Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0023418 (leukemia)
 93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe a cloning-expression vector system for selecting DNA fragments containing open reading frames (ORFs) and expressing them as beta-galactosidase (beta Gal) hybrid fusion proteins. The plasmid vector, pWS50, utilizes the very strong and easily regulated bacteriophage lambda promoter pL, and the efficient translation initiation signals of the N-terminal segment of the lambda cII gene. Fused distally to and out of translational phase with cII is the E. coli lacZ gene, lacking its own transcriptional and translational initiating signals. A unique restriction enzyme site (NruI) is located between the upstream regulatory sequences and the lacZ gene, which provides a cloning site for the insertion of blunt ended DNA fragments. In addition, there are two other unique restriction sites (NheI and BamHI) located in this region which can also be used as closing sites. If a DNA fragment does not contain any translation termination codons (i.e., an ORF), and is inserted correctly into the vector, the translational reading frame between cII and lacZ can be restored. Colonies containing these recombinants can be easily screened as LacZ+ on lactose indicator media. The beta-galactosidase fusion proteins produced from the LacZ+ recombinants are identified on sodium dodecyl sulfate polyacrylamide gels by their large size and high level of production. To test the ORF cloning-expression system, a segment of the human T-cell lymphotrophic virus type I envelope gene was cloned and expressed at high levels. The envelope-beta Gal fusion protein was recognized by antibodies in serum from a patient with adult T-cell leukemia.
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PMID:A plasmid vector for cloning and expression of gene segments: expression of an HTLV-I envelope gene segment. 288 44

To improve the applicability of immunotoxins (ITs), we have developed a new two-step indirect procedure. The target cells to be killed are first incubated with cell-specific mouse monoclonal antibodies (MAbs). After removal of excess unbound antibody, the cells are incubated in the presence of lactose with an indirect IT, a conjugate of whole abrin and sheep anti-mouse immunoglobulin (SAM) that binds only to cells having primary mouse MAbs on their surfaces. The SAM-abrin IT is affinity purified before use to remove molecules with exposed B-chain-binding sites; it was nontoxic in the absence of the specific mouse MAbs, demonstrating the specificity of the two-step method. We compared the indirect approach, using four different primary MAbs, with the conventional method, in which abrin is coupled directly to the mouse MAbs. In three human cell lines--the melanoma line FEMX, the Burkitt cell line Rael, and the leukemia cell line KM3--the cell kill, measured by a clonogenic assay, was consistently greater with the indirect than with the direct method. In the melanoma and Rael cells, the indirect method gave a higher cell kill than even native abrin. With a mixture of two different antibodies an additive effect was observed with the indirect but not with the direct method. The new approach greatly simplifies the therapeutic application in vitro of ITs, because it permits the use of different primary antibodies, singly or in mixtures, in conjunction with only one or a few general indirect ITs. In efforts to further improve the usefulness of the indirect method, other indirect ITs containing different toxin moieties are being examined. The possibility of employing the indirect principle in vivo is being explored.
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PMID:New indirect approach to the therapeutic use of immunotoxins. 325 65

We have synthesized a reagent for antibody directed cell targeting composed of the monoclonal antibody (MoAb) T101 linked to the potent toxin ricin. The immunotoxin (IT) was subsequently radiolabeled by a cyclic anhydride procedure with 90Yttrium (90Y) to construct a radioimmunotoxin (RIT) that may have potential for cancer therapy. We evaluated the reagent for selectivity in binding and protein synthesis inhibition (PSI) assays. The RIT selectively bound antigen positive leukemia T-cell lines, with minimal binding to antigen negative control lines. The IT inhibited 87% or greater protein synthesis activity at 1 microgram/ml and exhibited an IC50 (the dose inhibiting 50% activity) of 0.18 +/- 0.08 microgram/ml in the presence of lactose. RIT and nonlabeled IT showed comparable degrees of PSI at 1 microgram/ml and 10 micrograms/ml, suggesting that labeling had little overall effect on the activity of the immunoconjugate. However, indirect evidence showed that the galactose binding site of ricin was inhibited 10-fold by its exposure to 90Y. Control RIT were minimally inhibitory. IT labeled with 131Iodine (131I) by an iodine monochloride technique also retained its capability to selectively inhibit protein synthesis. When RIT were tested for potency in a clonogenic assay against human leukemia T-cell lines, they inhibited 3.61 logs of tumor cell growth at 10 micrograms/ml. This did not represent an improvement over the log elimination with radiolabeled antibody alone, which showed 4.19 log elimination of tumor cells. Our observation that the 90Y-labeled RIT and labeled antibody can selectively eliminate about four logs of tumor cells in an in vitro clonogenic assay is unique. The ability of RIT to kill several logs of tumor cells in vitro renders RIT interesting anti-tumor reagents.
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PMID:Human leukemia cell binding and killing by anti-CD5 radioimmunotoxins. 349 27

Murine leukemia cells (M1), in their undifferentiated state, have been characterized by the presence of cancer-associated lactoganglio-series glycolipids, one of which was identified as lactogangliotetraosylceramide (LcGg4) having a novel branching at the II-Gal of lactosylceramide through GlcNAc beta 1----3 and GalNAc beta 1----4 linkage, as shown below (Kannagi, R., Levery, S.B., and Hakomori, S. (1984) J. Biol. Chem., 259, 8444-8451): GalNAc beta 1----4 Gal beta 1----4Glc beta 1----1Cer GlcNac beta 1----3 Since this glycolipid is a very minor component, it has been difficult to obtain enough of the purified glycolipid for the preparation of a monoclonal antibody. We developed a method to chemically synthesize this glycolipid using a lactose unit, a ceramide unit, and two hexosamine donors as synthons and made the synthetic glycolipid available as an immunogen. The two monoclonal antibodies we obtained (YI328-18 and YI328-51, both IgG3) specifically recognized the novel branching structure and had no cross-reactivity with gangliotriaosylceramide or lactotriaosylceramide. Thus, the antibodies were found to be useful probes to detect lactogangliotetraosylceramide expressed in undifferentiated M1 leukemia cells, which disappears on induced differentiation. The results of this study indicate a new strategy to establish monoclonal antibody directed to novel minor glycolipid markers or their artificially designed analogs, employing chemically synthesized glycolipid antigens.
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PMID:Monoclonal antibodies directed to chemically synthesized lactogangliotetraosylceramide, a leukemia-associated antigen having a novel branching structure. 380 24

We have studied the potential use of immunotoxins (ITs) for therapeutic treatment of human tumors in an experimental model of human neoplasia. We tested intact ricin IT for its antitumor activity against established tumors. CEM, a human T-cell leukemia line expressing an Mr 67,000 cell surface antigen, and Daudi, a human B-cell lymphoma line which does not express the antigen, were found to be consistently tumorigenic in nude mice. ITs were synthesized using T101, a high-affinity monoclonal antibody reacting with the Mr 67,000 protein determinant and intact ricin. We have shown for the first time that established CEM solid tumors in nude mice will regress following intratumoral injection of T101-ricin IT, while Daudi tumors will not. Selective activity of T101-ricin is dependent on systemic i.v. administration of lactose and local intratumoral injection of the T101-ricin IT with lactose. Intact ricin ITs require the presence of lactose to block native ricin binding and render them antigen specific when linked to monoclonal antibody. Killing of target was cell specific since (a) nonspecific (irrelevant) ITs did not cause the regression of CEM tumors, and (b) injection of large amounts of free T101 antibody prior to T101-ricin IT blocked antitumor activity. Selectivity was not absolute, since regression occurred in one of six animals given irrelevant IT, and blocking was observed in two of four mice. Intratumoral IT treatment with 1 or 2 micrograms of T101-ricin IT plus lactose was not harmful to mice in contrast to intratumoral ricin treatment, which killed all treated tumor-bearing mice at a dose of 0.3 micrograms. Without i.v. injection of lactose, intratumoral injection of T101-ricin IT was also effective in eliminating established tumors. However, this treatment did not result in the selective elimination of tumor, since Daudi tumors also regressed following T101-ricin IT treatment. IT, made with ricin A chain only (T101-A chain IT), was also tested against established CEM tumors. We found that high dosages of T101-A chain IT did not destroy CEM tumors when injected intratumorally, even in the presence of activating agents such as NH4Cl or the carboxylic ionophore X-537 A. In contrast, in vitro experiments demonstrated that T101-A chain IT plus activating agents had potent and selective cytotoxic effect against CEM cells. We conclude that ITs are specifically toxic to established tumors. Although selectivity is not absolute, ITs exhibit potential as a new class of antitumor reagents.
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PMID:Cytotoxic effect of anti-Mr 67,000 protein immunotoxins on human tumors in a nude mouse model. 397 76

A highly efficient method for the generation of insertion mutations is described. The procedure involves the use of a 220-base-pair (bp) EcoRI fragment bearing the SuIII+ suppressor tRNA gene as an insertional mutagen. The plasmid DNA to be mutagenized is linearized by a variety of means, and the suppressor fragment is ligated into the site of cleavage. Successful insertion mutants can be readily detected in Escherichia coli carrying lac- amber mutations on MacConkey lactose plates; virtually 100% of the red colonies contain insertions of the fragment. Subsequent removal of the SuIII+ gene and recyclization leaves a 12-bp insertion if the original cleavage was blunt-ended and a 9-bp insertion if the original cleavage generated 3-bp cohesive termini. This technique, as well as conventional linker mutagenesis with decamer and dodecamer linkers, was used to generate a large library of insertion mutations in cloned DNA copies of the genome of Moloney murine leukemia virus. A number of viable mutants were isolated bearing 9-, 10-, and 12-bp insertions in various domains of the genome. The map positions of the viable mutations suggest that the viral long terminal repeats and portions of the gag and env genes are quite insensitive to alteration. Although most of the mutations were stable for many passages, some of the mutants lost the inserted DNA; we presume that the insertion was somewhat deleterious in these mutants and that continued passage of the virus selected for overgrowth by a revertant.
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PMID:Construction of mutants of Moloney murine leukemia virus by suppressor-linker insertional mutagenesis: positions of viable insertion mutations. 633 Jul 45

Two anti-Mr 65,000 protein (p65) murine monoclonal antibodies, T101 and VIII-1, were conjugated to intact ricin. Toxicity of the resulting immunotoxins (IT) was measured against leukemic cell lines treated alone and in the presence of excess bone marrow using a highly sensitive colony inhibition assay. Cells were pretreated with IT in the presence of lactose to block the native binding of ricin. The IT proved to be potent cytotoxins for the p65-positive cell lines, CEM and MOLT-4. Treatment with T101-ricin (1000 ng/ml) inhibited clonogenic activity of these lines by more than 5.1 logs. Less than 1 log of the inhibition at this dose was due to nonspecific killing by IT. Notably, the presence of excess bone marrow did not reduce IT toxicity against the leukemic populations. Comparison of IT concentrations which inhibited 50% of clonogenic activity showed that T101-ricin was 140- to 540-fold and VIII-1-ricin was 12- to 192-fold more toxic to p65-positive than to p65-negative cell lines. Neither unconjugated anti-p65 nor IT prepared with an irrelevant antibody inhibited clonogenic activity. Blocking of IT toxicity by unconjugated antibody further demonstrated that the antibody moiety of the IT directed the selective toxicity. We found that T101-ricin was more toxic for CEM cells than was VIII-1-ricin, even though blocking studies indicated that the two antibodies bind to proximal or identical epitopes. This report is unique in that an IT was shown to specifically eliminate greater than 99.99% of leukemic cells from human bone marrow. These findings indicate the utility of T101-ricin as an in vitro reagent for autologous bone marrow transplantation in treatment of T-cell leukemia.
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PMID:Elimination of clonogenic T-leukemic cells from human bone marrow using anti-Mr 65,000 protein immunotoxins. 637 99

Small bowel function before, during, and after treatment for acute lymphoblastic leukaemia was studied in 26 children. A significant impairment of D-xylose absorption was found during treatment. Permeability studies showed a significant decrease in mannitol and a significant increase in lactulose concentrations; five of 20 children tested had evidence of lactose malabsorption, three of whom were symptomatic. Intestinal function abnormalities were greater in children whose methotrexate treatments were separated by 7 day than by 16 day intervals. Only five (19%) children had no abnormal tests. Abnormalities of small bowel function may be treatment induced and this has implications for morbidity from gastrointestinal symptoms, impairment of the mucosal barrier, and malabsorption of both nutrients and drugs leading to malnutrition and suboptimal drug concentrations.
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PMID:Small bowel function in acute lymphoblastic leukaemia. 642 27

Conjugates of the monoclonal antibody anti-Thy 1:1 (OX7) and ricin have been constructed, using a thioether bond, such that the ricin no longer can bind Sepharose or asialofetuin. These conjugates were found to be very toxic to Thy 1:1 expressing AKR-A cells whereas they showed little toxicity to Thy 1:2 expressing EL4 cells. By comparison, OX7-ricin conjugates which retained galactose-binding capability were still highly toxic to EL4 cells and this toxicity was antagonized by lactose. A second conjugate made from the W3/25 monoclonal antibody was constructed. The W3/25-ricin conjugate which had lost Sepharose-binding capacity was toxic to W3/25 antigen-expressing rat T-leukaemia cells. This is in sharp contrast to the disulphide linked W3/25-ricin A-chain conjugate which is totally inactive, suggesting that the role of the B-chain in membrane transport of the A-chain into the cytosol is independent of galactose recognition.
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PMID:Antibody-ricin conjugates: a method of linkage which blocks the galactose binding site of ricin. 647 50

Nine children with acute lymphoblastic leukemia in remission were studied at the end of their three years' maintenance therapy. In the jejunal biopsy specimens significantly fewer intraepithelial lymphocytes and IgA-cells were observed in the patients than in the controls. However, the crypts were deeper than normal, and the mitotic activity of the crypt cells was markedly increased in all of the patients. Three children presented with decreased epithelial disaccharidase activities. One had abnormal D-xylose test and two abnormal lactose tolerance test. Although weight gain did not differ from the normal, height increase was retarded in all but one of the patients. We conclude that leukemia treatment leads to changes in the intestinal mucosa that might in part contribute to impairment of growth in the patients and to suppression of mucosal immunity in their jejunum.
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PMID:Jejunal mucosa after leukemia treatment in children. Increase of crypt mitotic activity, decrease of intraepithelial lymphocytes and IgA-cells. 659 37


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