Increased effectiveness of liposome-encapsulated doxorubicin in multidrug-resistant-transgenic mice compared with free doxorubicin.
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Animals↗
Biomedical subjects
I Pastan
Publications and source records attributed to I Pastan.
Translocation mediated by domain II of Pseudomonas exotoxin A: transport of barnase into the cytosol.
Pseudomonas exotoxin A (PE) is a protein toxin composed of three structural domains. Functional analysis of PE has revealed that domain I is the cell-binding domain and that domain III functions in ADP ribosylation. Domain II was originally designated as the translocation domain, mediating the transfer of domain III to the cytosol, because mutations in this domain result in toxin molecules with normal cell-binding and ADP-ribosylation activities but which are not cytotoxic. However, the results do not rule out the possibility that regions of PE outside of domain II also participate in the translocation process. To investigate this problem, we have now constructed a toxin in which domain III of PE is replaced with barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens. This chimeric toxin, termed PE1-412-Bar, is cytotoxic to a murine fibroblast cell line and to a murine hybridoma resistant to the ADP-ribosylation activity of PE. A mutant form of PE1-412-Bar with an inactivating mutation in domain II at position 276 was significantly less toxic. Because the cytotoxic effect of PE1-412-Bar was due to the ribonuclease-activity of barnase molecules which had been translocated to the cytosol, we conclude that domain II of PE is not only essential but also probably sufficient to carry out the translocation process.
ADP Ribose Transferases↗
Alteration of a protease-sensitive region of Pseudomonas exotoxin prolongs its survival in the circulation of mice.
Pseudomonas exotoxin A (PE) is a single-chain 66-kDa polypeptide that kills eukaryotic cells by ADP-ribosylation of translational elongation factor 2. PE is composed of three major structural domains whose functions are binding of cells (I), translocation (II), and ADP-ribosylation (III). Here we describe a protease cleavage target that is located near arginine-490 on the surface of domain III. We made several different types of mutations near arginine-490. Deletion of arginine-490 or replacement of arginine-490 and -492 with serine and lysine or with two lysines resulted in protease-resistant molecules that were fully cytotoxic and had normal ADP-ribosylation activity. However, the half-life in mouse blood of the PE delta 490 mutant was 24 min whereas that of PE was 13 min. Furthermore, two PE mutants that were protease-hypersensitive, PEGlu246,247,249 and PEGlu57,246,247,249 (in which glutamate residues replace basic residues at the indicated positions), had very short half-lives. These data indicate that protease sensitivity is an important determinant in the half-life of PE in the circulation and suggest that the half-life of other proteins may be prolonged by removal of protease sites. Deletion of arginine-492 or the replacement of amino acids 486-491 with three glycines markedly diminished ADP-ribosylation activity and cytotoxicity, indicating that this region of domain III is also important for catalytic activity.
ADP Ribose Transferases↗
Independent domain folding of Pseudomonas exotoxin and single-chain immunotoxins: influence of interdomain connections.
We have studied the refolding of completely unfolded and reduced Pseudomonas exotoxin (PE) and of recombinant single-chain immunotoxins made with monoclonal antibody B3 that are composed of a heavy-chain variable region connected by a flexible linker to the corresponding light-chain variable region (Fv), which is in turn fused to a truncated form of PE. We have found by direct activity assays that different functional domains of these multifunctional proteins fold independently with different kinetics. The ADP-ribosylation domain of PE and of the recombinant immunotoxin fold rapidly, whereas the assembly of the binding and/or translocation domains is regained more slowly. The complete refolding of native PE occurs more rapidly than the refolding of the recombinant immunotoxins. To determine the influence of the connector region between the B3(Fv) moiety and the toxin on the folding process of the recombinant immunotoxin B3(Fv)-PE38KDEL, we have made two different mutations in the peptide that connects the single-chain Fv domain to domain II of PE. These molecules show different folding kinetics, differences in their propensity to aggregate, and different yields of correctly folded molecules. A mutation that decreases aggregation increases the rate of formation and the yield of active immunotoxin molecules.
ADP Ribose Transferases↗
Interleukin-6 fused to a mutant form of Pseudomonas exotoxin kills malignant cells from patients with multiple myeloma.
IL-6-PE4E is a recombinant protein consisting of interleukin-6 (IL-6) fused to a mutant form of Pseudomonas exotoxin in which four basic amino acids are changed to glutamate (PE4E). The chimeric toxin has been previously shown to specifically kill malignant hepatic, prostatic, epidermoid, and myeloma cell lines in vitro. To explore the possible clinical utility of IL-6-PE4E, particularly as an agent for ex vivo purging of marrow for autologous bone marrow transplantation (ABMT), we tested malignant cells from patients with multiple myeloma for sensitivity to this chimeric toxin. Ficoll-purified bone marrow cells were incubated with and without IL-6-toxin for 2 to 3 days. Eight of the 15 myeloma patients had cells that were sensitive to IL-6-toxin as measured by a decrease in the level of protein synthesis. Cells from five patients were very sensitive to IL-6-PE4E, with 50% inhibition of protein synthesis (ID50) achieved at or below 6 ng/mL (7 x 10(-11) mol/L). Cells from three additional patients showed moderate sensitivity, with ID50s between 30 and 140 ng/mL. The remaining seven samples showed little or no sensitivity, with ID50s greater than or equal to 400 ng/mL. Normal bone marrow cells or normal BFU-E and CFU-GM were resistant to the IL-6-toxin even at 1,000 ng/mL. Neither IL-6, IL-2-PE4E, nor an enzymatically deficient mutant of IL-6-PE4E was cytotoxic toward the myeloma cells, indicating that the cytotoxic effect of IL-6-PE4E required the adenosine diphosphate-ribosylation function as well as the specific ligand. Our data suggest that IL-6-toxin could be effective in ex vivo marrow purging in selected multiple myeloma patients who are candidates for ABMT, and that this toxin should also be investigated further for in vivo therapy.
ADP Ribose Transferases↗
Expression and chromosomal localization of the gene for the human transcriptional repressor GCF.
GCF is a human transcriptional regulator that represses transcription of certain genes and is encoded by a 3-kilobase (kb) mRNA (Kageyama, R., and Pastan, I. (1989) Cell 59, 815-825). The expression of GCF was examined in a variety of clonal cell lines. The 3.0-kb GCF mRNA was found to be expressed at the highest level in HUT 102 cells (derived from a T-cell lymphoma). Elevated levels of the GCF mRNA were also noted in KATO III and AGS (gastric carcinomas), FEM-X (melanoma), and U266B1 (myeloma) cell lines. A human fibroblast cell line (WI38) did not express GCF mRNA, and no cross-hybridization to a mouse cell line (NIH 3T3) or monkey cell line (CV-1) could be detected. The GCF cDNA also hybridizes to RNA species of 4.5 and 1.2 kb. The 4.5-kb RNA has the same general expression pattern as the GCF mRNA. Hybridization of cellular RNA with various probes derived from the 3-kb cDNA revealed that the 4.5-kb RNA species only hybridizes to GCF cDNA probes from the extreme 5' end. By using single-stranded RNA probes, hybridization to the three RNA species was detected with the antisense probe for the 5' end (nucleotides 1-561). The single-stranded antisense probe for the region encompassing nucleotides 561-1692 hybridized to the 3.0- and 1.2-kb RNA species. The sense probes for these regions did not hybridize to these RNAs. The GCF gene was localized to a single locus, the chromosome 2 p11.1-11.2 region, by in situ hybridization. Treatment of human KB epidermoid carcinoma cells with phorbol 12-myristate 13-acetate (PMA) lead to a rapid induction of GCF RNA after 1 h and a decline to lower than control levels after 6 h. Epidermal growth factor receptor mRNAs were not increased by PMA until 2 h after treatment and were at their highest level only after GCF mRNAs were decreased. The 4.5- and 1.2-kb RNAs were also induced by PMA with the same kinetics as the GCF mRNA. These results show that the GCF gene is widely expressed in human tissues and cell lines and that the 4.5- and 1.2-kb RNAs have similar expression patterns.
Animals↗
Modulation of activity of the promoter of the human MDR1 gene by Ras and p53.
Drug resistance in human cancer is associated with overexpression of the multidrug resistance (MDR1) gene, which confers cross-resistance to hydrophobic natural product cytotoxic drugs. Expression of the MDR1 gene can occur de novo in human cancers in the absence of drug treatment. The promoter of the human MDR1 gene was shown to be a target for the c-Ha-Ras-1 oncogene and the p53 tumor suppressor gene products, both of which are associated with tumor progression. The stimulatory effect of c-Ha-Ras-1 was not specific for the MDR1 promoter alone, whereas a mutant p53 specifically stimulated the MDR1 promoter and wild-type p53 exerted specific repression. These results imply that the MDR1 gene could be activated during tumor progression associated with mutations in Ras and p53.
3T3 Cells↗
Reduced mRNA levels for the multidrug-resistance genes in cAMP-dependent protein kinase mutant cell lines.
We have previously shown that in Chinese hamster ovary (CHO) cells, a mutant cell line with a defective regulatory subunit (RI) for the cAMP-dependent protein kinase (Abraham et al: Mol. Cell. Biol., 7:3098-3106, 1987), and a transfectant cell line expressing the same mutant kinase, showed increased sensitivity to a number of drugs that are known to be substrates for the multidrug transporter (P-glycoprotein). In the current study we have investigated the mechanism by which cAMP-dependent protein kinase controls drug resistance. We report here that the sensitivity of the kinase defective CHO cell lines to multiple drugs results from decreased RNA levels for the multidrug-resistance gene. Similar results were obtained with mouse Y1 adrenal cells. Wild-type Y1 cells had high levels of P-glycoprotein due to expression of both the mdr1b and mdr2 genes, whereas the cAMP-dependent protein kinase mutant Kin 8 cells had decreased RNA levels for these genes. A Kin 8 transfectant with restored cAMP-dependent protein kinase activity recovered mdr expression, indicating a cause and effect relationship between the protein kinase mutations and mdr expression. No changes in nuclear run-off assays could be detected, suggesting a non-transcriptional mechanism of regulation. Wild-type Y1 cells are more drug sensitive despite having higher levels of P-glycoprotein than the mutant cells. This paradoxical result may be explained by the higher rate of synthesis of steroids by the wild-type Y1 cells, which appear to be inhibitors of P-glycoprotein transport activity.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
A method for increasing the yield of properly folded recombinant fusion proteins: single-chain immunotoxins from renaturation of bacterial inclusion bodies.
Many proteins produced in Escherichia coli accumulate in inclusion bodies. We have systematically evaluated the parameters that affect the refolding and renaturation of enzymatically active molecules from bacterial inclusion bodies containing a recombinant single-chain immunotoxin, B3(Fv)-PE38KDEL. This recombinant molecule is composed of the variable domains of monoclonal antibody B3 (B3(Fv)) fused to a truncated mutant form of Pseudomonas exotoxin A (PE38KDEL). This immunotoxin kills carcinoma cells in vitro, causes tumor regression in animal tumor models, and is being developed as an anti-cancer therapeutic agent (Brinkmann et al., 1991, Proc. Natl. Acad. Sci. USA 88, 8616-8620). Like many other recombinant proteins, B3(Fv)-PE38KDEL is produced in E. coli in inclusion bodies and must be denatured and refolded to become active. This requires correct folding, formation of native disulfide bonds, and the association of different domains. All these steps are strongly dependent on the renaturation conditions used. Optimum conditions of refolding were obtained by the addition of reduced and oxidized thiol reagents to promote disulfide bond formation and the addition of a labilizing agent such as L-arginine. Furthermore, the necessity to reactivate proteins at low protein concentrations due to its tendency to aggregate at high concentrations was overcome by a step-by-step addition of denatured and reduced protein into the refolding solution. This approach should be useful for the production of active forms of other recombinant proteins.
ADP Ribose Transferases↗
In vitro and in vivo suppression of interleukin-2-activated killer cell activity by chimeric proteins between interleukin-2 and Pseudomonas exotoxin.
The biological effects of IL-2 are mediated through high (complex of alpha and beta chain) or intermediate (beta chain) affinity IL-2 receptors. Previously, chimeric proteins composed of IL-2 and Pseudomonas exotoxin (IL-2-PE) were shown to be specifically cytotoxic to cells bearing IL-2 receptors. It has also been shown that IL-2-PE chimeric proteins can abrogate T cell-mediated immune response in vitro. In the current study, we have investigated the effects of IL-2-PE on LAK activity both in vivo and in vitro. We administered either IL-2-PE40 (comprised of IL-2 and 40-kDa portion of PE) or IL-2-PE66 (comprised of IL-2 and 66-kDa molecule of PE) to normal C57BL/6 mice for 3 or 8 days and LAK activity was assessed in various organs of mice. We found that IL-2-PE40 generated LAK activity in various compartments of mice and the level of activity was slightly lower than that observed with an equivalent amount of recombinant (r) IL-2 alone. However, IL-2-PE66 failed to generate LAK activity which would have been induced due to an equivalent concentration of rIL-2. IL-2-PE66 also did not induce LAK activity from the splenocytes during in vitro culture while IL-2-PE40 generated good LAK activity. An equivalent amount of IL-2 also generated potent LAK activity. The suppression of LAK activity by IL-2-PE66 was also evident in cells preactivated with IL-2; however, this inhibition was partial. The suppressive activity of IL-2-PE66 was shown to be mediated through IL-2 receptor interactions as excess amounts of rIL-2 were able to abrogate its effect. Both IL-2 toxins were equivalently cytotoxic to IL-2 receptor-bearing HUT 102 cells and both were able to compete from high and intermediate affinity IL-2 receptors. Taken together, our data indicate that IL-2-PE66 is highly cytotoxic to LAK cells while IL-2-PE40 is less cytotoxic. Thus, data from our study and from other published reports indicate that IL-2-PE66 is more potent immunosuppressive agent than IL-2-PE40.
ADP Ribose Transferases↗
Targeting growth factor receptors with fusion toxins.
Recombinant toxins which bind to growth factor receptors have been prepared and used to kill cells responsible for malignant or autoimmune disease. Our strategy has been to genetically fuse ligands to different forms of Pseudomonas exotoxin which due to mutations or deletions do not bind to normal cells. The resulting recombinant chimeric toxins, in concentrations often less than 1 ng/ml, selectively kill cells expressing the appropriate growth factor receptor. The ligand may be a growth factor, such as transforming growth factor alpha (TGF alpha), interleukin 6 (IL6) or interleukin 2 (IL2), or single chain antigen binding proteins, such as the variable heavy and light regions of the monoclonal antibody anti-Tac. These chimeric toxins kill not only established cell lines but also fresh tumor cells from patients and display anti-tumor activity toward human malignant tumors in nude mice. While clinical trials are beginning with some of these agents, work continues to improve the effectiveness of recombinant chimeric toxins, and to widen the scope of disorders which might be treated by this approach.
ADP Ribose Transferases↗
Multidrug resistance in human cancer.
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
Renaturation of a single-chain immunotoxin facilitated by chaperones and protein disulfide isomerase.
B3(Fv)-PE38KDEL, a recombinant immunotoxin, forms inclusion bodies when produced in Escherichia coli. In renaturation experiments, nonspecific aggregation of non-native polypeptide chains, and the formation of incorrect disulfide linkages lead to inactive molecules. To prevent these side reactions, we added molecular chaperones and protein disulfide isomerase (PDI) to the refolding buffer. Both DnaK and GroEL/S influenced the reactivation process. GroEL alone inhibited reactivation, but in the presence of ATP, GroEL and GroES significantly increased the yield of active protein. DnaK also increased the yield of properly folded protein and the stimulating effect of DnaK was also observed using immobilized DnaK, which can be used repeatedly without significant loss of activity. PDI, which catalyzes disulfide bridging of proteins, also stimulated reactivation of the immunotoxin. Under optimum conditions, reactivation yields in the presence of PDI were about twice that obtained with nonenzymatic disulfide bond formation. Furthermore, DnaK and PDI were additive when renaturation was performed in the presence of both proteins.
Antibodies, Monoclonal↗
Pseudomonas exotoxin: recombinant conjugates as therapeutic agents.
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ADP Ribose Transferases↗
Double minute chromosomes carrying the human multidrug resistance 1 and 2 genes are generated from the dimerization of submicroscopic circular DNAs in colchicine-selected KB carcinoma cells.
This study characterizes amplified structures carrying the human multidrug resistance (MDR) genes in colchicine-selected multidrug resistant KB cell lines and strongly supports a model of gene amplification in which small circular extrachromosomal DNA elements generated from contiguous chromosomal DNA regions multimerize to form cytologically detectable double minute chromosomes (DMs). The human MDR1 gene encodes the 170-kDa P-glycoprotein, which is a plasma membrane pump for many structurally unrelated chemotherapeutic drugs. MDR1 and its homolog, MDR2, undergo amplification when KB cells are subjected to stepwise selection in increasing concentrations of colchicine. The structure of the amplification unit at each step of drug selection was characterized using both high-voltage gel electrophoresis and pulsed-field gel electrophoresis (PFGE) techniques. An 890-kb submicroscopic extrachromosomal circular DNA element carrying the MDR1 and MDR2 genes was detected in cell line KB-ChR-8-5-11, the earliest step in drug selection in which conventional Southern/hybridization analyses detected MDR gene amplification. When KB-ChR-8-5-11 was subjected to stepwise increases in colchicine, this circular DNA element dimerized as detected by PFGE with and without digestion with Not 1, which linearizes the 890-kb amplicon. This dimerization process, which also occurred at the next step of colchicine selection, resulted in the formation of cytologically detectable DMs revealed by analysis of Giemsa-stained metaphase spreads.
Chromosomes, Human↗
Monoclonal antibody K1 reacts with epithelial mesothelioma but not with lung adenocarcinoma.
Immunoperoxidase histochemical staining of cryostat sections from human tumor tissues revealed that a murine monoclonal antibody (MAb), K1, can distinguish epithelial mesotheliomas from lung adenocarcinomas. All of 15 epithelial-type mesotheliomas and all four mixed type mesothelioma samples, but none of 23 lung adenocarcinomas with different degrees of histologic differentiation demonstrated reactivity with antibody K1. Of the cell populations in each mesothelioma tested, 80% to 100% showed strong and homogeneous staining with MAb K1. Immunofluorescence analysis of live cultured cells from an epithelioid mesothelioma (H-meso) and several lung carcinoma cell lines as well as a pleural effusion of a patient with mesothelioma also showed selective reactivity of K1 with the mesothelioma cells. These data indicate that K1 can be useful as a mesothelial cell marker for the differential pathological diagnosis of the epithelial form of mesothelioma; K1 may also be useful in the study of the pathogenesis, immunodiagnosis, and immunotherapy of epithelial-type and mixed-type human malignant mesothelioma.
Adenocarcinoma↗
Recombinant toxins as novel therapeutic agents.
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Animals↗
In vitro effects of a recombinant toxin targeted to the fibroblast growth factor receptor on rat vascular smooth muscle and endothelial cells.
The dominant mechanism responsible for restenosis after angioplasty is believed to be the activation of medial smooth muscle cells (SMCs), leading to their proliferation, migration to the subintima, and further proliferation. To develop novel strategies that might inhibit or prevent restenosis, we previously used a chimeric toxin composed of transforming growth factor-alpha (which targets the epidermal growth factor receptor) and mutated Pseudomonas exotoxin to preferentially recognize and kill rapidly proliferating, versus quiescent, vascular SMCs. We have recently cloned and expressed a recombinant gene encoding Pseudomonas exotoxin with a mutated (nonfunctional) cell recognition domain fused with the ligand acidic fibroblast growth factor, termed aFGF-PE66(4Glu)KDEL; thus, this recombinant toxin targets the fibroblast growth factor receptor. In the present study, we evaluated the relative effects of this chimeric toxin on quiescent versus rapidly proliferating vascular SMCs and also determined whether aFGF-PE66(4Glu)KDEL exerted different effects on SMCs versus endothelial cells. Rapidly proliferating SMCs (grown in 10% fetal bovine serum) were very sensitive to the cytotoxic effects of aFGF-PE66(4Glu)KDEL, whereas cytotoxicity was significantly less when the SMCs were in a quiescent state (grown in medium supplemented with 0.5% fetal bovine serum). The chimeric toxin was also significantly less cytotoxic against endothelial cells. Competition studies using excess acidic fibroblast growth factor indicated that the cytotoxic effects are specifically mediated by the fibroblast growth factor receptor. Thus, the present studies suggest a potentially expanded role of recombinant toxin therapy in restenosis: multiple receptors can be targeted, and cytotoxic effects, at least in vitro, can be preferentially directed to rapidly proliferating vascular SMCs, with relative sparing of vascular endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)
ADP Ribose Transferases↗