Search PubMed⌕ Search

Biomedical subjects

I Pastan

Publications and source records attributed to I Pastan.

At least 73 records · Page 4Linked to original sources
PubMed1999

Tissue-specific alternative splicing of the CSE1L/CAS (cellular apoptosis susceptibility) gene.

CSE1L/CAS (CAS) is a nuclear transport factor that plays a role in proliferation and apoptosis. The CAS gene consists of 25 exons. mRNA homologous over its entire length to the yeast homologue CSE1 is the predominant transcript in proliferating tissues. Additional mRNAs are generated by alternative splicing in a tissue-specific manner. An extended 3'-end is found in fetal and adult brain. A mRNA containing the 5'-end of CAS up to position 690 and an alternative 3'-end is expressed in trachea and encodes a truncated Ran-binding domain. Fetal liver expresses a mRNA with deletions of a central portion of CAS and additional sequences encoded by the last intron. SW480 colon cancer cells express another approximately 1500-base mRNA. Western blot analyses of various human tissues and immunohistology of mouse embryos show a correlation of CAS transcripts and CAS protein in different tissues. CAS isoforms may control nuclear transport of tissue-specific proteins.

Alternative Splicing↗
PubMed1999

Apoptosis induced by Pseudomonas exotoxin: a sensitive and rapid marker for gene delivery in vivo.

The rapid progress in gene therapy has expanded our ability to alter genetic structure, necessitating the development of methods for detecting the activity of new vectors. The central concept of a reporter gene is simple: it is a defined nucleotide sequence, which when introduced into a biological system, yields a readily measurable phenotype on expression. This provides a convenient parameter that is correlated to the molecular events associated with genetic expression. In this study we demonstrate that Pseudomonas exotoxin A (PE) can serve as a sensitive reporter gene to detect gene expression in single cells of mouse lung on cationic liposome delivery of PE-encoding DNA in vivo. Furthermore, we show that PE expression can be detected as early as 2 hr after systemic gene delivery in lungs of recipient mice. We compared PE with the widely used beta-galactosidase gene for this purpose. PE induces apoptosis that can be detected by TdT end labeling of DNA fragments (TUNEL assay) Since few expressed PE molecules are necessary to trigger the apoptosis cascade, the minimal amount of PE-encoding plasmid DNA needed for detection of apoptotic cells after systemic delivery was 0.1 microg per animal compared with at least 1 microg for the beta-galactosidase-encoding plasmid DNA. The maximum number of apoptotic cells detected in lungs was about 15-20 times higher than the maximum number of beta-galactosidase-positive cells. Specificity of apoptosis due to PE expression on delivery of the PE-encoding plasmid was shown by prevention of the apoptotic cascade by the caspase inhibitor Z-VAD-fmk.

ADP Ribose Transferases↗
PubMed1999

Novel genes in the PAGE and GAGE family of tumor antigens found by homology walking in the dbEST database.

We have developed a computer-based screening strategy to search the dbEST database to find differentiation antigens that are expressed by cancers arising in nonessential normal tissues such as prostate, breast, and ovary (G. Vasmatzis et al., Proc. Natl. Acad. Sci. USA, 95: 300-304, 1998). Here, we report the identification of three new members of the GAGE/ PAGE family, termed XAGEs. XAGE-1 and XAGE-2 are expressed in Ewing's sarcoma, rhabdomyosarcoma, a breast cancer, and a germ cell tumor. We also describe the relationship of the XAGEs to the GAGE/ PAGE family. XAGE-1 and XAGE-2 should be evaluated as possible targets for vaccine-based therapies of cancer.

Amino Acid Sequence↗
PubMed1999

Complete regression of human B-cell lymphoma xenografts in mice treated with recombinant anti-CD22 immunotoxin RFB4(dsFv)-PE38 at doses tolerated by cynomolgus monkeys.

RFB4(dsFv)-PE38 is a recombinant immunotoxin in which the variable light domain (V(L)) is disulfide bonded via cysteine residues to the variable heavy domain (V(H)), which in turn is fused to PE38, a mutant form of Pseudomonas exotoxin A. RFB4 binds to CD22, which is a differentiation antigen expressed on the majority of B-cell leukemias and lymphomas. To examine the potential efficacy of RFB4(dsFv)-PE38 when administered at a dose schedule appropriate for phase I testing, mice bearing CA46 human CD22+ Burkitt's lymphoma xenografts were treated on alternate days i.v. for 3 doses (QOD x 3). Complete regressions were observed in 80% and 100% of mice treated with 200 and 275 microg/kg QOD x 3, respectively. The higher dose was 27% of the LD50 and 34% of the LD10 in mice. Because RFB4(dsFv)-PE38 is stable at 37 degrees C, it could also be given by continuous infusion using pumps placed in the peritoneal cavity; complete regressions also resulted from this mode of administration. To study toxicology, a pilot toxicology study of RFB4(dsFv)-PE38 was undertaken in cynomolgus monkeys, which like humans but unlike mice have CD22, which binds RFB4. Doses of 100 and 500 microg/kg i.v. QOD x 3 were well tolerated, indicating that a dose that cured tumors in mice was tolerated by primates. Based on these preclinical results, RFB4(dsFv)-PE38 is being developed for the treatment of patients with CD22-positive leukemias and lymphomas.

ADP Ribose Transferases↗
PubMed1999

A two-step selection approach for the identification of ligand-binding determinants in cytokine receptors.

We have developed a novel cell-based method for the isolation and selection of mutant cytokine receptors with defects in ligand binding and applied it to the human interleukin-4 receptor. The experimental procedure is based upon the functional heterologous expression of receptor mutants in eukaryotic cells followed by a two-step selection procedure. Positive selection for cells that express receptor variants is achieved by means of an agonistic antibody that mediates cell survival through receptor dimerization. An IL-4-coupled toxin is subsequently used to select against cells expressing wild-type receptors. Cells expressing mutant receptors that are unable to bind the cytotoxic ligand survive and can be amplified. The procedure allows the isolation of rare receptor variants from cell pools containing predominantly wild-type cells. This method, which should be equally applicable to similar receptor systems, was used to demonstrate the importance of a critical charged amino acid residue in the human IL-4 receptor alpha-subunit for IL-4-induced receptor activation.

Animals↗
PubMed1999

111Indium-labeled monoclonal antibody K1: biodistribution study in nude mice bearing a human carcinoma xenograft expressing mesothelin.

The monoclonal antibody (MAb) K1 is a murine IgG1 that recognizes mesothelin, a differentiation antigen present on mesothelium which is highly expressed on cancers derived from mesothelium, including most ovarian cancers and epithelioid mesotheliomas. MAb K1 was conjugated to 2-(p-isothiocyanatobenzyl)-cyclohexyl- diethylenetriaminepentaacetic acid and labeled with 111In. The biodistribution of 111In-K1 was studied in athymic nude mice bearing 2 s.c. tumors, one expressing a stably transfected plasmid encoding mesothelin and one composed of the parental untransfected A431 epidermoid carcinoma cells which do not express mesothelin. Tumor-bearing mice were given an i.v. injection of 111In-K1 and killed at different time points to determine the uptake of radiolabeled antibody. Significantly higher uptake was seen in antigen-positive tumors at all time points, with peak values at 72 hr (52.9% vs. 8% of the injected dose/g tissue for antigen-positive and antigen-negative tumors, respectively). Uptake in antigen-positive tumors was higher than the blood level at all time points, and the tumors contained a high level of the radiolabeled MAb even at 7 days (28.6% of the injected dose/g tumor).

Animals↗
PubMed1999

Use of an antibody against the soluble interleukin 2 receptor alpha subunit can modulate the stability and biodistribution of interleukin-2.

The authors have previously reported that the soluble serum form of the alpha subunit of the IL-2 receptor (sIL-2Ralpha), whose natural half-life is approximately 40 min, survived much longer in the circulation when bound by a specific antibody. In the present study, the authors evaluated the extent to which sIL-2Ralpha protected IL-2 in freshly collected serum using biochemical analyses, and a functional CTLL-2 assay. In particular, sIL-2Ralpha protected IL-2 from forming complexes with alpha(2)-macroglobulin and from inactivation in vitro. In addition, the authors demonstrated that the anti-IL-2Ralpha monoclonal antibody 7G7/B6, which does not inhibit the binding of IL-2 to its binding site on sIL-2Ralpha, protected IL-2 from degradation and inactivation in vivo in the presence of sIL-2Ralpha. Both(125)I-labelled and unlabelled IL-2 were injected into mice preinjected with humanized anti-Tac (hTac) or 7G7/B6 and sIL-2Ralpha, or sIL-2Ralpha alone. Using size-exclusion HPLC, ELISA, and CTLL-2 cell proliferation assays, we observed that the presence of 7G7/B6 led to formation of complexes with sIL-2Ralpha and increased the serum levels of IL-2 more than 3- to 40-fold those of groups receiving IL-2 alone, sIL-2Ralpha, or hTac. Taken as a whole, these results suggest that the complex of 7G7/B6 and sIL-2Ralpha not only prolongs the survival of IL-2 in vivo, but also maintains the bioactivity of IL-2. The use of antibodies against endogenous soluble receptors could increase the in vivo survival of cytokines, protect their bioactivity and thereby facilitate their clinical use in the treatment of various malignancies and AIDS.

Antibodies, Monoclonal↗
PubMed1999

An scFv phage clone that binds to human beta2-microglobulin.

Using phage display technology, a novel mouse scFv phage clone D1 was generated from mice immunized with single-chain HLA-A2, the human MHC class I molecules. D1 recognized a determinant in beta2-microglobulin. It reacted with beta2-microglobulin associated with the heavy chain of HLA-A2, recombinant beta2-microglobulin made in Escherichia coli or beta2-microglobulin prepared from human urine. The heavy chain variable region of D1 belongs to gene family VII and the light chain variable region belongs to the kappa chain gene family XI according to the Kabat database.

Amino Acid Sequence↗
PubMed1999

Expression profile of saccharide epitope CaMBr1 in normal and neoplastic tissue from dogs, cats, and rats: implication for the development of human-derived cancer vaccines.

CaMBr1 is a blood group-related tumour-associated antigen, whose pattern of expression provides a therapeutic window for passive or active immunotherapy and points to the promise of a vaccine against carcinomas overexpressing this antigen. In this context, an animal model that closely mimics the human situation would be extremely useful. We, therefore, utilised the murine monoclonal antibody MBr1, which defines CaMBr1, as a useful probe to detect the molecule targeted for vaccine development on canine and feline spontaneous breast and uterus tumours and on their normal counterparts, and on rat normal tissues and carcinoma cell lines. Immunoperoxidase staining of cryostat sections revealed homogeneous CaMBr1 expression only in normal feline uterus and a uterus papilloma, whereas MBr1 reactivity was very weak and heterogeneous in normal (1/3 and 1/3) and tumour (1/10 and 1/6) breast tissues from dogs and cats, respectively. In contrast, the data obtained in rat tissues were reproducible in the strains tested and showed that CaMBr1 was expressed in all epithelial tissues of the digestive tract, although with variable intensities. Monoclonal antibody staining appeared to correspond to membrane-bound structures as well as mucinous secretions. Similarly, secretion products of lactating mammary glands expressed CaMBr1. The spectrum of expression on rat digestive tract was broader than that in humans but the specificity of MBr1 reactivity was confirmed by competition assay with a synthetic tetrasaccharide that mimics the CaMBr1 antigen. On FACS analysis, only one of two clonal derivatives of the rat breast carcinoma line RAMA 25 expressed CaMBr1, and a negative cell subset was evident in repeated experiments. By contrast, both colon carcinoma lines, DHD/K12 and CC531, showed staining with MBr1, albeit at different levels of intensity, and no evidence of a negative subset. The cell line CC531 maintained or even increased CaMBr1 expression levels following transplantation in syngeneic immunocompetent animals. Our data suggest the usefulness of the rat as a test model for vaccines against human cancers overexpressing the CaMBr1 antigen.

Animals↗
PubMed1999

A Lewis(y) epitope mimicking peptide induces anti-Lewis(y) immune responses in rabbits and mice.

Lewis(y) carbohydrate antigens are abundant on the surface of many carcinomas. Mab B3 directed against this carbohydrate antigen has been used to make an immunotoxin that is very cytotoxic to cancer cells expressing the Lewis(y) antigen. Mab B3 was also used to screen a phage-displayed peptide library and identified a peptide mimicking the Lewis(y) epitope. In this report, we demonstrate that the Lewis(y) epitope-mimicking peptide induces anti-Lewis(y)immune responses in both rabbits and mice. In addition, Lewis(y) antigens induce anti-peptide immune responses. These results indicate that carbohydrate-mimicking peptides provide a novel strategy to elicit immune responses for tumor-associated carbohydrates.

Animals↗
PubMed1999

Interleukin-4 receptor-directed cytotoxin therapy of AIDS-associated Kaposi's sarcoma tumors in xenograft model.

The elusive and enigmatic origin of AIDS-associated Kaposi's sarcoma (AIDS-KS) makes it a complex tumor and therefore difficult to treat. Here we demonstrate that AIDS-KS cells express surface interleukin-4 (IL-4) receptors, and that IL-4 toxin (IL-4(38-37)-PE38KDEL) is specifically cytotoxic to these cells. Intratumoral, intraperitoneal and intravenous administration of IL-4 toxin in nude mice with established subcutaneous AIDS-KS tumors caused considerable anti-tumor activity in a dose-dependent manner, with highest dose producing durable complete responses. Metabolic changes, including cachexia and lymphopenia, induced by KS tumors were prevented by IL-4 toxin treatment. This report establishes IL-4(38-37)-PE38KDEL as an experimental therapeutic agent for the treatment of AIDS-KS.

ADP Ribose Transferases↗
PubMed1999

Improving antibody affinity by mimicking somatic hypermutation in vitro.

In vivo affinity maturation of antibodies involves mutation of hot spots in the DNA encoding the variable regions. We have used this information to develop a strategy to improve antibody affinity in vitro using phage display technology. In our experiment with the antimesothelin scFv, SS(scFv), we identified DNA sequences in the variable regions that are naturally prone to hypermutations, selected a few hot spots encoding nonconserved amino acids, and introduced random mutations to make libraries with a size requirement between 10(3) and 10(4) independent clones. Panning of the hot spot libraries yielded several mutants with a 15- to 55-fold increase in affinity compared with a single clone with a fourfold increased affinity from a library in which mutagenesis was done outside the hot spots. The strategy should be generally applicable for the rapid isolation of higher-affinity mutants of Fvs, Fabs, and other recombinant antibodies from antibody phage libraries that are small in size.

Antibody Affinity↗
PubMed1999

Prevention of graft-versus-host disease (GVHD) by elimination of recipient-reactive donor T cells with recombinant toxins that target the interleukin 2 (IL-2) receptor.

Graft-versus-host disease (GVHD), due to the presence of recipient-reactive T cells, limits the usefulness of bone marrow transplantation (BMT) and is a major contributor to patient mortality. To prevent GVHD, murine and human T cells were activated by antigen or mitogens and treated with a genetically engineered form of Pseudomonas exotoxin A (PE) directed against the IL-2 receptor. Treatment with the chimeric toxin eliminated alloreactive cytotoxic T lymphocytes (CTL) as determined by cytotoxicity and mixed lymphocyte culture assays. Precursor frequencies of alloreactive cytotoxic T cells and proliferative T cells were reduced up to 100-fold as shown by limiting dilution assays. Flow cytometric analyses revealed that treatment with the chimeric toxin completely eliminated CD25+ cells from the cultures. Toxin treatment had no significant effect on hematopoietic stem and progenitor cells as determined in vitro by colony-forming assays and in vivo by long-term hematopoietic recovery after 950 rad irradiation. Toxin treatment decreased GVHD in transplanted mice to less than 10% (as compared to 88% in untreated controls). Thus, it is possible to prevent life-threatening GVHD after BMT by using a CD25 receptor-directed toxin to eliminate host-reactive T cells from bone marrow grafts.

ADP Ribose Transferases↗
PubMed1999

Domain exchangeability between the multidrug transporter (MDR1) and phosphatidylcholine flippase (MDR2).

Multidrug resistance (MDR) mediated by P-glycoprotein (MDR1) is clinically significant. Understanding how MDR1 substrate specificity is determined will help to overcome MDR to improve cancer treatment. One potential approach to achieve this goal is to study chimeras of MDR1 and its homolog MDR2 (also called MDR3), which has been identified as a phosphatidylcholine flippase. With an approach involving exchanging homologous segments of MDR1 and MDR2 and site-directed mutagenesis, we previously demonstrated MDR1 residues Q330, V331, and L332 in transmembrane domain 6 (TM6) are essential for multidrug transport activity; substituting these residues allows the N-terminal transmembrane region of MDR2 to support MDR1 activity. To further determine the exchangeability between MDR1 and MDR2, we constructed additional MDR1/MDR2 chimeras. We found that the N-terminal half of MDR1 and MDR2 was mostly exchangeable except for a few residues in TM6. However, this degree of exchangeability was not found in the C-terminal half of MDR1 and MDR2. In addition, with substitution of MDR1 residues 318-332 (TM6) and 937-994 (TM11-12), MDR2 had relatively normal affinity for MDR1 substrates, but it did not have multidrug transporter activity. These results suggest that the inability of MDR2 to transport most MDR1 drugs efficiently may be due to failure of those drugs to stimulate ATPase and activate transport as well as to decreased drug binding.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

Studies of human MDR1-MDR2 chimeras demonstrate the functional exchangeability of a major transmembrane segment of the multidrug transporter and phosphatidylcholine flippase.

P-glycoprotein (P-gp), encoded by the MDR1 gene, is a plasma membrane transporter which effluxes a large number of structurally nonrelated hydrophobic compounds. The molecular basis of the broad substrate recognition of P-gp is not well understood. Despite the 78% amino acid sequence identity of the MDR1 and MDR2 transporter, MDR2, which has been identified as a phosphatidylcholine transporter, does not transport most MDR1 substrates. The structural and functional differences between MDR1 and MDR2 provide an opportunity to identify the residues essential for the broad substrate spectrum of MDR1. Using an approach involving exchanging homologous segments of MDR1 and MDR2 and site-directed mutagenesis, we have demonstrated that MDR1 residues Q330, V331, and L332 in transmembrane domain 6 are sufficient to allow an MDR2 backbone in the N-terminal half of P-gp to transport several MDR1 substrates, including bisantrene, colchicine, vinblastine, and rhodamine-123. These studies help define some residues important for multidrug transport and indicate the close functional relationship between the multidrug transporter (MDR1) and phosphatidylcholine flippase (MDR2).

3T3 Cells↗
PubMed1999

Biochemical, cellular, and pharmacological aspects of the multidrug transporter.

Considerable evidence has accumulated indicating that the multidrug transporter or P-glycoprotein plays a role in the development of simultaneous resistance to multiple cytotoxic drugs in cancer cells. In recent years, various approaches such as mutational analyses and biochemical and pharmacological characterization have yielded significant information about the relationship of structure and function of P-glycoprotein. However, there is still considerable controversy about the mechanism of action of this efflux pump and its function in normal cells. This review summarizes current research on the structure-function analysis of P-glycoprotein, its mechanism of action, and facts and speculations about its normal physiological role.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

125I-labeled anti-epidermal growth factor receptor-vIII single-chain Fv exhibits specific and high-level targeting of glioma xenografts.

A single-chain antibody fragment, MR1(scFv), with specific binding to epidermal growth factor receptor-vIII (EGFRvIII), was produced, radiolabeled, and evaluated for biodistribution in human glioma-bearing athymic mice. The mutant receptor EGFRvIII has a deletion in its extracellular domain that results in the formation of a new, tumor-specific antigen found in glioblastomas, breast carcinomas, and other tumors. The scFv molecule, designed as V(H)-(Gly4-Ser)3-V(L), was expressed in Escherichia coli in inclusion body form; recovered scFv fragments were properly refolded in redox-shuffling buffer. Size-exclusion chromatography of purified scFv demonstrated a protein monomer of Mr 26,000. Labeling was performed using N-succinimidyl 5-[125I]iodo-3-pyridinecarboxylate (SIPC) or Iodogen to specific activities of 0.5-2.0 mCi/mg, with yields of 35-50% and 45-70%, respectively. The immunoreactive fraction (IRF) of the labeled MR1(scFv) was 65-80% when SIPC was used and 50-55% when Iodogen was used. The affinity (K(A)) of MRI(scFv) for EGFRvIII was 4.3 x 10(7) +/- 0.1 x 10(7) M(-1) by BIAcore analysis, and it was 1.0 x 10(8) +/- 0.1 x 10(8) M(-1) and by Scatchard analysis versus EGFRvIII-expressing cells. After incubation at 37 degrees C for 24 h, the binding affinity was maintained, and the IRF was maintained at 60-70%. The specificity of MR1(scFv) for EGFRvIII was demonstrated in vitro by incubation of radiolabeled MR1(scFv) with the EGFRvIII-expressing U87MG.deltaEGFR cell line in the presence or absence of competing unlabeled MR1(scFv) or anti-EGFRvIII MAbs L8A4 and H10. In biodistribution studies using athymic mice bearing s.c. U87MG.deltaEGFR tumor xenografts, animals received intratumoral or i.v. infusions of paired-label [125I]SIPC-MR1(scFv) and [131I]SIPC-anti-Tac(scFv) as a control. When given by the intratumoral route, MR1(scFv) retained high tumor uptakes of 85% injected dose per gram of tissue at 1 h and 16% injected dose per gram of tissue at 24 h following administration. Specific: control scFv tumor uptake ratios of more than 20:1 at 24 h demonstrated specific localization of MR1(scFv). The excellent tumor retention of MR1(scFv), combined with its rapid clearance from normal tissues, resulted in high tumor:normal organ ratios.

Amino Acid Sequence↗