal that the 2'- and 5'-hydroxyl groups are not necessary for the recognition of nucleosides by this transporter. However, the 3'-hydroxyl appears to be important. Michaelis-Menten constants (Km) were calculated for the influx at 0 degrees C of deoxycytidine (220 +/- 116 microM) and adenosine (45 +/- 24 microM), with respective Vmax values of 13 +/- 4 and 11 +/- 2 pmol min-1 (10(6) cells)-1. Only 12-26% of [3H]thymidine influx occurred through this transporter, the remainder entering the cells through a thymine/uracil-specific transporter described previously. Thymidine exhibited a Ki of 205 +/- 90 microM against [3H]deoxycytidine influx.

Adenosine↗

PubMed1992

A low-affinity nucleobase transporter in the protozoan parasite Giardia intestinalis.

A membrane transporter with general affinity for purine and pyrimidine bases has been identified in Giardia intestinalis trophozoites by measuring cellular influx of [3H]adenine, [3H]guanine and [3H]thymine at 0 degrees C. The base transporter is distinct from the thymine/uracil-specific (type 1) and broad-specificity (type 2) nucleoside transporters of G. intestinalis. Influx of each labelled base was retarded by unlabelled bases, with inhibition in the order: hypoxanthine greater than adenine greater than thymine greater than uracil. The IC50 values for these bases (measured for [3H]adenine influx) were 0.46 mM, 1.15 mM, 1.52 mM and 2.28 mM, respectively. Nucleosides did not inhibit base influx (less than or equal to 15% inhibition at 2 mM, a 400-fold molar excess, at which concentration [3H]nucleoside influx was inhibited by greater than 95%). The Michaelis-Menten constant (Km), calculated for adenine and thymine influx at 0 degrees C, was 1.44 +/- 0.08 mM and 1.61 +/- 0.37 mM, respectively, with corresponding Vmax of 383 +/- 16 and 498 +/- 112 pmol min-1 (10(6) cells)-1. The data demonstrate the existence of a low-affinity, facilitative base transporter with no detectable affinity for nucleosides. The inability of uridine or thymidine to significantly reduce the rate of thymine influx indicates that the previously described thymine/uracil-specific (type 1) thymidine transporter cannot transport thymine, despite its affinity for the base.

Adenine↗

PubMed1992

The MTT cell viability assay for cytotoxicity testing in multidrug-resistant human leukemic cells.

The MTT cell viability assay is widely used in determining drug sensitivity profiles for patients with hematological malignancies and in primary screening of potential chemotherapeutic drugs. Because the multidrug resistance (MDR) phenotype is associated with these malignancies, and since many vital dyes are effluxed from MDR expressing cells, we have investigated whether the MDR phenotype interferes with the MTT assay. In CCRF-CEM and K562 human leukemic cell lines and drug-resistant sub-lines developed from them, comparison of the MTT assay with other cell viability assays showed significant variation in IC50 concentrations, although the resistance relative to the sensitive parent cell was correlated. Inclusion of verapamil, an inhibitor of drug efflux activity, had no effect on the MTT assay.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

Characteristics of thymidine transport in Giardia intestinalis trophozoites.

The transport of thymidine by the protozoan parasite Giardia intestinalis was examined at 0 degrees C. This temperature prevented attachment of the cells to vessel walls, so that a rapid sampling technique could be used. Thymidine influx (distinguished from gross uptake) was readily measurable at 0 degrees C and was specific and saturable. The transporter appears to be a facilitative carrier, exhibiting a high affinity for thymidine (Km = 50 microM). Thymine and uracil were the most effective inhibitors (Ki = 30 microM and 45 microM, respectively), followed by thymidine, deoxyuridine and uridine (Ki = 64-96 microM). Cytosine, cytidine and deoxycytidine were not inhibitory, even at high concentrations. The data indicate that the oxygen at position 4 of the pyrimidine ring is essential for recognition by the transporter, whereas the 5-methyl group of thymine is unimportant. The furanose ring appears not to be recognized, since D-ribose was non-inhibitory and uridine and deoxyuridine were equally inhibitory but less so than uracil and thymine. This carrier probably mediates the transport of uracil, as well as uridine and thymidine, although influx of the base remains to be measured.

Animals↗

PubMed1989

Atypical multidrug resistance in CCRF-CEM cells selected for high level methotrexate resistance: reactivity to monoclonal antibody C219 in the absence of P-glycoprotein expression.

A series of CCRF-CEM sublines selected for extreme resistance to methotrexate has been shown previously to exhibit cross resistance to a number of agents belonging to the multidrug resistance phenotype (J.Natl.Cancer Inst.1989; 81, 1250-1254). The role of the mdr1 gene and its product (P-glycoprotein) in this atypical pattern of multidrug resistance has now been investigated. Southern and Northern analyses failed to demonstrate any amplification, rearrangement or over-expression of the mdr1 gene in the drug-resistant cells. Similarly, monoclonal antibodies MRK16 and JSB1 revealed no increase in the amount of P-glycoprotein present. By contrast, monoclonal antibody C219 detected a 170 kDa protein in all sublines, and in highest concentration in the most resistant cells. The results raise the possibility that a novel, C219-reactive protein may mediate resistance to both methotrexate and members of the multidrug resistance family.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1989

Atypical multidrug resistance in a therapy-induced drug-resistant human leukemia cell line (LALW-2): resistance to Vinca alkaloids independent of P-glycoprotein.

Near diploid leukemic T-cells (LALW-2), exposed to cytotoxic drugs only as a consequence of therapy administered to the donor patient, have been maintained by serial xenograft in nude mice. In comparison with the leukemic line CCRF-CEM, using a growth inhibition assay, LALW-2 cells were resistant to Vinca alkaloids and actinomycin D (relative resistance, 200-fold or more), were slightly resistant to Adriamycin (relative resistance, 4-fold), and showed no resistance to daunorubicin or teniposide. By comparison, a vincristine-resistant CEM subline developed in our laboratory (CEM/VCR R) was resistant to all these agents by at least 30-fold. The VCR R subline served as a positive control, confirming the previously reported correlation between multidrug resistance and amplification of the P-glycoprotein gene. Comparison of CEM, CEM/VCR R, and LALW-2 cells establish that the P-glycoprotein gene was not amplified or overexpressed in the LALW-2 cells; neither could the gene product be detected by immunoblotting in extracts from these cells. The LALW-2 cells were further distinguished from CEM/VCR R cells due to the lack of increased vincristine efflux by the xenografted cells, an effect readily demonstrable in the CEM/VCR R cells. However, although LALW-2 cells efflux vincristine at the same rate as CCRF-CEM cells, the xenografted cells exhibited a reduced rate of vincristine accumulation. Uptake of daunorubicin by LALW-2 cells was not distinguished from that by CEM cells, consistent with similar 50% inhibitory dose levels for this drug in both cell populations, and differentiating both from CEM/VCR R cells. Thus, clinical resistance in this case appears to be an "atypical" form of multidrug resistance specifically distinguished by resistance to Vinca alkaloids and actinomycin D occurring in the absence of increased amounts of P-glycoprotein and manifesting decreased drug uptake.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1989

Comparison of western blot analysis and immunocytochemical detection of P-glycoprotein in multidrug resistant cells.

A sensitive immunocytochemical technique was developed to detect a 170,000 dalton cell membrane glycoprotein (P-gp) in cell lines resistant to vincristine and vinblastine with varying degrees of resistance. P-gp was shown very clearly using the C219 monoclonal antibody and immunocytochemical detection with either antialkaline phosphate or peroxidase-antiperoxidase with silver gold intensification. There was good correlation between the results obtained with immunocytochemical detection of P-gp in single cells and Western blot analysis. The technique is easily performed and can detect P-gp in relatively small numbers of cells that Western blot analysis could miss and is suitable for clinical application.

ATP Binding Cassette Transporter, Subfamily B, Mem↗