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Biomedical subjects

I Pastan

Publications and source records attributed to I Pastan.

At least 415 records · Page 23Linked to original sources
PubMed1990

Genomic organization of the human multidrug resistance (MDR1) gene and origin of P-glycoproteins.

The MDR1 gene, responsible for multidrug resistance in human cells, encodes a broad specificity efflux pump (P-glycoprotein). P-glycoprotein consists of two similar halves, each half including a hydrophobic transmembrane region and a nucleotide-binding domain. On the basis of sequence homology between the N-terminal and C-terminal halves of P-glycoprotein, we have previously suggested that this gene arose by duplication of a primordial gene. We have now determined the complete intron/exon structure of the MDR1 gene by direct sequencing of cosmid clones and enzymatic amplification of genomic DNA segments. The MDR1 gene includes 28 introns, 26 of which interrupt the protein-coding sequence. Although both halves of the protein-coding sequence are composed of approximately the same number of exons, only two intron pairs, both within the nucleotide-binding domains, are located at conserved positions in the two halves of the protein. The other introns occur at different locations in the two halves of the protein and in most cases interrupt the coding sequence at different positions relative to the open reading frame. These results suggest that the P-glycoprotein arose by fusion of genes for two related but independently evolved proteins rather than by internal duplication.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Interdomain hydrolysis of a truncated Pseudomonas exotoxin by the human immunodeficiency virus-1 protease.

The specificity of HIV-1 (human immunodeficiency virus-1) protease has been evaluated relative to its ability to cleave the three-domain Pseudomonas exotoxin (PE66) and related proteins in which the first domain has been deleted or replaced by a segment of CD4. Native PE66 is not hydrolyzed by the HIV-1 protease. However, removal of its first domain produces a molecule which is an excellent substrate for the enzyme. The major site of cleavage in this truncated exotoxin, called LysPE40, occurs in a segment that connects its two major domains, the translocation domain (II), and the ADP-ribosyltransferase (III). This interdomain region contains the sequence ...Asn-Tyr-Pro-Thr... which is similar to that surrounding the scissile Tyr-Pro bond in the gag precursor polyprotein, a natural substrate of the HIV-1 protease. Nevertheless, it is not this sequence that is recognized and cleaved by the enzyme, but one 6 residues away, ...Ala-Leu-Leu-Glu... in which the Leu-Leu peptide bond is hydrolyzed. A second, slower cleavage takes place at the Leu-Ala bond 3 residues in from the NH2 terminus of LysPE40. When domain I of PE66 is replaced by a segment comprising the first two domains of CD4, the resulting chimeric protein is hydrolyzed at the same Leu-Leu bond by HIV-1 protease. Enzyme activities toward synthetic peptides modeled after the sequences defined above in LysPE40 are in complete accord, relative to specificity, kinetics, and pH optimum, with results obtained in the hydrolysis of the parent protein. These findings demonstrate that ideas concerning the specificity of the HIV-1 protease that are based solely upon its processing of natural viral polyproteins can be expanded by evaluation of other multidomain proteins as substrates. Moreover, it would appear that it is not a particular conformation, but sequence and accessibility that play the dominant role in defining sites in a protein substrate that are susceptible to hydrolysis by the enzyme.

ADP Ribose Transferases↗
PubMed1990

Genetic basis of multidrug resistance of tumor cells.

Multidrug resistance in animal cells is defined as the simultaneous resistance to a variety of compounds which appear to be structurally and mechanistically unrelated. One type of multidrug resistance is characterized by the decreased accumulation of hydrophobic natural product drugs, a phenotype which is mediated by an ATP-dependent integral membrane multidrug transporter termed P-glycoprotein or P170. The gene coding for P170 is called MDR. The nucleotide-binding domain of P-glycoprotein shares sequence homology with a family of bacterial permease ATP-binding components. In addition, P170 as a whole is structurally very similar to a number of prokaryotic and eukaryotic proteins believed to be involved in transport activities. This review summarizes our current knowledge of the molecular biology and clinical significance of MDR expression and P-glycoprotein transport activity, as well as some theories about the function of this protein in normal cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Expression of the multidrug resistance gene in myeloid leukemias.

The human multidrug-resistance gene (MDR1) encodes an energy-dependent multidrug efflux protein responsible for the cross-resistance of cultured cells to natural product chemotherapeutic agents such as the anthracyclines and vinca alkaloids. RNA transcript levels were measured in leukemia cells obtained from 15 adult acute nonlymphocytic leukemia (ANLL) cases and 15 cases of chronic myelogenous leukemia (CML). Expression of MDR1 RNA was common in ANLL, and appears to be most frequent in leukemic cells of patients with the poorest response to chemotherapy. Expression of the MDR1 gene was not detectable in the peripheral white blood cells of any of the CML cases during the chronic phase, but was detectable in the immature cells present during this phase of the disease. The cells of the three blastic crisis patients contained detectable levels of MDR1 RNA. These studies support the idea that expression of the MDR1 gene contributes to drug resistance in ANLL, and may play a role in some instances in the drug-resistance of CML in blastic crisis. In contrast, studies of the level of expression of anionic glutathione transferase and DNA polymerase B failed to show any relationship between the RNA transcript levels of these enzymes and responsiveness to chemotherapy.

Adult↗
PubMed1990

Purification of calf thymus RNA polymerase II for in vitro transcription studies.

A procedure for the purification of a large amount of RNA polymerase II (RNAP II) from calf thymus is described. This procedure results in an approximately 1400-fold purification with 40% yield of enzyme in 60 h. The partially purified enzyme is highly suitable for in vitro transcription studies in a cell-free system utilizing HeLa S-100. This method for RNAP II purification would find significant applications in in vitro studies for the analysis of factors modulating eukaryotic transcription.

Animals↗
PubMed1990

Pseudomonas exotoxin contains a specific sequence at the carboxyl terminus that is required for cytotoxicity.

Pseudomonas exotoxin (PE), a single-chain polypeptide toxin of 613 amino acids, consists of three functional domains: an amino-terminal receptor-binding domain, a middle translocation domain, and a carboxyl-terminal ADP-ribosylation domain. Deletion of as few as 2 or as many as 11 amino acids from the carboxyl terminus of PE does not affect ADP-ribosylation activity but produces noncytotoxic molecules. Deletions and substitutions between positions 602 and 611 of PE show that the last 5 amino acids of PE are very important for its cytotoxic action. The carboxyl-terminal sequence of PE is Arg-Glu-Asp-Leu-Lys. Mutational analysis indicates that a basic amino acid at 609, acidic amino acids at 610 and 611, and a leucine at 612 are required for full cytotoxic activity. Lysine at 613 can be deleted or replaced with arginine but not with several other amino acids. Mutant toxins are able to bind normally to target Swiss mouse 3T3 cells and are internalized by endocytosis, but apparently they do not penetrate into the cytosol. A PE molecule that ends with Lys-Asp-Glu-Leu, which is a well defined endoplasmic reticulum retention sequence [Munro, S. and Pelham, R. B. (1987) Cell 48, 899-907], is fully cytotoxic, suggesting that a common factor may be involved in intoxication of cells by PE and retention of proteins in the lumen of the endoplasmic reticulum. Sequences similar to those at the carboxyl end of PE are also found at the end of Cholera toxin A chain and Escherichia coli heat-labile toxin A chain.

ADP Ribose Transferases↗
PubMed1990

Transforming growth factor alpha-Pseudomonas exotoxin fusion protein prolongs survival of nude mice bearing tumor xenografts.

Transforming growth factor alpha (TGF alpha)-Pseudomonas exotoxin 40 (PE40) is a chimeric protein consisting of an N-terminal TGF alpha domain fused to a C-terminal 40-kDa segment of the Pseudomonas exotoxin A protein. TGF alpha-PE40 exhibits the receptor-binding activity of TGF alpha and the cell-killing activity of PE40. These properties make TGF alpha-PE40 an effective cytotoxic agent for cells that possess epidermal growth factor receptors (EGFR). However, the utility of this protein as an anticancer agent has been unclear because many normal tissues express EGFR and may be damaged by exposure to TGF alpha-PE40. To address this issue, we injected nude mice with a lethal inoculum of either A431 or HT29 human tumor cells that possess EGFR or with Chinese hamster ovary (CHO) tumor cells that lack EGFR. Animals were treated with a derivative of TGF alpha-PE40 in which the cysteine residues are replaced by alanine, termed "TGF alpha-PE40 delta cys," or with saline once a day for 5 days. Mice bearing EGFR+ tumor cells lived significantly (P less than 0.001) longer when treated with TGF alpha-PE40 delta cys compared with saline-treated controls (median survival: A431 cells, 51.5 vs. 25.5 days; HT29 cells, 101 vs. 47.5 days). TGF alpha-PE40 delta cys did not prolong the survival of mice bearing tumor cells that lack EGFR (median survival: CHO cells, 15.5 vs. 19.5 days). The only toxicity to normal tissues was mild periportal hepatic necrosis. These studies indicate that a therapeutic window exists in vivo for the use of some growth factor-toxin fusion proteins as anticancer agents.

ADP Ribose Transferases↗
PubMed1990

The recombinant immunotoxin anti-Tac(Fv)-Pseudomonas exotoxin 40 is cytotoxic toward peripheral blood malignant cells from patients with adult T-cell leukemia.

Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin containing the heavy and light variable regions of the anti-Tac monoclonal antibody fused to a mutant form of Pseudomonas exotoxin (PE). Anti-Tac binds to the p55 subunit of the human interleukin 2 (IL-2) receptor, and anti-Tac(Fv)-PE40 kills human or monkey cell lines that contain either the intact IL-2 receptor or its p55 subunit alone. To assess the usefulness of anti-Tac(Fv)-PE40 in treatment of IL-2 receptor-positive leukemia, we tested peripheral blood mononuclear cells from six patients with adult T-cell leukemia. In each of the six patients, anti-Tac(Fv)-PE40 was extremely cytotoxic to the malignant cells. Metabolic activity and sensitivity of the fresh cells improved when a small amount of IL-2 (10 units per ml) was present during incubation. The toxin concentration necessary to inhibit protein synthesis by 50% after 16-hr incubation of cells with immunotoxin varied from 1.6 to 16 ng/ml (2.5-25 x 10(-11) M). In every case, binding was by means of the Tac antigen because anti-Tac(Fv)-PE40 cytotoxicity was prevented by adding excess anti-Tac antibody. Moreover, anti-Tac alone or an inactive mutant of anti-Tac(Fv)-PE40 without ADP-ribosylation activity had very little cytotoxic activity. Peripheral blood mononuclear cells from normal controls, from a patient with Tac-negative leukemia, and from adult T-cell leukemia patients without significant peripheral blood involvement were not sensitive to anti-Tac(Fv)-PE40. These results indicate that anti-Tac(Fv)-PE40 is a potent cytotoxin against adult T-cell leukemia cells in vitro and warrants clinical testing.

ADP Ribose Transferases↗
PubMed1990

Elimination of infectious human immunodeficiency virus from human T-cell cultures by synergistic action of CD4-Pseudomonas exotoxin and reverse transcriptase inhibitors.

We have previously described a recombinant protein, designated CD4(178)-PE40, consisting of the human immunodeficiency virus (HIV) envelope glycoprotein-binding region of human CD4 linked to the translocation and ADP-ribosylation domains of Pseudomonas aeruginosa exotoxin A. By virtue of its affinity for gp120 (the external subunit of the HIV envelope glycoprotein), the hybrid toxin selectively binds to and kills HIV-1-infected human T cells expressing surface envelope glycoprotein and also inhibits HIV-1 spread in mixed cultures of infected and uninfected cells. We now report that CD4(178)-PE40 and reverse transcriptase inhibitors exert highly synergistic effects against HIV-1 spread in cultured human primary T cells. Furthermore, combination treatment can completely eliminate infectious HIV-1 from cultures of human T-cell lines. This conclusion is based on protection of a susceptible cell population from HIV-induced killing, complete inhibition of virus protein accumulation, and elimination of HIV DNA (as judged by quantitative polymerase chain reaction analysis). The results highlight the therapeutic potential of treatment regimens involving combination of a virostatic drug that inhibits virus replication plus an agent that selectively kills HIV-infected cells.

ADP Ribose Transferases↗
PubMed1990

A recombinant single-chain immunotoxin composed of anti-Tac variable regions and a truncated diphtheria toxin.

To kill human or primate cells expressing the p55 subunit of the interleukin 2 receptor, we have constructed a single-chain immunotoxin. DNA sequences encoding the first 388 amino acids of diphtheria toxin (DT) were fused to DNA elements encoding the antigen-binding portion (variable region or Fv) of the anti-Tac monoclonal antibody. The antigen-binding portion consists of 116 amino acids of the heavy-chain variable region connected by a 15-amino acid linker to 106 amino acids of the variable region of the light chain. The single-chain immunotoxin DT388-anti-Tac(Fv) was expressed in Escherichia coli and found in inclusion bodies. The monomeric form was then purified to near homogeneity with a high yield (3-5 mg/liter). Monomeric DT388-anti-Tac(Fv) was highly cytotoxic to cell lines bearing the p55 subunit of the human interleukin 2 receptor but not to cells without this subunit. DT388-anti-Tac(Fv) was also very effective in killing proliferating human T cells produced in a mixed leukocyte reaction.

Animals↗
PubMed1990

A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins.

We have devised a strategy based on polymerase chain reaction (PCR) for the rapid cloning of functional antibody genes as single-chain immunotoxins. RNA from a hybridoma producing an antibody (OVB3) that reacts with ovarian cancer cells was used as a template to make the first strand of a cDNA. Then a second strand was synthesized and amplified by using two sets of DNA primers that (i) hybridized to the ends of the light- and heavy-chain variable regions, (ii) encoded a linker peptide, and (iii) contained appropriate restriction enzyme sites for cloning. After 30 cycles of PCR, the DNA fragments containing sequences encoding the light- and heavy-chain variable regions were cloned into an Escherichia coli expression vector containing a portion of the Pseudomonas exotoxin gene. Clones encoding recombinant single-chain immunotoxins were expressed in E. coli and the protein product was assessed for its ability to bind to or kill cells bearing the OVB3 antigen. By using this approach it should be possible to rapidly clone the functional variable region sequences of many different antibodies from hybridoma RNA.

Amino Acid Sequence↗
PubMed1990

Recombinant CD4-Pseudomonas exotoxin hybrid protein displays HIV-specific cytotoxicity without affecting MHC class II-dependent functions.

The present study describes several in vitro activities of CD4(178)-PE40, a recombinant protein containing a portion of human CD4 linked to active regions of Pseudomonas aeruginosa exotoxin A. Using assays for cell viability, we demonstrate that the hybrid toxin displays highly selective cytotoxicity for HIV-infected T lymphocytes. In a latently infected human T-cell line which is inducible for HIV expression, toxin sensitivity is observed only upon virus induction. At concentrations which readily kill HIV-infected T cells, CD4(178)-PE40 has no observable cytotoxic effects on uninfected human cell lines expressing surface major histocompatibility complex (MHC) Class II molecules, and does not interfere with cellular responses known to be dependent on functional association between CD4 and MHC Class II molecules.

ADP Ribose Transferases↗
PubMed1990

Retroviral transfer of a chimeric multidrug resistance-adenosine deaminase gene.

A fusion between a selectable multidrug resistance (MDR1) cDNA and an adenosine deaminase (ADA) cDNA concomitantly confers multidrug resistance and ADA activity on transfected cells. We have produced a Harvey murine sarcoma virus-derived, replication-defective, recombinant retrovirus to transduce this chimeric MDR-ADA gene efficiently into a great variety of cells. Infection with the MDR-ADA retrovirus conferred the multidrug resistance phenotype on drug-sensitive cells, therefore allowing selection in the presence of colchicine. Colchicine-resistant cells synthesized large amounts of a membrane-associated 210-kDa MDR-ADA fusion protein that preserved both MDR and ADA functional activities. To monitor expression of the chimeric gene in vivo, Kirsten virus-transformed NIH cells were infected with the MDR-ADA retrovirus, and after drug-selection, injected into athymic nude mice. Tumors developed that contained the bifunctionally active MDR-ADA fusion protein. When these mouse tumor cells were placed in tissue culture without the selecting drug, they did not lose the bifunctionally active MDR-ADA fusion protein. The replication-defective, recombinant MDR-ADA retrovirus should be useful to stably introduce the chimeric MDR-ADA gene into a variety of cell types for biological experiments in vitro and in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

In situ localization of the human multidrug-resistance gene mRNA using thymine-thymine dimerized single-stranded cDNA.

In order to detect the mRNA transcribed from the multidrug-resistance gene (MDR1), thymine-thymine (T-T) dimerized single-stranded DNA probes have been utilized for hybridization with mRNA either on nitrocellulose filters or in cells and tissues. S1 nuclease digestion rather than sonication was used to obtain short T-T dimerized single-stranded DNA (300-400 bases) so that they could penetrate well into the cytoplasm. The hybridized T-T DNA was detected immunohistochemically using rabbit anti-T-T DNA antibody (Ab) and peroxidase-labeled goat anti-rabbit IgG Ab. Employing this system, MDR1 mRNA could be localized clearly in the human multidrug-resistant cell lines K562/ADM, CEM/VLB, 2780AD, and KBC4 cells as well as in human fetal kidney and gastric carcinoma. Furthermore, our system successfully detected the expression of MDR1 mRNA in cell lines of increasing resistance. These results paralleled results obtained at the protein level by immunohistochemistry. The analysis of MDR1 RNA expression by this in situ hybridization technique should be useful in the study of normal human tissues and tumor samples expressing the MDR1 gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Low pH-induced changes in Pseudomonas exotoxin and its domains: increased binding of Triton X-114.

Pseudomonas exotoxin (PE), which is composed of three structural domains, is a 66-kilodalton protein secreted by P. aeruginosa that is cytotoxic for mammalian cells. After binding to cell surface receptors and internalization into low-pH endocytic vesicles, PE or an active fragment kills mammalian cells by translocating across an intracellular membrane to the cytoplasm and shutting down protein synthesis. To investigate possible conformational changes associated with the translocation process, full-length PE or recombinant proteins containing the PE cell recognition domain, translocation domain, enzymatic domain, or translocation plus enzymatic domains were incubated with Triton X-114 at pH values ranging from 3.0 to 7.0. The truncated forms used were intact domains that had been expressed in Escherichia coli and subsequently purified. Previous studies (K. Sandvig and J. O. Moskaug, Biochem. J. 245:899-901, 1987) had shown that full-length PE bound more Triton X-114 at a low pH than at a physiologic pH. Therefore, we investigated whether this increased binding was due to a global change in PE or a change within a particular domain. Results showed that all the truncated toxin proteins displayed a similar pH-dependent entry into the detergent phase as native PE, with a transition point of 4.2 for PE and 4.4 to 4.5 for the truncated toxins. The isoelectric points of the recombinant proteins were measured and indicate that, at a low pH (5.0), the cell recognition domain bears a net positive charge, the translocation domain bears a net negative charge, and the enzymatic domain bears no charge. The results suggest that upon acidification in the endosome, PE becomes globally hydrophobic and is converted into a translocation-competent form.

ADP Ribose Transferases↗
PubMed1990

Proteases from human immunodeficiency virus and avian myeloblastosis virus show distinct specificities in hydrolysis of multidomain protein substrates.

The virally encoded proteases from human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) have been compared relative to their ability to hydrolyze a variant of the three-domain Pseudomonas exotoxin, PE66. This exotoxin derivative, missing domain I and referred to as LysPE40, is made up of a 13-kilodalton NH2-terminal translocation domain II connected by a segment of 40 amino acids to enzyme domain III of the toxin, a 23-kilodalton ADP-ribosyltransferase. HIV protease hydrolyzes two peptide bonds in LysPE40, a Leu-Leu bond in the interdomain region and a Leu-Ala bond in a nonstructured region three residues in from the NH2-terminus. Neither of these sites is cleaved by the AMV enzyme; hydrolysis occurs, instead, at an Asp-Val bond in another part of the interdomain segment and at a Leu-Thr bond in the NH2-terminal region of domain II. Synthetic peptides corresponding to these cleavage sites are hydrolyzed by the individual proteases with the same specificity displayed toward the protein substrate. Peptide substrates for one protease are neither substrates nor competitive inhibitors for the other. A potent inhibitor of HIV type 1 protease was more than 3 orders of magnitude less active toward the AMV enzyme. These results suggest that although the crystallographic models of Rous sarcoma virus protease (an enzyme nearly identical to the AMV enzyme) and HIV type 1 protease show a high degree of similarity, there exist structural differences between these retroviral proteases that are clearly reflected by their kinetic properties.

ADP Ribose Transferases↗
PubMed1990

Cell-specific toxicity of a chimeric protein composed of interleukin-6 and Pseudomonas exotoxin (IL6-PE40) on tumor cells.

IL6-PE40 is a chimeric toxin composed of human interleukin-6 (IL6) linked by a peptide bond to PE40, a form of Pseudomonas exotoxin (PE) devoid of its cell recognition domain. To identify cancer cell lines with high numbers of IL6 receptors and to assess the usefulness of IL6-PE40 as a possible anticancer agent, we evaluated the toxicity of IL6-PE40 on a variety of tumor cell lines and demonstrated that certain human myeloma and hepatoma cell lines were particularly sensitive. IL6 binding to selected hepatoma and myeloma cell lines were determined by using [125I]IL6. IL6 receptor mRNA levels were measured by polymerase chain reactions. When comparisons were made among different hepatoma cell lines, the sensitivity to IL6-PE40 correlated with the number of IL6 receptors. However, the hepatoma line PLC/PRF/5, which contains 2,300 IL6 receptors, was more sensitive to IL6-PE40 (amount of protein required to inhibit protein synthesis by 50% was 5 ng/ml) than both the myeloma cell lines U266 and H929 (for both cell lines, the 50% inhibitory dose was 8 ng/ml), which contain 15,500 and 16,500 IL6 receptors, respectively. RNA analysis confirmed that the sensitivity of these cells to IL6-PE40 and the amount of IL6 receptor RNA detected did not correlate. These data suggest that factors in addition to the number of IL6-binding sites contribute to the sensitivity of cells to IL6-PE40.

ADP Ribose Transferases↗