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K D Tew

Publications and source records attributed to K D Tew.

At least 73 records · Page 4Linked to original sources
PubMed1994

cDNA and deduced amino acid sequences of a human colon dihydrodiol dehydrogenase.

A 1.24 kbp cDNA encoding a human colon HT29 cell dihydrodiol dehydrogenase of 323 amino acid residues (M(r) 36,735) has been isolated and sequenced. The deduced amino acid sequence was 97%, 81%, and 76% identical to human liver dihydrodiol dehydrogenase (also called bile acid binder), human liver chlordecone reductase, and bovine lung prostaglandin f synthase, respectively. It was 98% identical in 990 bp overlap with the nucleotide sequence of human liver pseudochlordecone reductase. Northern blots revealed that dihydrodiol dehydrogenase(s) were markedly induced in HT29 colon cells by treatment with the Michael acceptor agent ethacrynic acid.

Alcohol Oxidoreductases↗
PubMed1994

Regulation of human dihydrodiol dehydrogenase by Michael acceptor xenobiotics.

A human oxidoreductase (H-37) that is overexpressed in ethacrynic acid-resistant HT29 colon cells (Ciaccio, P. J., Stuart, J.E., and Tew, K.D. (1993) Mol. Pharmacol. 43, 845-853) has been identified as a dihydrodiol dehydrogenase. Translated protein from a dihydrodiol dehydrogenase cDNA isolated from a library prepared from ethacrynic acid-resistant HT29 cell poly(A+) RNA was recognized by anti-H-37 IgG and was identical in molecular weight with H-37. The isolated cDNA was identical in both nucleotide and amino acid sequences with the recently cloned liver dihydrodiol dehydrogenase (Stolz, A., Hammond, L., Lou, H., Takikawa, H., Ronk, M., and Shively, J.E. (1993) J. Biol. Chem. 268, 10448-10457). Using this cDNA as probe, we have examined its induction by Michael acceptors. The steady state dihydrodiol dehydrogenase mRNA level in the ethacrynic acid-resistant line was increased 30-fold relative to that of wild-type cells. Twenty-four hour treatment of wild-type cells with ethacrynic acid or dimethyl maleate increased mRNA 10-fold and 5-fold, respectively. These changes are accompanied by both increased protein expression and increased NADP-dependent 1-acenaphthenol oxidative activity in cell cytosol. In gel shift assays, compared to wild type controls, increased binding of NAD(P)H quinone oxidoreductase human antioxidant response element (hARE) DNA to redox labile protein complexes present in treated and resistant cell nuclear extract was observed. Ethacrynic acid induced CAT activity 2-fold in Hepa1 cells stably transfected with NAD(P)H quinone oxidoreductase hARE-tk-CAT chimeric gene construct. Thus, dihydrodiol dehydrogenase protein is inducible by de novo synthesis from mRNA by structurally related monofunctional inducer Michael acceptors. Altered in vitro binding of nuclear protein to the hARE is indirect evidence for the involvement of an element similar to hARE in the regulation of dihydrodiol dehydrogenase by these agents.

Alcohol Oxidoreductases↗
PubMed1994

P-glycoprotein binding and modulation of the multidrug-resistant phenotype by estramustine.

BACKGROUND: Previous preclinical studies of combinations of estramustine and vinblastine or paclitaxel (Taxol) have shown that it is possible to achieve a greater than additive cytotoxicity with these antimicrotubule drug combinations. Phase II studies in hormone-refractory prostate cancer have demonstrated clinical antitumor activity of sufficient magnitude to stimulate further laboratory and clinical studies of these drugs combinations. PURPOSE: Our purpose was to characterize the interactions of estramustine with P-glycoprotein and to determine its effects. METHODS: Standard laboratory techniques were used to study the effects of estramustine on intracellular drug concentrations, cytotoxicity, and induction of messenger RNA (mRNA) for the MDR1 (also known as PGY1) gene. Using a photoaffinity analogue of estramustine 17-0-[[2-[3-(4-azido-3-[125I]-iodophenyl) propionamido]ethyl]-carbamyl]estradiol-3-N-bis(2-chloroethyl)ca rba mate ([125I]AIPP-estramustine), binding to the membrane proteins of human ovarian (SKOV3) and their multidrug-resistant counterpart SKVLB1 cells was studied. Southern-blot analysis was performed on DNA extracted from human prostate carcinoma wild-type DU145, estramustine-resistant cell line (E4), and SKVLB1 cells. RESULTS: Membrane fractions from SKOV3 and SKVLB1 cells were analyzed for proteins that could be photoaffinity labeled with [125I]AIPP-estramustine. Competitive inhibition of this binding was achieved with excess concentrations of (in order of efficacy) estramustine, vinblastine, verapamil, progesterone, and to a lesser degree, by paclitaxel but not with estramustine phosphate, estradiol, and estriol. SKVLB1 cells accumulated much less [3H]vinblastine and [3H]paclitaxel than did SKOV3 cells. Estramustine caused a concentration-dependent enhancement of drug accumulation in the SKVLB1 cells to a maximum of approximately 12-fold. No effect of estramustine was apparent for the wild-type SKOV3 cells. In comparison with verapamil, estramustine was less effective as a modulator; however estramustine demonstrated good chemosensitizing activity in combination with actinomycin D and vinblastine. Neither short-term, low-dose no longer-term, higher concentration were found to produce measurable transcript (mRNA for the MDR1 gene levels. Such data suggest that, at least levels. Such data suggest that, at least for two distinct human cell line (SKOV3 and DU145), estramustine does not induce the overexpression of the MDR1 gene. CONCLUSION: It is apparent from the P-glycoprotein data that estramustine interacts with this efflux pump, altering intracellular drug accumulation. Overall, the nonempiric basis for including estramustine in clinical protocols that contain other multidrug-resistant drugs is strengthened by the present data.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Pharmakokinetics and bioavailability study of ethacrynic acid as a modulator of drug resistance in patients with cancer.

Ethacrynic acid (EA) is an inhibitor of the glutathione S-transferases (GSTs), a family of detoxification enzymes the expression of which has been associated with resistance to several classes of anticancer drugs. We performed a two-way randomized crossover study to investigate the pharmacokinetics and bioavailability of EA and describe any toxicities of EA associated with i.v. administration. We administered EA (100 mg) either by the p.o. or i.v. route on days 1 and 2 for pharmacokinetic analysis. After i.v. administration, plasma EA disappearance was biphasic in seven patients and monophasic in two patients with a terminal half-life of 30 and 8 min, respectively. Mean total body clearance was high; 1405 ml/min in patients described by using a one-compartment model and 611 ml/min in those patients described by a two-compartment model. After p.o. administration, peak EA plasma concentrations were less than 10% of i.v. EA and the absolute bioavailability was less than 21% (range, 7-35%). The urinary output as a result of EA treatment was equal following either route of administration and together with the large first-pass effect suggests that a metabolite(s) may be the active diuretic agent(s). Burning at the injection site was the only toxicity unique to the i.v. route of EA administration. We concluded that the systemic availability of EA after p.o. administration is low and variable. This finding supports the potential utility of the i.v. route of administration for the treatment of drug resistance.

Aged↗
PubMed1994

Interaction of an estramustine photoaffinity analogue with cytoskeletal proteins in prostate carcinoma cells.

To identify specific drug targets of the antimitotic drug estramustine, a photoaffinity analogue, 17-O-[[2-[3-(4-azido-3-[125I] iodophenyl)propionamido]ethyl]carbamyl]estradiol-3-N-bis(2- chloroethyl)carbamate, was synthesized and reacted in competition assays with cytoskeletal protein preparations. By attaching the photoaffinity ligand to the 17 beta-position of the steroid D-ring, the cytotoxic properties of the drug were maintained. In cytoskeletal protein preparations from human prostate carcinoma cells (DU 145) or a clonally selected, estramustine-resistant cell line (E4), the major microtubule-associated protein (MAP) present was MAP4. In both cytoskeletal fractions and reconstituted microtubules, 17-O-[[2-[3-(4-azido-3-[125I]iodophenyl)propionamido] ethyl]carbamyl]estradiol-3-N-bis(2-chloroethyl)carbamate bound to both MAP4 and tubulin. From competition assays, the apparent binding constant for MAP4 from DU 145 cells was 15 microM. Similar calculations for tubulin gave values of 13 microM (bovine brain), 19 microM (DU 145 wild-type cells), and 25 microM (E4 cells). The identification of these cytoskeletal proteins as specific drug targets provides a direct explanation for the antimicrotubule and antimitotic effects of estramustine.

Affinity Labels↗
PubMed1993

Analysis of glyoxalase-I from normal and tumor tissue from human colon.

Glyoxalase-I (Gly-I) is part of the glyoxalase system which converts methylglyoxal to D-lactic acid via an S-D-lactoylglutathione intermediate. This glutathione (GSH)-binding protein was purified from human colon tumors and corresponding normal tissue. The GSH-affinity purified fraction from normal human colon tissue showed enzyme activity of 30.6 +/- 11.5 mumol/min per mg protein, with methylglyoxal as substrate. Corresponding fractions from carcinomas showed significantly elevated Gly-I activity of 54.5 +/- 15 mumol/min per mg protein. Polyclonal antibodies made against human Gly-I cross-reacted weakly with mouse liver Gly-I but not with yeast Gly-I. Isoelectric points of Gly-I from human, mouse and yeast were determined to be 4.6, 4.9 and 7.0, respectively, by horizontal IEF. Immunohistochemical analysis confirmed the increase of Gly-I in human colon carcinoma in 16 out of 21 samples when compared to corresponding normal tissue. The elevated levels of Gly-I in colon tumors may be an indicator of the enhanced proliferative status of the neoplastic condition.

Animals↗
PubMed1993

Cloning and characterization of human colon glyoxalase-I.

Glyoxalase-I cDNA clones were isolated from a human colon cDNA library using polyclonal antibodies raised against the protein purified from human colon tissue. Positive clones were purified, subcloned, and their nucleotide sequence determined. The glyoxalase-I cDNA encodes a 184-amino acid protein with a predicted molecular weight of 20,774, corresponding to the monomeric subunit weight of the purified protein from human colon glyoxalase-I. The human enzyme showed 51% homology at the nucleotide level and 42% at the amino acid level with bacterial glyoxalase-I. Transfection of COS-1 cells with the 622-base pair cDNA containing the entire coding region cloned into a pMT2 vector produced an immunoreactive protein and an approximate 180-fold increase in glyoxalase-I enzyme activity as determined with methylglyoxal as a substrate. Transfection of a truncated cDNA lacking 94 base pairs of the 5'-coding sequence also produced an approximately 15-kDa immunoreactive protein, but with no detectable increase in enzyme activity. Northern analysis of the RNA showed an approximately 12-fold increase of the 2.2-kilobase glyoxalase-I transcript in carcinoma when compared to normal colon tissue from the same patient. Examination of colon carcinomas for the amplification of the glyoxalase-I gene by Southern blot analysis revealed no change in gene copy number. These results suggest induction of the glyoxalase-I gene expression in colon carcinomas.

Amino Acid Sequence↗
PubMed1993

Time course of glutathione S-transferase elevation in Walker mammary carcinoma cells following chlorambucil exposure.

Resistance of Walker 256 rat mammary carcinoma cells to chlorambucil has been shown to be accompanied by a specific increase in the A2-2 subunit of glutathione S-transferase (GST) (Buller et al., Mol Pharmacol 31: 575-578, 1987). Analysis of the time course of GST activity following chlorambucil exposure revealed a 7.5- and 3-fold elevation on day 7 post-treatment in Walker-sensitive (WS) and Walker-resistant (WR) cells, respectively. Flow activated cell sorting (FACS) analyses using antibodies specific for rat liver cytosolic GST supported these results and demonstrated the heterogeneous response of WS cells to chlorambucil exposure. The range of GST levels in drug-treated cells was very broad as compared to that of untreated cells. Transcripts for each class of GST (alpha, mu and pi) were quantified for days 1-9 post-treatment from densitometric scans of RNA slot blots. Elevations in GST alpha RNA preceded increases in GST activity (day 7) in both WS and WR cells. Because fluctuations in GSTA1-1 transcripts were not observed, it was concluded that the increased expression of the alpha class must be attributed to increases in GSTA2-2 transcripts. Amplification of the GST genes in drug-treated cells was not present. These results support the role of GSTA2-2 in the detoxification of chlorambucil. The time course of the cellular response to chlorambucil suggests that the elevation of GSTA2-2 transcripts following alkylating agent exposure may represent only one component of a series of events which collectively confer protection and lead to the establishment of drug resistance.

Animals↗
PubMed1993

Overproduction of a 37.5-kDa cytosolic protein structurally related to prostaglandin F synthase in ethacrynic acid-resistant human colon cells.

We report the initial identification of a 37.5-kDa putative aldoketo reductase in human colon carcinoma cells. An aminoterminal trypsin fragment was sequenced and found to be identical to bovine prostaglandin F synthase in 19 of 21 amino acids. Levels of this cytosolic human aldo-keto reductase, assessed by immunoblots using polyclonal antibodies raised against this protein, increased 30-fold in cells resistant to the Michael reaction acceptor ethacrynic acid and increased with time and ethacrynic acid concentration after treatment of wild-type cells. Induction of the reductase appeared to be cell type and drug specific. It was induced by the Michael reaction acceptors dimethyl maleate, t-butylhydroquinone, and hydroquinone but not by the nitrogen mustard chlorambucil. Ethacrynic acid and dimethyl maleate induced the reductase in a second human colon cell line but not in human prostate cells. NADPH-dependent metabolism of aldoketo reductase substrates by cytosol from colon but not prostate cells was enhanced 2-3-fold when cells were grown in the presence of either ethacrynic acid or dimethyl maleate. The discrepancy between induced reductase activity and protein levels may be due to the multiplicity of constitutively expressed NADPH-dependent reductases that compete for substrate. Ethacrynic acid-resistant cells exhibited low levels of cross-resistance to Adriamycin, mitomycin C, and the bovine prostaglandin F synthase substrates phenylglyoxal and prostaglandin D2. Thus, significant overexpression of a human aldo-keto reductase structurally related to bovine prostaglandin F synthase may result from exposure of cells to Michael reaction acceptors and may give rise to an enhanced capacity to metabolize exogenous and endogenous substrates, thereby contributing to the drug-resistant phenotype.

20-Hydroxysteroid Dehydrogenases↗
PubMed1992

Combined antimicrotubule activity of estramustine and taxol in human prostatic carcinoma cell lines.

Estramustine (EM) and taxol, two antimicrotubule agents with distinct and apparently opposing mechanisms of action, were found to be effective in combination in the preclinical treatment of EM-resistant and sensitive, wild-type human prostatic carcinoma cell lines. Estramustine combined with 1 nM taxol (concentration 100-fold less than that measured in plasma of patients treated with taxol) produced greater than additive effects on the inhibition of cell survival of both wild-type and EM-resistant cells. When taxol was used with another microtubule-destabilizing drug, vinblastine, no significantly increased cytotoxicity was observed. Other effects on wild-type and EM-resistant cells produced by the combination of EM and taxol included (a) an increased proportion of the cells in the S phase of the cell cycle; (b) no mitotic block; and (c) an increase in the percentage of micronucleated cells from a control value of less than 1% to greater than 20% after drug treatment. Immunofluorescent microscopic analysis of the effect of this drug combination on the mitotic spindle apparatus revealed specific examples of aberrant mitotic figures, including multiple asters, cells with two distinct spindles, and tripolar spindles able to traverse mitosis and complete cytokinesis. These data provide supportive preclinical evidence for the potential development of an EM/taxol combination clinical regimen either for prostate or other cancers.

Alkaloids↗
PubMed1992

Increased levels of glutathione S-transferase pi transcript as a mechanism of resistance to ethacrynic acid.

Subpopulations of HT 29 human colon carcinoma cells (HT/M and HT/S) were selected for resistance to the glutathione S-transferase (GST) inhibitor ethacrynic acid (EA). Both clones displayed a 2-fold resistance to the selection agent and required its constant presence for the maintenance of the resistant phenotype. Purification and characterization of GST isoforms showed similar profiles in the wild-type (WT) and EA-resistant clones, with microheterogeneous forms of the pi isoenzyme detected in each case. Metabolism of EA in vitro in the presence of GSH and the isolated GST from each cell line was characterized by a biphasic disappearance of the parent drug; the initial rate at which each of these enzymes metabolized EA was similar. These enzymes also displayed similar Km values for 1-chloro-2,4-dinitrobenzene. However, the amount of GST isolated per total cellular protein was 3.0-fold in HT/M and 1.6-fold in HT/S relative to WT in the continuous presence of EA. Under these conditions GST activity was increased by 2.3-fold in HT/M and 3.2-fold in HT/S as were GSH levels (2.7- and 4.1-fold for HT/M and HT/S respectively). When EA was removed, enzyme activity and GSH concentrations decreased to values similar to those of the WT. Slot-blot and Southern analyses of the DNA gave no evidence of GST-pi-gene amplification or rearrangement. However, RNA analyses by both slot-blot and Northern studies indicate a 2.5-3.5-fold elevation in the GST pi transcript in the EA-resistant population. Results from these studies indicate that: (1) maintenance of the EA-resistant phenotype requires constant presence of the agent; (2) the 2-fold resistance to EA can be quantitatively related to a 2-3-fold increase in GST activity and amount which appears to be the result of a 2.5-3.5-fold elevation in GST transcript; (3) EA, a Michael-reaction acceptor, can induce GST at the transcriptional level.

Cell Division↗
PubMed1992

Preclinical and clinical perspectives on the use of estramustine as an antimitotic drug.

A variety of cell biological, pharmacological, crystallographic and clinical approaches have indicated that the antimitotic drug estramustine has interesting and unusual properties. Although designed as an alkylating agent, the marked stability of the carbamate linkage to the steroid carrier molecule prevents the formation of alkylating intermediates. The affinity of the parent molecule for microtubule associated proteins and the concomitant antimicrotubule activity have cytotoxic consequences in tumor cells. Both preclinical and clinical studies of estramustine in combination with other antimicrotubule agents have shown that this approach has great potential to achieve therapeutic advantage, especially in disease states such as hormone refractory prostate cancer.

Animals↗
PubMed1992

Heterogeneity of glutathione S-transferase enzyme and gene expression in ovarian carcinoma.

Expression of the three major cytosolic classes of glutathione S-transferases (GST; Pi, Alpha and Mu) was examined by 2D gel analysis and Western blotting of biopsies from 26 patients diagnosed with ovarian carcinoma. In contrast to other tissues, at least one 'constitutive' subunit from each of the three major cytosolic GST classes was expressed. In most cases, pi appeared to be the major form present, although levels of alpha and mu subunit expression were approximately equal to pi in some patients. There was no detectable effect of prior chemotherapy on enzyme activity. Mean transferase activity for primary carcinoma was 79.9 +/- 11.9 (mean +/- SEM; nmol min-1 mg-1), with three pair-matched normal tissues showing minor decreases in transferase activity. One sample, in which a 32% increase in tumour enzyme activity was noted, was from a patient with primary disease and was associated with marked overexpression of a relatively basic form of alpha which was absent from the matching normal tissue, but present in 20% of all tumours examined. RFLP analysis of genomic tumour DNA using a human mu class cDNA probe indicated that at least two of the three mu forms (the 'constitutive' form and one other) observed in ovarian tissue were allelic variants, as a one-to-one correlation was observed between the presence of two Hind III fragments at 13.1 and 2.2 kb and expression of a second, more basic, variable form. This latter form was positively identified as the mu class subunit mu based on Southern analysis and was seen to be present in 40% of the samples examined. However, in the absence of mu expression, at least one other mu class subunit probably corresponding to GST psi, was seen to be present. Thus, at least in ovarian tissues, absence of the mu subunit does not necessarily imply a lack of ability to metabolize mu substrates, as psi has similar catalytic activity. A third mu subunit, probably corresponding to GST phi based on its relatively acidic pI, was also noted in 72% of samples examined, but has unknown substrate specificity. Increased expression of both alpha and mu forms may be of relevance to disease diagnosis and drug response.

Adult↗
PubMed1992

Molecular conformation of estramustine and two analogues.

The crystal and molecular structures of estramustine and two of its analogues have been determined by X-ray crystallographic techniques (a total of three different compounds). The compounds studied are estramustine [1,3,5(10)-estratriene-3,17 beta-diol-3-N,N-bis(2'- chloroethyl)carbamate] and its monohydrate, estromustine [17-oxo-1,3,5(10)-estratriene-3-yl-N,N-bis(2'-chloroethyl)carbamate], and 17-oxo-5-androsten-3 beta-yl-N,N-bis(2'-chloroethyl)carbamate. Three views of estramustine were obtained from the study of its two crystal forms. The main structural features found are as follows: (a) the geometries of the steroid moieties are closely similar to those of the parent steroids, (b) the bonds around the nitrogen atom of the nitrogen mustard grouping lie approximately in a plane in each structure, (c) the plane through the carbon atoms of the steroid A-ring lies approximately perpendicular to the plane through the carbamate atoms in each structure, (d) the carbonyl C-O of the carbamate points to the alpha side of the steroid moiety in each structure, and (e) one chlorine atom of the nitrogen mustard grouping makes a close contact [3.13 A], in each structure, to the nitrogen atom. Hydrogen bonding to the carbamate appears to occur from the alpha side of the steroid; there is no hydrogen bonding to the nitrogen atom of the carbamate group. These structural data provide some steric explanations for the resistance of the carbamate to enzymatic hydrolysis. The long in vivo half-life of the intact estramustine molecule is a result of this stability. This is responsible for the absence of alkylating ability and the propensity of the drug to bind microtubule-associated proteins and express an antimitotic mechanism of action.

Estramustine↗
PubMed1991

Phase I study of thiotepa in combination with the glutathione transferase inhibitor ethacrynic acid.

The glutathione transferases comprise a family of isoenzymes, one or more of which are involved in the conjugation of alkylating agents to glutathione (GSH). Increased GSH transferase activity has been shown to underlie acquired resistance to several alkylating agents. Ethacrynic acid inhibits the isoenzymes of GSH transferase with 50% inhibitory concentration values ranging from 0.3 to 6.0 microM and has been shown to restore sensitivity to alkylating agents in drug-resistant animal tumor models. We entered 27 previously treated patients with advanced cancer on a study of ethacrynic acid (25 to 75 mg/m2 p.o. every 6 h for 3 doses) and thiotepa (30 to 55 mg/m2 i.v. 1 h after the second dose of ethacrynic acid). The major toxicity of ethacrynic acid was diuresis, which was observed at every dose level; in addition, severe metabolic abnormalities occurred at 75 mg/m2. At 50 mg/m2, the diuretic effects were manageable. Myelosuppression was the most important effect of the combination. Two of seven courses of ethacrynic acid, 50 mg/m2, and thiotepa, 55 mg/m2, were associated with grade 3 or 4 neutropenia and/or thrombocytopenia. Nausea/vomiting greater than or equal to grade 2 was observed in 16% of courses. GSH transferase activity was assayed spectrophotometrically in the peripheral mononuclear cells of all patients. At each dose level, activity decreased following ethacrynic acid administration, with recovery by 6 h. Administration of ethacrynic acid, 50 mg/m2, resulted in a mean nadir of transferase activity of 37% of control. The pharmacokinetics of thiotepa and its principal metabolite TEPA were studied in 23 patients. The plasma disappearance of thiotepa fit a two-compartment open model with a terminal half-life of approximately 2 h. Plasma TEPA levels peaked at a mean of 2.16 h following thiotepa administration. The harmonic mean terminal half-life of TEPA was 10.4 h, and the TEPA area under the curve (AUC) did not increase with increasing thiotepa dose. The AUC of thiotepa was approximately twice, and the clearance about one-half, of the values obtained in a previous study of single agent thiotepa. The AUC of TEPA was lower than that previously observed. The data suggest that ethacrynic acid inhibits enzymes involved in the metabolic disposition of thiotepa, including its oxidative desulfuration to TEPA. The severity of the platelet toxicity was correlated with the AUC of thiotepa, but not with that of TEPA. This combination of thiotepa and ethacrynic acid will be tested further in Phase II trials.

Adult↗
PubMed1991

Interaction of forskolin with the P-glycoprotein multidrug transporter.

Forskolin and 1,9-dideoxyforskolin, an analogue that does not activate adenylyl cyclase, were tested for their ability to enhance the cytotoxic effects of adriamycin in human ovarian carcinoma cells, SKOV3, which are sensitive to adriamycin and express low levels of P-glycoprotein, and a variant cell line, SKVLB, which overexpresses the P-glycoprotein and has the multidrug resistance (MDR) phenotype. Forskolin and 1,9-dideoxyforskolin both increased the cytotoxic effects of adriamycin in SKVLB cells, yet had no effect on SKOV3 cells. Two photoactive derivatives of forskolin have been synthesized, 7-O-[[2-[3-(4-azido-3- [125I]iodophenyl)propionamido]ethyl] carbamyl]-7-deacetylforskolin, 125I-7-AIPP-Fsk, and 6-O-[[2-[3-(4-azido-3- [125I]iodophenyl)propionamido]ethyl]carbamyl]forskolin, 125I-6-AIPP-Fsk, which exhibit specificity for labeling the glucose transporter and adenylyl cyclase, respectively (Morris et al., 1991). Both photolabels identified a 140-kDa protein in membranes from SKVLB cells whose labeling was inhibited by forskolin and 1,9-dideoxyforskolin. There was no specific labeling of proteins in membranes from the SKOV3 cells. The overexpressed 140-kDa protein in SKVLB membranes was identified as the P-glycoprotein by immunoblot analysis and immunoprecipitation using anti-P-glycoprotein antiserum. Total inhibition of photolabeling of the P-glycoprotein was observed with verapamil, nifedipine, diltiazem, and vinbalastine, and partial inhibition was observed with colchicine and cytochalasin B. Forskolin was less effective at inhibiting the photolabeling of the P-glycoprotein than 1,9-dideoxyforskolin or a lipophilic derivative of forskolin. The data are consistent with forskolin binding to the P-glycoprotein analogous to that of other chemosensitizing drugs that have been shown to partially reverse MDR.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP Binding Cassette Transporter, Subfamily B, Mem↗