Biomedical subjects

I Pastan

Publications and source records attributed to I Pastan.

At least 55 records · Page 3Linked to original sources

PubMed2000

Site-specific expression of transferrin receptor by human colon cancer cells directly correlates with eradication by antitransferrin recombinant immunotoxin.

We determined the efficacy of HB21(Fv)PE40, a single-chain immunotoxin made by fusing the variable regions of a monoclonal antibody directed at the human transferrin receptor (TfR) with a truncated mutant of Pseudomonas exotoxin (PE), against metastatic human colon carcinoma KM12L4 cells growing in the liver or subcutis of nude mice. Organ-specific modulation of TfR expression was examined by immunohistochemistry and flow cytometry using anti-human CD71 antibody. KM12L4 cells expressed human TfR and were lysed in vitro by HB21(Fv)PE40 but not LMB-7 (a control immunotoxin specific for a Lewis Y-related carbohydrate antigen). KM12L4 cells growing in the liver expressed higher levels of TfR than cells growing s.c. Systemic administration of HB21(Fv)PE40 eliminated KM12L4 liver metastasis, whereas administration of LMB-7 did not. Treatment of mice with HB21(Fv)PE40 only delayed the growth of s.c. tumors. KM12L4 cells recovered from liver metastases, expressed higher levels of TfR, and were more sensitive to lysis by HB21(Fv)PE40 than KM12L4 cells recovered from s.c. tumors. Indeed, collectively, the data show that the expression level of the TfR by human colon cancer cells is modulated by the organ microenvironment which can be advantageous for the use of therapeutic immunotoxins.

Animals↗

PubMed2000

Pharmacokinetics of 111In- and 125I-labeled antiTac single-chain Fv recombinant immunotoxin.

UNLABELLED: The use of immunotoxins for cancer therapy is an attractive strategy that exploits the targeting specificity of monoclonal antibodies and their fragments as well as the exquisite toxicity of the toxins. However, few studies of immunotoxins have evaluated their biodistribution in vivo. Previous studies have used 125I for tracing immunotoxin biodistribution in mice. Because the immunotoxin works only when it is internalized and because of known problems with quick dehalogenation after internalization of antibodies, we decided to use 111In, which has greater intracellular retention than iodine. METHODS: To trace the in vivo pharmacokinetics of the immunotoxin in mice, we labeled the antiTac(Fv)-PE38 with 111ln and compared it with 125I-labeled antiTac(Fv)-PE38. We successfully labeled antiTac(Fv)-PE38 with 111In at up to 2.96 GBq/mg. A 3- to 4-fold decrease in cytotoxicity was observed for both radiolabeled preparations. We evaluated the internalization of 111In- and 125I-labeled antiTac(Fv)PE38 into ATAC4 cells (Tac-positive) as well as their biodistribution and pharmacokinetics in vivo in mice. In addition, some mice receiving these reagents were co-infused with 30 mg L-lysine to inhibit renal accumulation. RESULTS: Significantly more 111In- than 125I-labeled antiTac(Fv)-PE38 accumulated in the ATAC4 cells (20% versus 5% of initial surface-bound radioactivity; P < 0.001). In vivo, significantly more 111In- than 125I-labeled antiTac(Fv)-PE38 accumulated in the kidney (119 versus 31 percentage injected dose per gram [%ID/g]; P < 0.001). The tumor accumulation of 111In-labeled antiTac(Fv)-PE38 at 96 h was 13-fold greater than that of 125I-labeled antiTac(Fv)-PE38 (1.4 versus 0.1 %ID/g; P < 0.001). No antiTac(Fv)-PE38 was excreted into the urine in its intact form unless lysine was co-infused. Co-injected lysine reduced the renal accumulation of 111In-labeled antiTac(Fv)-PE38 by 62%. CONCLUSION: We evaluated the biodistribution, pharmacokinetics, and catabolism of 111In-labeled antiTac(Fv)-PE38 and found that it differed from 125I-labeled antiTac(Fv)PE38. These studies suggest that 111In-labeled antiTac(Fv)-PE38 can be used to trace the fate of antiTac(Fv)-PE38 in humans.

Animals↗

PubMed2000

Cytotoxic activity of disulfide-stabilized recombinant immunotoxin RFB4(dsFv)-PE38 (BL22) toward fresh malignant cells from patients with B-cell leukemias.

Chemical conjugates of anti-CD22 monoclonal antibodies and toxins have been used to treat CD22+ hematological malignancies. A new anti-CD22 recombinant immunotoxin RFB4(dsFv)-PE38, composed of the Fv portion of the monoclonal antibody RFB4 fused to a truncated form of Pseudomonas exotoxin A, is being developed to target CD22+ tumor cells. To explore the potential clinical utility of this recombinant toxin in treating patients with B-cell malignancies, the fresh cells of patients were incubated ex vivo with RFB4(dsFv)-PE38. Specific cytotoxicity was demonstrated in the malignant cells of 25 of 28 patients with a variety of B-cell malignancies, including acute and chronic lymphocytic leukemias and large cell, mantle cell, and follicular lymphomas. The IC50S, the concentrations necessary for 50% inhibition of protein synthesis, were 3-10 ng/ml in five patients and 10-50 ng/ml in seven patients. Cytotoxicity correlated with cell death upon direct examination of the malignant cells. Significant cytotoxicity was observed with cells containing as few as 350 CD22 sites/cell. A more active derivative of RFB4(dsFv)-PE38, RFB4(dsFv)-PE38KDEL, was produced and was slightly to more than 10-fold more cytotoxic toward patient cells and about twice as toxic to mice. Thus, RFB4(dsFv)-PE38 was specifically cytotoxic toward malignant cells from patients with B-cell leukemias. These data support the testing of RFB4(dsFv)-PE38 in patients with CD22+ leukemias and lymphomas, which is presently under way.

Antibodies, Monoclonal↗

PubMed2000

Imaging and phase I study of 111In- and 90Y-labeled anti-LewisY monoclonal antibody B3.

B3 is a murine monoclonal antibody (mAb) that recognizes a LewisY carbohydrate antigen present on the surface of many carcinomas. An imaging and Phase I trial was performed to study the ability of 111In-mAb B3 to image known metastasis and determine the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), kinetics, and biodistribution of 90Y-mAb B3. Patients (n = 26) with advanced epithelial tumors that express the LewisY antigen were entered. All patients received 5 mCi of 111In-mAb B3 for imaging. 90Y-mAb B3 doses were escalated from 5 to 25 mCi in 5-mCi increments. 111In-mAb B3 and 90Y-mAb B3 were coadministered over a 1-h infusion. Definite tumor imaging was observed in 20 of 26 patients. Sites imaged included lung, liver, bone, and soft tissues. The MTD of 90Y-mAb B3 was determined to be 20 mCi. The DLTs were neutropenia and thrombocytopenia. Tumor doses ranged from 7.7 to 65.1 rad/mCi. 111In- and 90Y-mAb B3 serum pharmacokinetics (n = 23) were found to be similar. The amount of B3 administered (5, 10, and 50 mg) did not alter the pharmacokinetics. Bone marrow biopsies (n = 23) showed 0.0038+/-0.0016% of injected dose/gram for 111In-mAb B3 compared to 0.0046+/-0.0017% of injected dose/gram for 90Y-mAb B3 (P = 0.009). When given to patients with carcinomas that express the LewisY antigen, 111In-mAb B3 demonstrated good tumor localization. The MTD of 90Y-mAb B3 is 20 mCi, with myelosuppression as the DLT. Higher doses of radioactivity need to be delivered to achieve an antitumor effect. Humanized mAb B3 is being developed for evaluation in radioimmunotherapy. A clinical trial to explore the use of higher doses of 90Y-mAb B3 with autologous stem cell support is planned.

Adult↗

PubMed2000

Intratumoral administration of recombinant circularly permuted interleukin-4-Pseudomonas exotoxin in patients with high-grade glioma.

Human glioblastoma but not normal brain cells express numerous receptors for the cytokine interleukin (IL)-4. To target these receptors, we have investigated the safety and activity of directly infusing IL-4(38-37)-PE38KDEL, a chimeric protein composed of circularly permuted IL-4 and a truncated form of Pseudomonas exotoxin (PE), into recurrent malignant high-grade gliomas. IL-4(38-37)-PE38KDEL (IL-4-toxin) was infused over a 4-8-day period into gliomas of nine patients by one to three stereotactically placed catheters. No apparent systemic toxicity occurred in any patient. The infusion of IL-4-toxin in six of nine patients showed glioma necrosis as evidenced by diminished gadolinium enhancement on magnetic resonance imaging. Seven of nine patients underwent craniotomy because of increased intracranial pressure at 16-101 days after the beginning of infusion. In six of these seven patients, partial-to-extensive tumor necrosis with edema was confirmed pathologically. No histological evidence of neurotoxicity to normal brain was identified in any patient. Two patients were not operated on; by magnetic resonance imaging, one showed mottled gadolinium enhancement, and the other showed extensive necrosis of tumor leading to complete remission; this patient remains disease-free > 18 months after the procedure. We conclude that direct glioma injection of IL-4(38-37)-PE38KDEL is safe without systemic toxicity. Local toxicity seemed attributable mainly to tumor necrosis or occasionally to the volume of infusion. Histological evidence of toxicity to normal brain was not observed and in many patients, could be pathologically excluded. Additional patients are being treated to determine the maximal tolerated concentration and volume of IL-4(38-37)-PE38KDEL.

Adult↗

PubMed2000

Immunotoxins with increased activity against epidermal growth factor receptor vIII-expressing cells produced by antibody phage display.

Recombinant immunotoxins are fusion proteins composed of Fv regions of antibodies and bacterial or plant toxins that are being developed for the targeted therapy of cancer. MR1(Fv)-PE38 is a single-chain recombinant immunotoxin that targets a mutant form of the epidermal growth factor receptor (EGFR), EGFRvIII, that is frequently overexpressed in malignant glioblastomas. We have used random complementarity determining region (CDR) mutagenesis to obtain mutants of MR1(Fv) with an increased affinity for EGFRvIII and an increased activity when converted to a recombinant immunotoxin. Initially, nine residues of heavy chain CDR3 were randomly mutagenized, and several mutants with increased binding affinity were isolated. All mutations were located at amino acids 98 and 99, which correspond to a DNA hot spot, a DNA sequence that mutates at high frequency during natural antibody maturation. A specific region of variable region of antibody light chain CDR3 was mutagenized that corresponded to a hot spot and a mutant (MR1-1) with an additional increase in affinity, and cytotoxic activity was isolated. These studies show that targeting hot spots in the CDRs of Fvs is an effective approach to obtaining Fvs with increased affinity. The increased affinity of MR1-1(Fv) makes it an attractive candidate for the targeted therapy of glioblastomas.

ADP Ribose Transferases↗

PubMed2000

Human breast carcinoma cells express type II IL-4 receptors and are sensitive to antitumor activity of a chimeric IL-4-Pseudomonas exotoxin fusion protein in vitro and in vivo.

BACKGROUND: Human breast carcinoma cell lines express high-affinity interleukin-4 receptors (IL-4R). We examined the expression and structure of these receptors on primary and cultured breast carcinoma cell lines and normal breast epithelial cells. We also tested the antitumor activity in vitro and in vivo of a fusion protein comprised of circular permuted IL-4 and truncated Pseudomonas exotoxin, termed IL-4(38-37)-PE38KDEL. MATERIALS AND METHODS: Eight different primary cell cultures and cell lines of human breast carcinomas were examined for the expression of IL-4R by radiolabeled binding, reverse transcription polymerase chain reaction (RT-PCR) and Northern analyses, and subunit structure by crosslinking studies. The antitumor activity of IL-4 toxin was tested in vitro by cytotoxicity assays and in vivo in a xenograft model in immunodeficient animals. RESULTS: 125I-IL-4 specifically bound to primary cell cultures and cell lines with a Kd ranging between 0.2 and 1 nM. Breast tumor cells were found to express IL-4R beta and IL-13R alpha' chains, but not IL-2R gamma c chain. These cells were highly sensitive to the cytotoxic effect of IL-4(38-37)-PE38KDEL. The IC50 (concentration inhibiting protein synthesis by 50%) ranged between approximately 0.005-1.5 nM. A normal breast epithelial cell culture was not sensitive to the cytotoxic activity of IL-4(38-37)-PE38KDEL. MDA-MB231 human breast carcinoma cell line formed a rapidly growing tumor in nude mice. Intratumor and intraperitoneal administration of IL-4(38-37)-PE38KDEL caused a dose dependent regression of established tumors. A control toxin, anti-Tac(Fv)-PE38KDEL, targeted to the IL-2 receptor alpha chain did not cause regression of these tumors. CONCLUSIONS: These results suggest that IL-4(38-37)-PE38KDEL may be a useful agent for targeting of IL-4 receptor positive human breast carcinomas and further studies should be performed to explore fully its potential.

ADP Ribose Transferases↗

PubMed1999

Analysis of cloned Fvs from a phage display library indicates that DNA immunization can mimic antibody response generated by cell immunizations.

BACKGROUND: Generation and cloning of antibodies against cell surface antigens can be simplified by combining DNA immunization which enables generation of antibodies against a protein in its natural configuration without the need for any protein purification step and antibody phage display which due to its immense screening power and physical coupling between the phenotype and genotype of antibodies simplifies the cloning of antibody genes. OBJECTIVES: Since DNA immunization is expected to elicit antibodies against a protein in its natural configuration, we wanted to see if it can mimic the antibody response generated by cell immunization. STUDY DESIGN: A phage display library made from splenic mRNA of a mouse immunized with mesothelin cDNA was panned on mesothelin-positive cells. The single-chain Fvs (scFvs) selected were then analyzed. RESULTS: We obtained several anti-mesothelin scFvs. One of these Fvs is almost identical to the Fv of a monoclonal antibody that was previously obtained from a hybridoma in which the mice were immunized with a mesothelin-positive ovarian cancer cell line. Another Fv was found to be specific for mesothelin present on human cells. CONCLUSION: Our results indicate that an antibody phage display library made from spleens of DNA-immunized mice is a rapid and efficient alternative to cell immunization for obtaining antibodies against different epitopes of a membrane antigen that is very difficult to purify in a native form.

3T3 Cells↗

PubMed1999

Reduction of the nonspecific animal toxicity of anti-Tac(Fv)-PE38 by mutations in the framework regions of the Fv which lower the isoelectric point.

Anti-Tac(Fv)-PE38, also called LMB-2, is a very active recombinant immunotoxin that has produced eight responses, including a durable clinical complete remission in a recently completed phase I trial of leukemias and lymphomas. Dose escalation was limited by liver toxicity. We have noted that the Fv of anti-Tac has an isoelectric point (pI) of 10.2. We hypothesize that the overall positive charge on the Fv portion of anti-Tac(Fv)-PE38 contributes to nonspecific binding to liver cells and results in dose-limiting liver toxicity. We have used a mouse model to investigate the basis of this toxicity and found that lowering the pI of the Fv of anti-Tac from 10.2 to 6. 82 by selective mutation of surface residues causes a 3-fold decrease in animal toxicity and hepatic necrosis. This change in pI did not significantly alter the CD25 binding affinity, the cytotoxic activity toward target cells, or antitumor activity, resulting in a 3-fold improvement in the therapeutic index. If this decreased toxicity occurs in humans, it should greatly increase the clinical utility of this immunotoxin.

ADP Ribose Transferases↗

PubMed1999

Responses in refractory hairy cell leukemia to a recombinant immunotoxin.

We report major responses in 4 of 4 patients with hairy cell leukemia (HCL) who have recently been treated on a phase I trial with the recombinant immunotoxin LMB-2. The immunotoxin, designed to target CD25(+) malignancies, is composed of the Fv portion of the anti-Tac (anti-CD25) antibody, fused to a 38-kD truncated form of Pseudomonas exotoxin A, and has previously been called anti-Tac(Fv)-PE38. All 4 HCL patients were resistant to standard and salvage therapies for HCL, including 2-chlorodeoxyadenosine (CdA) and interferon alpha, and all patients responded to LMB-2 after a single cycle. One patient treated with 2 cycles had a complete remission (CR), with regression of HCL cells from the blood and marrow and resolution of splenomegaly and pancytopenia. As is typical for patients in CR after treatment with CdA, minimal residual disease was detectable by flow cytometry of the bone marrow aspirate. This patient has not relapsed after 11 months. Three other patients had 98% to 99.8% reductions in malignant circulating cells. These results represent a proof of principal that targeted therapy with recombinant Fv-containing proteins can be clinically useful. LMB-2 may be an effective new therapy for patients with chemotherapy-resistant CD25(+) HCL.

ADP Ribose Transferases↗

PubMed1999

Identification of three new alternate human kallikrein 2 transcripts: evidence of long transcript and alternative splicing.

In a search for prostate-specific genes in the human expressed sequence tag (EST) database, we identified a seemingly unique EST cluster C81. Experimental data linked C81 to the human hKLK2 gene that encodes a prostate specific serine protease-human glandular kallikrein (hK2). We uncovered a full-length hKLK2 cDNA corresponding to a 3.0 kb hKLK2 mRNA by PCR and sequence analysis. The 3.0 kb transcript accounts for about 25% of the hKLK2 transcripts as compared to the previously known 1.5 kb transcript. We also identified a third spliced form of the hKLK2 gene produced by alternative splicing between intron III and exon 4. This spliced form was detected in normal prostate, prostate cancer and the prostate adenocarcinoma cell line LNCaP. The identification of long hKLK2 transcript and an alternative spliced form of the hKLK2 gene indicates that regulation of the gene is complex.

Alternative Splicing↗

PubMed1999

Both ATP sites of human P-glycoprotein are essential but not symmetric.

Human P-glycoprotein (P-gp) is a cell surface drug efflux pump that contains two nucleotide binding domains (NBDs). Mutations were made in each of the Walker B consensus motifs of the NBDs at positions D555N and D1200N, thought to be involved in Mg(2+) binding. Although the mutant and wild-type P-gps were expressed equivalently at the cell surface and bound the drug analogue [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) comparably, neither of the mutant proteins was able to transport fluorescent substrates nor had detectable basal nor drug-stimulated ATPase activities. The wild-type and D1200N P-gps were labeled comparably with [alpha-(32)P]-8-azido-ATP at a subsaturating concentration of 2.5 microM, whereas labeling of the D555N mutant was severely impaired. Mild trypsin digestion, to cleave the protein into two halves, demonstrated that the N-half of the wild-type and D1200N proteins was labeled preferentially with [alpha-(32)P]-8-azido-ATP. [alpha-(32)P]-8-Azido-ATP labeling at 4 degrees C was inhibited in a concentration-dependent manner by ATP with half-maximal inhibition at approximately 10-20 microM for the P-gp-D1200N mutant and wild-type P-gp. A chimeric protein containing two N-half NBDs was found to be functional for transport and was also asymmetric with respect to [alpha-(32)P]-8-azido-ATP labeling, suggesting that the context of the ATP site rather than its exact sequence is an important determinant for ATP binding. By use of [alpha-(32)P]-8-azido-ATP and vanadate trapping, it was determined that the C-half of wild-type P-gp was labeled preferentially under hydrolysis conditions; however, the N-half was still capable of being labeled with [alpha-(32)P]-8-azido-ATP. Neither mutant was labeled under vanadate trapping conditions, indicating loss of ATP hydrolysis activity in the mutants. In confirmation of the lack of ATP hydrolysis, no inhibition of [(125)I]IAAP labeling was observed in the mutants in the presence of vanadate. Taken together, these data suggest that the two NBDs are asymmetric and intimately linked and that a conformational change in the protein may occur upon ATP hydrolysis. Furthermore, these data are consistent with a model in which binding of ATP to one site affects ATP hydrolysis at the second site.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Retroviral transfer of human MDR1 gene to hematopoietic cells: effects of drug selection and of transcript splicing on expression of encoded P-glycoprotein.

Protection of hematopoietic cells of patients undergoing anticancer chemotherapy by MDR1 gene transfer is currently being studied in clinical trials. From animal studies, it has been suggested that aberrant splicing due to cryptic donor and acceptor sites in the MDR1 cDNA could be a major reason for failure to obtain high-level expression of P-glycoprotein in bone marrow. We investigated effects of drug selection on protein expression levels and on splicing of MDR1 transcripts in murine bone marrow cells (BMCs) in vitro. To this end, retroviruses were generated through an identical plasmid, pHaMDR1/A, introduced into different packaging cells. GP + E86- but not PA317-derived producer cells were found to express truncated in addition to full-length message. In BMCs transduced with GP + E86-derived viruses, both messages were increased after treatment with colchicine or daunomycin. Similar results were obtained with NIH 3T3 fibroblasts. However, transduced and drug-selected BMCs displayed the spliced transcript even if the respective PA317-derived producer cells contained no truncated RNA as detected in transduced NIH 3T3 fibroblasts. Short-term drug selection in BMCs transduced with either ecotropic or amphotropic retroviruses resulted in a striking increase in P-glycoprotein expression. Thus, aberrant splicing failed to abrogate P-glycoprotein expression in BMCs. We also studied a vector in which MDR1 was coexpressed with glucocerebrosidase, using an internal ribosomal entry site. Although chemoprotection was less efficient than with pHaMDR1/A, augmentation of protein expression was observed at low selecting drug concentrations. Our study shows that drug selection can partially compensate for inefficient transduction of hematopoietic cells, and may help to develop strategies by which unstable expression of transduced genes can be overcome.

3T3 Cells↗

PubMed1999

Pharmacokinetics and antitumor activity of a bivalent disulfide-stabilized Fv immunotoxin with improved antigen binding to erbB2.

We have generated a stable bivalent Fv molecule [(dsFv)2] of the anti-erbB2 monoclonal antibody e23 in which the V(H) and V(L) domains of the Fv are linked to each other by a disulfide bond and the two Fvs are connected by a 15-amino acid linker (T. K. Bera et al., J. Mol. Biol., 281: 475-483, 1998). The e23 (dsFv)2 molecule is linked to a truncated form of Pseudomonas exotoxin (PE38) to generate a bivalent disulfide-stabilized immunotoxin e23 (dsFv)2-PE38. Compared to the monovalent immunotoxin, the (dsFv)2 immunotoxin showed greatly increased cytotoxicity to four cancer cell lines expressing low levels of erbB2 but not to four other cell lines with high erbB2 expression. e23 (dsFv)2-PE38 was administered i.v. to mice, and its half-life was determined. The t(1/2)alpha and t(1/2)beta were 20 and 325 min, respectively, whereas the corresponding values for the monovalent dsFv immunotoxin were shorter, 6 and 52 min. The antitumor activities of the monovalent and bivalent immunotoxin were compared using mice bearing A431 tumors. Despite the fact that e23 (dsFv)2-PE38 was 13-fold more active than e23 dsFv-PE38 on A431 cells in cell culture, its antitumor activity in mice was <2-fold that of the monovalent immunotoxin. These data show that a large increase in avidity does not always lead to an increase in cytotoxic activity. Furthermore, in one of the cases in which cytotoxic activity in vitro was greatly enhanced, there was only a small increase in antitumor activity.

Animals↗

PubMed1999

High expression of a specific T-cell receptor gamma transcript in epithelial cells of the prostate.

We have identified expression of T-cell receptor gamma chain (TCRgamma) mRNA in human prostate and have shown that it originates from epithelial cells of the prostate and not from infiltrating T-lymphocytes. In contrast, the T-cell receptor delta chain (TCRdelta) gene is silent in human prostate. The major TCRgamma transcript in prostate has a different size than the transcript expressed in thymus, spleen, and blood leukocytes. It is expressed in normal prostate epithelium, adenocarcinoma of the prostate, and the prostatic adenocarcinoma cell line LNCaP. The RNA originates from an unrearranged TCRgamma locus, and it is initiated within the intronic sequence directly upstream of the Jgamma1.2 gene segment. The prostate-specific TCRgamma transcript consists of the Jgamma1.2 and Cgamma1 gene segments, and it has an untranslated sequence including a polyadenylation signal and poly(A) sequence at the 3'end. The finding that prostate epithelial cells express a high level of a transcript from a gene that was thought to by exclusively expressed by T-lymphocytes is highly unexpected.

Amino Acid Sequence↗

PubMed1999

The extracellular loop between TM5 and TM6 of P-glycoprotein is required for reactivity with monoclonal antibody UIC2.

P-glycoprotein (P-gp), encoded by the MDR1 gene, is a plasma membrane transporter which confers resistance to many chemotherapeutic drugs. Monoclonal antibodies raised against P-gp have been used as tools to study P-gp topology and activity. Monoclonal antibody UIC2 recognizes a functional conformation of P-gp on the cell surface and blocks P-gp-mediated drug transport. Knowledge about the UIC2 epitope and the mechanism of its inhibitory effects may be helpful for understanding P-gp structure and developing P-gp inhibitors. In the present work, using several chimeras of MDR1 and MDR2, we found that the native sequence of the predicted extracellular loop between transmembrane domains (TM) 5 and 6 of P-gp is necessary, but not sufficient, for UIC2 reactivity. In addition, UIC2 reactivity is also affected by mutations in TM6, a region known to be involved in interactions of P-gp with substrates. These observations suggest that residues in the extracellular loop between TM5 and TM6 are directly involved in the display of the UIC2 epitope. Since TM6 has been shown to be actively involved in drug transport process, the proximity of this region to TM6 may help to explain why UIC2 binding is sensitive to the functional state of P-gp and why binding of UIC2 inhibits P-gp-mediated drug transport.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1999

Engraftment of MDR1 and NeoR gene-transduced hematopoietic cells after breast cancer chemotherapy.

To determine whether the multidrug resistance gene MDR1 could act as a selectable marker in human subjects, we studied engraftment of peripheral blood progenitor cells (PBPCs) transduced with either MDR1 or the bacterial NeoR gene in six breast cancer patients. This study differed from previous MDR1 gene therapy studies in that patients received only PBPCs incubated in retroviral supernatants (no nonmanipulated PBPCs were infused), transduction of PBPCs was supported with autologous bone marrow stroma without additional cytokines, and a control gene (NeoR) was used for comparison with MDR1. Transduced PBPCs were infused after high-dose alkylating agent therapy and before chemotherapy with MDR-substrate drugs. We found that hematopoietic reconstitution can occur using only PBPCs incubated ex vivo, that the MDR1 gene product may play a role in engraftment, and that chemotherapy may selectively expand MDR1 gene-transduced hematopoietic cells relative to NeoR transduced cells in some patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

A single amino acid residue contributes to distinct mechanisms of inhibition of the human multidrug transporter by stereoisomers of the dopamine receptor antagonist flupentixol.

Both cis and trans isomers of the dopamine receptor antagonist flupentixol inhibit drug transport and reverse drug resistance mediated by the human multidrug transporter P-glycoprotein (Pgp) with a stereoselective potency. The rate of ATP hydrolysis by Pgp and photoaffinity labeling of Pgp with the substrate analogue [125I]iodoarylazidoprazosin ([125I]IAAP) are modulated by each isomer in an opposite manner, suggesting different mechanisms for the inhibitory effect on drug transport. In this study we demonstrate that substitution of a single phenylalanine residue at position 983 (F983) with alanine (F983A) in putative transmembrane (TM) region 12 selectively affects inhibition of Pgp-mediated drug transport by both isomers of flupentixol. In F983A the stimulatory effect of cis(Z)-flupentixol and the inhibitory effect of trans(E)-flupentixol on ATP hydrolysis and [125I]IAAP labeling were significantly altered. This indicates that F983 contributes to inhibition of drug transport by both isomers of flupentixol and plays an important role in stimulation and inhibition of ATP hydrolysis and [125I]IAAP labeling by cis(Z)- and trans(E)-flupentixol, respectively. The near-wild-type level of drug transport by the F983A Pgp mutant dissociates susceptibility to inhibition by flupentixol from drug translocation, indicating the allosteric nature of the flupentixol interaction. The inhibitory effects of cyclosporin A on drug transport, drug-stimulated ATP hydrolysis, and [125I]IAAP labeling as well as the stimulatory effect of verapamil on ATP hydrolysis by Pgp were minimally affected by substitution of F983, suggesting no global alteration in the structural and functional integrity of the mutant. Taken together, our data suggest that distinct mechanisms of inhibition of Pgp-mediated drug transport by the cis and trans isomers of flupentixol are mediated through a common site of interaction.

ATP Binding Cassette Transporter, Subfamily B, Mem↗