Flutamide induced lupus.
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Adenocarcinoma↗
Biomedical subjects
L M Glode
Publications and source records attributed to L M Glode.
In vitro expression of the diphtheria toxin A-chain gene under the control of human chorionic gonadotropin gene promoters as a means of directing toxicity to ovarian cancer cell lines.
OBJECTIVE: Our goal was to determine whether toxicity of the diphtheria toxin A-chain gene regulated by the human chorionic gonadotropin promoter can be directed to malignant ovarian cell lines. STUDY DESIGN: Plasmids containing diphtheria toxin A-chain gene linked to the regulatory elements of the metalloergothioneine and human chorionic gonadotropin promoters were transfected into the cell lines. Expression of diphtheria toxin A-chain gene was determined by the inhibition of a cotransfected luciferase reporter gene. RESULTS: Cytotoxicity of the diphtheria toxin A-chain gene is shown in a dose-responsive manner. Transfection of a plasmid expressing the diphtheria toxin A-chain gene controlled by a constitutive promoter readily inhibits protein synthesis. Specific inhibition of luciferase protein synthesis occurs in ovarian cancer cells transfected with the diphtheria toxin A-chain gene under the control of the human chorionic gonadotropin promoters when compared with normal ovarian epithelial cells or fibroblasts. CONCLUSIONS: These data demonstrate the preferential expression of the diphtheria toxin A-chain gene, regulated by the human chorionic gonadotropin promoter, to ovarian cancer cell lines. This provides an avenue for targeting such cells for suicide, toxin, or cytokine genes.
Carcinoma↗
Current challenges of gene therapy for prostate cancer.
Gene therapy for prostate cancer faces hurdles similar to those being encountered for other cancers and nonmalignant processes. The greatest obstacle is the identification of efficient delivery systems, since numerous animal models and cell culture systems have shown potential efficacy when most cells express the introduced genetic material. Early prostate cancers are easily accessible to gene vector introduction, and the predictable metastatic patterns of this cancer may offer additional advantages for gene therapy. This article reviews gene vectors and gene products, as well as ongoing trials of gene therapy that have recently begun in prostate cancer.
Animals↗
Macrophage colony-stimulating factor complementary DNA: a candidate for gene therapy in metastatic melanoma.
BACKGROUND: At present, there is no highly effective treatment for metastatic melanoma. Innovative approaches aimed at inducing a more effective immune response against tumors have shown promising results in animal models. One approach involves the genetic modification of tumor cells so that they produce cytokines that stimulate an immune response. PURPOSE: The aim of this study was to determine the effectiveness of cytokine gene therapy for metastatic melanoma in a murine melanoma model. METHODS: B16F10 murine melanoma cells, which readily metastasize to the lungs, were transduced with a retroviral vector containing genes encoding neomycin resistance and human macrophage colony-stimulating factor (M-CSF). The presence of M-CSF messenger RNA in transduced cells was examined by coupled reverse transcription and polymerase chain reaction. Concentrations of soluble M-CSF in cell culture supernatants were determined by enzyme-linked immunosorbent assays (ELISAs). A clonal cell line, designated N+/CSF+, that expressed and secreted M-CSF was identified. Another clonal cell line, designated N+/CSF-, did not secrete M-CSF at levels detectable by ELISA. B16F10, N+/CSF-, and N+/CSF+ cells, individually or in combination, were injected intravenously or subcutaneously into C57BL/6 mice; we then evaluated the tumorigenicity and metastatic behavior of the cells, as well as the immune responses and survival of the mice. The immune responses assayed were the cytotoxic T lymphocyte (CTL) and peritoneal exudate cell (PEC) tumoricidal activities. RESULTS: Injection of B16F10 cells into the tail vein of C57BL/6 mice led to the establishment of lung metastases by week 2 and death by week 8. Injection of the N+/CSF+ or N+/CSF- cells led to the establishment of lung metastases that were detected at 2 and 3 weeks, respectively; however, these metastatic lesions were eliminated, and the animals had survival rates similar to those of the noninjected control mice. Injection of mice with a mixture of B16F10 and N+/CSF- cells resulted in the development of metastatic disease and 0% survival at 8 weeks, whereas mice that had been given an injection of a mixture of B16F10 and N+/CSF+ cells had an 80% survival rate at 8 weeks and survived at least two times longer (P = .007). The CTL and PEC tumoricidal activities in animals given an injection of N+/CSF+ cells suggested that monocytes and lymphocytes were responsible for the observed antitumor response. CONCLUSION: These findings suggest that the expression of M-CSF by genetically modified melanoma cells caused an effective antitumor immune response in host C57BL/6 mice and, thus, prolonged survival over that observed in the control mice.
Animals↗
Gene therapy for B-cell lymphoma in a SCID mouse model using an immunoglobulin-regulated diphtheria toxin gene delivered by a novel adenovirus-polylysine conjugate.
Despite advances in conventional therapy, many lives continue to be lost to common forms of B-cell cancers, including leukemias, lymphomas and multiple myeloma. We propose a novel approach to therapy of such cancers using controlled expression of a diphtheria toxin gene (DT-A) to kill malignant cells. We have previously demonstrated selective killing of various cell types, in vitro and in vivo, by cell-specific, transcriptionally controlled expression of this gene. Organ-specific ablation in otherwise healthy transgenic mice has convincingly demonstrated the exquisite specificity achievable by this technique. In the studies now described, DT-A was delivered in vitro and in vivo using a novel gene delivery system employing DNA physically attached to the exterior of adenovirus. After demonstrating the efficacy of gene delivery to Epstein-Barr virus transformed human B-cells in vitro, in vivo work was performed using a SCID mouse model for B-cell lymphoma, in which protection against tumor was observed. The concepts of tissue-regulated toxin gene therapy, and this novel adenovirus gene delivery system are discussed.
Adenoviridae↗
Phase II evaluation of coumarin (1,2-benzopyrone) in metastatic prostatic carcinoma.
The unavailability of effective treatment of metastatic hormone refractory prostatic carcinoma warrants trials of new and promising treatments. Coumarin is an investigational new drug that has produced objective tumor regression in some patients with metastatic renal cell carcinoma and malignant melanoma. Coumarin has shown activity against prostatic carcinoma in the Dunning R-3327 rat prostatic adenocarcinoma model. Forty-eight patients with metastatic hormone naive (5 stage D1 and 10 stage D2) or hormone refractory (33 stage D3) prostatic carcinoma of average age 67.6 years (range 46-86) and ECOG performance status of 2 or better were given 3 grams coumarin daily by mouth and evaluated monthly for toxicity and response by rigid criteria in a multicenter trial. Toxicity was limited to asymptomatic SGOT elevations in 3 patients and nausea and vomiting in 4 patients that required cessation of therapy in 2. Eligibility and protocol violations removed 6 additional patients from response evaluation. There were no complete responses. Partial responses (3 of 40 patients, 8%) occurred in 2 patients with bidimentionally measurable disease and 1 patient with disease evaluable by bone scan and elevated prostate specific antigen and prostatic acid phosphatase. The remaining patients progressed after 1 to 12 (average 4.4) months. Coumarin is a relatively nontoxic drug that may warrant further trials in a subset of patients with prostatic carcinoma.
Adult↗
Inhibition of HIV production in cells containing an integrated, HIV-regulated diphtheria toxin A chain gene.
Construction of a DNA plasmid that expresses a diphtheria toxin A chain (DT-A) gene under control of human immunodeficiency virus (HIV-1) proteins Tat and Rev has been described. Here the generation of HeLa cell clones containing integrated, HIV-regulated DT-A sequences is reported. Five such clones were identified by their decreased expression of a luciferase reporter gene transiently cotransfected with Tat- and Rev-encoding plasmids. The decreased luciferase expression most probably was due to activation of the integrated DT-A gene because higher luciferase activity could be restored by introducing either DT antitoxin or a gene encoding a mutant, DT-resistant elongation factor 2 (the intracellular target for DT-A). Analysis by polymerase chain reaction (PCR) indicated that all clones expressed DT-A encoding RNA. The clones were then transfected with an HIV proviral clone and were tested for HIV production; all five clones demonstrated substantially impaired HIV production compared with parental HeLa cells, as shown by p24 assays of culture supernatants. Our success in generating these cell lines indicates that extremely low basal expression has been achieved in view of the high cellular lethality of DT-A. HIV-regulated expression of DT-A may be applicable as a gene therapy approach for the acquired immune deficiency syndrome (AIDS), to bring about selective suicide of HIV-infected cells before production of viral progeny.
Cloning, Molecular↗
Expression of diphtheria toxin A-chain in mature B-cells: a potential approach to therapy of B-lymphoid malignancy.
Specifically targeted expression of a toxin gene potentially represents a novel approach to cancer therapy. With a view to the ablation of B-cell malignancies, we have constructed a plasmid, designated pTHA71, which expresses the A-chain of diphtheria toxin (DT-A) with high efficiency and specificity in transfected, mature B-lymphoid cells. The construction incorporated immunoglobulin (Ig) kappa light chain gene regulatory sequences, including a kappa promoter, small intron, partial constant region exon, and 3'-flanking sequence (but lacking a known enhancer). These sequences conferred substantially more efficient expression of DT-A in mature B-cells than was seen from constructs that included only Ig promoters and enhancers. When transfected into the 70Z/3 murine pre-B-cell line, pTHA71 was only expressed efficiently if the cells were induced to express their endogenous, rearranged Ig kappa gene by prior exposure to lipopolysaccharide. The insertion of the enhancer from the Ig kappa large intron into pTHA71, generating pTHA81, did not markedly influence the level of DT-A expression in 70Z/3 cells. The observed absence of expression in pre-B-cells suggests that DT-A constructs similar to pTHA71 might be used for the therapeutic ablation of malignant B-cells of mature stages, while sparing normal progenitor cells.
Animals↗
Expression of the diphtheria toxin A-chain coding sequence under the control of promoters and enhancers from immunoglobulin genes as a means of directing toxicity to B-lymphoid cells.
Previous results have shown that cells can be killed by the expression of an introduced gene encoding diphtheria toxin A-fragment (DT-A) and that killing can be targeted using tissue-specific transcriptional regulatory elements. Here, we describe expression plasmids containing the DT-A gene linked with promoters and enhancers from immunoglobulin heavy chain or kappa-light chain genes. When these plasmids were transfected into cultured cells, DT-A was expressed in B-lymphoid cells but not detectably in HeLa cells or fibroblasts. A high specificity for B-cells was confirmed by assaying for luciferase reporter gene expression from a plasmid containing an analogous combination of immunoglobulin heavy chain regulatory elements. A plasmid containing an immunoglobulin-kappa promoter and enhancer was substantially less active in expressing DT-A in a pre-B-cell line than in B-lymphoma cells, suggesting the possibility of targeting DT-A expression to mature, malignant B-cells while sparing normal B-cell progenitors. By means of viral delivery vehicles, the constructs described might be applied in gene therapy for B-cell leukemias or lymphomas.
Animals↗
Activation of a diphtheria toxin A gene by expression of human immunodeficiency virus-1 Tat and Rev proteins in transfected cells.
Expression of a gene encoding the diphtheria toxin A (DT-A) fragment, controlled by tissue specific regulatory elements, has previously been used to kill selected cell populations. Here, we have examined the feasibility of controlling DT-A expression using regulatory systems from the human immunodeficiency virus (HIV-1) genome. Plasmids were constructed which express either DT-A or, as a model system, the luciferase (luc) reporter gene, under control of HIV-1 long terminal repeat (LTR) sequences (-167 to +80). While trans-activation by expression of the viral protein Tat was demonstrated, significant basal expression was observed. To reduce basal expression, cis-acting negative regulatory elements from the env region of the HIV-1 genome were inserted in the 3' untranslated region of both the luc and DT-A constructs. This dramatically reduced basal expression from the HIV LTR, and now both viral regulatory proteins Tat and Rev were required for maximal trans-activation. Such regulation of DT-A expression might be therapeutically applied to selectively kill HIV-infected cells in acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC).
Diphtheria Toxin↗
Construction and expression of plasmids containing mutated diphtheria toxin A-chain-coding sequences.
We previously demonstrated that cells can be killed through transfection of an expression plasmid that encodes the diphtheria toxin A-chain fragment (DT-A). This report describes the construction of expression plasmids containing three mutant DT-A-coding sequences substituting glutamic acid 148 with aspartic acid, serine, or glutamine which are known to have 100- to 300-fold-reduced ADP-ribosylation activity measured in vitro. The toxicity of these constructs was determined in cotransfection experiments using HeLa and 293 cells with a luciferase expression plasmid as the reporter. Dose responses were compared for the three new DT-A mutant plasmids and for the corresponding plasmids containing wild-type DT-A and the previously characterized tox 176 mutant. The dose required to produce 50% inhibition of control luciferase expression in 293 embryonic kidney cells for the five plasmids ranged from 0.01 micrograms for wild-type DT-A to 1.2 micrograms for the least toxic plasmid, which replaces glutamic acid 148 with glutamine. In conclusion, a wide range of DT-A toxicity can be achieved by using plasmid expression vectors that encode different DT-A mutations.
Diphtheria Toxin↗
Targeted ablation of pituitary gonadotropes in transgenic mice.
LH, FSH, and TSH are heterodimeric glycoprotein hormones composed of a common alpha-subunit and unique beta-subunits. The alpha-subunit is produced in two distinct specialized cell types of the pituitary gland: gonadotropes, which synthesize LH and FSH, and thyrotropes, which synthesize TSH. We have demonstrated that 313 base pairs of the bovine-alpha subunit promoter direct expression of diphtheria toxin A chain specifically to the gonadotropes in transgenic mice. Animals carrying this transgene generally exhibit reproductive failure and lack of gonadal differentiation, consistent with gonadotrope ablation. Lack of gonadotrope activity was verified by RIA and immunohistochemical staining for LH. The phenotype of these transgenic mice is nearly identical to mice homozygous for the spontaneous mutation, hpg, which is due to a deletion in the gene encoding GnRH. Thyrotrope function was judged normal based on overall growth of the animals, appearance of their thyroids, T4 levels measured by RIA, and immunohistochemical staining for TSH. The ablation of gonadotropes but not thyrotropes suggests that separate cis-acting elements are necessary for expression of the alpha-subunit gene in these two cell types. Pituitary content of ACTH and GH was apparently normal, while PRL synthesis and storage were reduced. Thus, in a pituitary almost completely devoid of gonadotropes, most other pituitary functions were normal. This suggests that most pituitary cells are able to differentiate independently of terminal gonadotrope differentiation and can function in the absence of paracrine signaling provided by gonadotropes.
Adrenocorticotropic Hormone↗
Sequence of cDNA for rat cystathionine gamma-lyase and comparison of deduced amino acid sequence with related Escherichia coli enzymes.
A cDNA clone for cystathionine gamma-lyase was isolated from a rat cDNA library in lambda gt11 by screening with a monospecific antiserum. The identity of this clone, containing 600 bp proximal to the 3'-end of the gene, was confirmed by positive hybridization selection. Northern-blot hybridization showed the expected higher abundance of the corresponding mRNA in liver than in brain. Two further cDNA clones from a plasmid pcD library were isolated by colony hybridization with the first clone and were found to contain inserts of 1600 and 1850 bp. One of these was confirmed as encoding cystathionine gamma-lyase by hybridization with two independent pools of oligodeoxynucleotides corresponding to partial amino acid sequence information for cystathionine gamma-lyase. The other clone (estimated to represent all but 8% of the 5'-end of the mRNA) was sequenced and its deduced amino acid sequence showed similarity to those of the Escherichia coli enzymes cystathionine beta-lyase and cystathionine gamma-synthase throughout its length, especially to that of the latter.
Amino Acid Sequence↗
Protection of rat spermatogenic epithelium from damage induced by procarbazine chemotherapy.
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Animals↗
Production of diphtheria toxin CRM228 in B. subtilis.
The gene coding for a nontoxic diphtheria toxin (DT), tox228, was isolated from lysogenic Corynebacterium diphtheriae and cloned into pBR322. A mature form of the tox228 gene, lacking its signal sequence, was expressed in Bacillus subtilis using a B. amyloliquefaciens alpha-amylase secretion vector. To test the possibility of producing partially deleted DT molecules, which could be used for cell-directed toxin conjugates, a truncated form lacking 151 amino acids from the C-terminus of the DT was generated by oligonucleotide mutagenesis. Both the truncated and intact DT were efficiently secreted into the culture medium. During prolonged cultivation, the truncated form was less stable than the intact DT molecule.
Amino Acid Sequence↗
Cystathionine metabolism in neuroblastoma.
Cystathioninuria is a frequent and highly specific marker of neuroblastoma, but the etiology of this abnormality has not been well studied. To investigate this phenomenon, we analyzed 27 human neuroblastoma tissue specimens for the presence of cystathionine synthase and cystathionase. Levels of cystathionine synthase varied from undetectable to 622 pmol/mg protein, but no specimen had cystathionase measurable by rocket radioimmunoassay or catalytic assay. In addition, we assayed neuroblastoma cell lines exposed to a variety of differentiating agents: butyric acid, dimethyl sulfoxide, serum-free medium, or sodium citrate to induce differentiation. In each case we were unable to demonstrate cystathionase induction. These data are consistent with the hypothesis that neuroblastomas have a biochemical block in the transsulfuration enzymes at the level of cystathionase and that expression of cystathionine synthase in the absence of cystathionase may account for the presence of cystathioninuria in patients with neuroblastoma.
Cell Differentiation↗
Genetic ablation: targeted expression of a toxin gene causes microphthalmia in transgenic mice.
Lineage-specific regulatory elements can be used to direct expression of a variety of genes to specific tissues in transgenic mice. If the hybrid constructs contain a gene encoding a cytotoxic gene product, then genetic ablation of a specific cell lineage can be achieved. We have generated six transgenic mice by introducing into fertilized eggs the mouse gamma 2-crystallin promoter fused to the coding region of the diphtheria toxin A-chain gene. Three of these mice and all the transgenic offspring analyzed were microphthalmic. The lenses of these mice displayed considerable heterogeneity: some were almost normal morphologically but reduced in size, whereas others were grossly aberrant and deficient in nuclear fiber cells. These studies indicate that programmed ablation of specific cell types can be stably transmitted through the germ line.
Animals↗
Long-term suppression of luteinizing hormone, follicle-stimulating hormone and testosterone by daily administration of leuprolide.
The chronic effect of long-term injections of leuprolide on the hypothalamic, pituitary and gonadal axes have been studied in men with advanced prostatic cancer. The possibility of transient acute changes in luteinizing hormone, follicle-stimulating hormone and testosterone after each daily injection was studied in 31 patients treated for more than 1 year. No evidence of escape from daily 1.0 mg. doses was noted. No pituitary responsiveness was observed at any time point examined. Thus, daily administration of 1.0 mg. leuprolide acetate subcutaneously produces durable, complete suppression of gonadotropins and testosterone for prolonged periods.
Follicle Stimulating Hormone↗