Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-1,2-benzopyrone (INH2BP)

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Abstract

Bovine aortic cndothelial cells were converted to a highly tumorigenic cell line by transfection with Ha-ras and stimulation with thrombin. Sustained pretreatment with a non-cytotoxic concentration (600 μM) of 5-iodo-6-amino-1,2-benzopyrone (INH2BP), a lipophilic ligand of poly(ADP-ribose) polymerase, abrogated in vivo tumorigenicity, of 105 cells per inoculum an effect which developed progressively during 2 to 6 weeks of drug treatment. The initial action of the drug was cytostasis, consisting of an arrest in prophase, extreme cell enlargement consistent with cytoplasmic hypertrophy, as seen by EM, and dramatic morphologic changes. Although neither DNA, RNA or protein syntheses are directly affected by INH2BP, apparently newly synthesized cellular DNA is degraded by endonucleases, which are upregulated by the inhibition of their poly-ADP-ribosylation. The drug treated cells exhibited greatly increased respiration and aerobic glycolysis, due to an augmentation of glycolytic and respiratory enzymes in enlarged cells. These responses to the drug were reversible in cell cultures following drug removal, within 5-10 days drug exposure but the progressive loss of tumorigenicity in nude mice that developed after 3-6 weeks of drug exposure of cells, prior to inoculation to nude mice, was not reversible in vivo. Drug treatment produced a sustained 70-80% inhibition of pADPRT in intact cells at 600 μM extracellular concentration of INH2BP. The prerequisite for the abrogation of tumorigenicity was the maintenance of pADPRT inhibition. The arrest of cell multiplication and a large decrease of Topo I, especially of Topo II and MAP kinase activities occurred without loss of enzyme protein as assayed in cell extracts of drug-treated cells. However INH2BP had no direct effect on these enzymes. Drug treatment down-regulated DNA-methyltransferase, PKC, ODC proteins, diminished cyclin A protein, but the hypophosphorylated form of Rb protein was significantly augmented. None of the enzymatic components of signal pathways so far studied, were directly affected by INH2BP. The inhibition of pADPRT by INH2BP coincided with an induction or activation of alkaline phosphatase and leucyl and glutamyl peptidase. The pADPRT content or the expression of pADPRT gene were not influenced by drug treatment, but the expression of ras gene was completely absent in nontumorigenic drug-treated cells, without a loss of ras gene from genomic DNA. Telomerase activity was not directly influenced by INH2BP treatment when assayed in diluted cell extracts, but the addition of homogeneous which explains the lack of tumorigenicity of drug-pretreated cancer cells in nude mice, is subject to further experimental analysis. The coincidence of Topo I and II inactivation induced by INH2BP (Table II) partly explains the inhibition of cell multiplication without actual interference with pathways of DNA synthesis. A number of highly toxic anticancer drugs inhibit topoisomerases (62) yet INH2BP accomplishes the same without apparent toxicity. When given orally at a daily dose of 2 g/kg INH2BP for a week is non-toxic with a 100% bioavailability as determined by [14C]-INH2BP distribution, and tissue levels are produced that exceed the concentration required to be effective on E-ras 20 cells in cell culture, therefore in vivo application of INH2BP appears feasible. The possible use of INH2BP in clinical oncology might be in cases of disseminated metastases, which may be inactivated by mechanisms described in this report. This treatment differs from traditional direct cytocidal methods of chemotherapy, and may fit into the category of biological response modification, a prediction made on the basis of theoretical considerations of cell cycle control in cancer (63).

Original languageEnglish
Pages (from-to)239-252
Number of pages14
JournalInternational Journal of Oncology
Volume8
Issue number2
Publication statusPublished - 1996

Fingerprint

Endothelial Cells
Cell Line
Growth
Pharmaceutical Preparations
Nude Mice
DNA
ras Genes
5-iodo-6-amino-1,2-benzopyrone
Cell Extracts
Proteins
Enzymes
Cell Culture Techniques
Cell Proliferation
Cell Enlargement
Cyclin A
Prophase
Retinoblastoma Protein
Medical Oncology
Poly(ADP-ribose) Polymerases
Poisons

Keywords

  • 2-benzopyrone
  • 5-iodo-6-amino-1
  • Endothelial cell line
  • Ras gene

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

@article{3c22b17e8a3b4cccb0aacc0ed2141adc,
title = "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-1,2-benzopyrone (INH2BP)",
abstract = "Bovine aortic cndothelial cells were converted to a highly tumorigenic cell line by transfection with Ha-ras and stimulation with thrombin. Sustained pretreatment with a non-cytotoxic concentration (600 μM) of 5-iodo-6-amino-1,2-benzopyrone (INH2BP), a lipophilic ligand of poly(ADP-ribose) polymerase, abrogated in vivo tumorigenicity, of 105 cells per inoculum an effect which developed progressively during 2 to 6 weeks of drug treatment. The initial action of the drug was cytostasis, consisting of an arrest in prophase, extreme cell enlargement consistent with cytoplasmic hypertrophy, as seen by EM, and dramatic morphologic changes. Although neither DNA, RNA or protein syntheses are directly affected by INH2BP, apparently newly synthesized cellular DNA is degraded by endonucleases, which are upregulated by the inhibition of their poly-ADP-ribosylation. The drug treated cells exhibited greatly increased respiration and aerobic glycolysis, due to an augmentation of glycolytic and respiratory enzymes in enlarged cells. These responses to the drug were reversible in cell cultures following drug removal, within 5-10 days drug exposure but the progressive loss of tumorigenicity in nude mice that developed after 3-6 weeks of drug exposure of cells, prior to inoculation to nude mice, was not reversible in vivo. Drug treatment produced a sustained 70-80{\%} inhibition of pADPRT in intact cells at 600 μM extracellular concentration of INH2BP. The prerequisite for the abrogation of tumorigenicity was the maintenance of pADPRT inhibition. The arrest of cell multiplication and a large decrease of Topo I, especially of Topo II and MAP kinase activities occurred without loss of enzyme protein as assayed in cell extracts of drug-treated cells. However INH2BP had no direct effect on these enzymes. Drug treatment down-regulated DNA-methyltransferase, PKC, ODC proteins, diminished cyclin A protein, but the hypophosphorylated form of Rb protein was significantly augmented. None of the enzymatic components of signal pathways so far studied, were directly affected by INH2BP. The inhibition of pADPRT by INH2BP coincided with an induction or activation of alkaline phosphatase and leucyl and glutamyl peptidase. The pADPRT content or the expression of pADPRT gene were not influenced by drug treatment, but the expression of ras gene was completely absent in nontumorigenic drug-treated cells, without a loss of ras gene from genomic DNA. Telomerase activity was not directly influenced by INH2BP treatment when assayed in diluted cell extracts, but the addition of homogeneous which explains the lack of tumorigenicity of drug-pretreated cancer cells in nude mice, is subject to further experimental analysis. The coincidence of Topo I and II inactivation induced by INH2BP (Table II) partly explains the inhibition of cell multiplication without actual interference with pathways of DNA synthesis. A number of highly toxic anticancer drugs inhibit topoisomerases (62) yet INH2BP accomplishes the same without apparent toxicity. When given orally at a daily dose of 2 g/kg INH2BP for a week is non-toxic with a 100{\%} bioavailability as determined by [14C]-INH2BP distribution, and tissue levels are produced that exceed the concentration required to be effective on E-ras 20 cells in cell culture, therefore in vivo application of INH2BP appears feasible. The possible use of INH2BP in clinical oncology might be in cases of disseminated metastases, which may be inactivated by mechanisms described in this report. This treatment differs from traditional direct cytocidal methods of chemotherapy, and may fit into the category of biological response modification, a prediction made on the basis of theoretical considerations of cell cycle control in cancer (63).",
keywords = "2-benzopyrone, 5-iodo-6-amino-1, Endothelial cell line, Ras gene",
author = "P. Bauer",
year = "1996",
language = "English",
volume = "8",
pages = "239--252",
journal = "International Journal of Oncology",
issn = "1019-6439",
publisher = "Spandidos Publications",
number = "2",

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TY - JOUR

T1 - Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-1,2-benzopyrone (INH2BP)

AU - Bauer, P.

PY - 1996

Y1 - 1996

N2 - Bovine aortic cndothelial cells were converted to a highly tumorigenic cell line by transfection with Ha-ras and stimulation with thrombin. Sustained pretreatment with a non-cytotoxic concentration (600 μM) of 5-iodo-6-amino-1,2-benzopyrone (INH2BP), a lipophilic ligand of poly(ADP-ribose) polymerase, abrogated in vivo tumorigenicity, of 105 cells per inoculum an effect which developed progressively during 2 to 6 weeks of drug treatment. The initial action of the drug was cytostasis, consisting of an arrest in prophase, extreme cell enlargement consistent with cytoplasmic hypertrophy, as seen by EM, and dramatic morphologic changes. Although neither DNA, RNA or protein syntheses are directly affected by INH2BP, apparently newly synthesized cellular DNA is degraded by endonucleases, which are upregulated by the inhibition of their poly-ADP-ribosylation. The drug treated cells exhibited greatly increased respiration and aerobic glycolysis, due to an augmentation of glycolytic and respiratory enzymes in enlarged cells. These responses to the drug were reversible in cell cultures following drug removal, within 5-10 days drug exposure but the progressive loss of tumorigenicity in nude mice that developed after 3-6 weeks of drug exposure of cells, prior to inoculation to nude mice, was not reversible in vivo. Drug treatment produced a sustained 70-80% inhibition of pADPRT in intact cells at 600 μM extracellular concentration of INH2BP. The prerequisite for the abrogation of tumorigenicity was the maintenance of pADPRT inhibition. The arrest of cell multiplication and a large decrease of Topo I, especially of Topo II and MAP kinase activities occurred without loss of enzyme protein as assayed in cell extracts of drug-treated cells. However INH2BP had no direct effect on these enzymes. Drug treatment down-regulated DNA-methyltransferase, PKC, ODC proteins, diminished cyclin A protein, but the hypophosphorylated form of Rb protein was significantly augmented. None of the enzymatic components of signal pathways so far studied, were directly affected by INH2BP. The inhibition of pADPRT by INH2BP coincided with an induction or activation of alkaline phosphatase and leucyl and glutamyl peptidase. The pADPRT content or the expression of pADPRT gene were not influenced by drug treatment, but the expression of ras gene was completely absent in nontumorigenic drug-treated cells, without a loss of ras gene from genomic DNA. Telomerase activity was not directly influenced by INH2BP treatment when assayed in diluted cell extracts, but the addition of homogeneous which explains the lack of tumorigenicity of drug-pretreated cancer cells in nude mice, is subject to further experimental analysis. The coincidence of Topo I and II inactivation induced by INH2BP (Table II) partly explains the inhibition of cell multiplication without actual interference with pathways of DNA synthesis. A number of highly toxic anticancer drugs inhibit topoisomerases (62) yet INH2BP accomplishes the same without apparent toxicity. When given orally at a daily dose of 2 g/kg INH2BP for a week is non-toxic with a 100% bioavailability as determined by [14C]-INH2BP distribution, and tissue levels are produced that exceed the concentration required to be effective on E-ras 20 cells in cell culture, therefore in vivo application of INH2BP appears feasible. The possible use of INH2BP in clinical oncology might be in cases of disseminated metastases, which may be inactivated by mechanisms described in this report. This treatment differs from traditional direct cytocidal methods of chemotherapy, and may fit into the category of biological response modification, a prediction made on the basis of theoretical considerations of cell cycle control in cancer (63).

AB - Bovine aortic cndothelial cells were converted to a highly tumorigenic cell line by transfection with Ha-ras and stimulation with thrombin. Sustained pretreatment with a non-cytotoxic concentration (600 μM) of 5-iodo-6-amino-1,2-benzopyrone (INH2BP), a lipophilic ligand of poly(ADP-ribose) polymerase, abrogated in vivo tumorigenicity, of 105 cells per inoculum an effect which developed progressively during 2 to 6 weeks of drug treatment. The initial action of the drug was cytostasis, consisting of an arrest in prophase, extreme cell enlargement consistent with cytoplasmic hypertrophy, as seen by EM, and dramatic morphologic changes. Although neither DNA, RNA or protein syntheses are directly affected by INH2BP, apparently newly synthesized cellular DNA is degraded by endonucleases, which are upregulated by the inhibition of their poly-ADP-ribosylation. The drug treated cells exhibited greatly increased respiration and aerobic glycolysis, due to an augmentation of glycolytic and respiratory enzymes in enlarged cells. These responses to the drug were reversible in cell cultures following drug removal, within 5-10 days drug exposure but the progressive loss of tumorigenicity in nude mice that developed after 3-6 weeks of drug exposure of cells, prior to inoculation to nude mice, was not reversible in vivo. Drug treatment produced a sustained 70-80% inhibition of pADPRT in intact cells at 600 μM extracellular concentration of INH2BP. The prerequisite for the abrogation of tumorigenicity was the maintenance of pADPRT inhibition. The arrest of cell multiplication and a large decrease of Topo I, especially of Topo II and MAP kinase activities occurred without loss of enzyme protein as assayed in cell extracts of drug-treated cells. However INH2BP had no direct effect on these enzymes. Drug treatment down-regulated DNA-methyltransferase, PKC, ODC proteins, diminished cyclin A protein, but the hypophosphorylated form of Rb protein was significantly augmented. None of the enzymatic components of signal pathways so far studied, were directly affected by INH2BP. The inhibition of pADPRT by INH2BP coincided with an induction or activation of alkaline phosphatase and leucyl and glutamyl peptidase. The pADPRT content or the expression of pADPRT gene were not influenced by drug treatment, but the expression of ras gene was completely absent in nontumorigenic drug-treated cells, without a loss of ras gene from genomic DNA. Telomerase activity was not directly influenced by INH2BP treatment when assayed in diluted cell extracts, but the addition of homogeneous which explains the lack of tumorigenicity of drug-pretreated cancer cells in nude mice, is subject to further experimental analysis. The coincidence of Topo I and II inactivation induced by INH2BP (Table II) partly explains the inhibition of cell multiplication without actual interference with pathways of DNA synthesis. A number of highly toxic anticancer drugs inhibit topoisomerases (62) yet INH2BP accomplishes the same without apparent toxicity. When given orally at a daily dose of 2 g/kg INH2BP for a week is non-toxic with a 100% bioavailability as determined by [14C]-INH2BP distribution, and tissue levels are produced that exceed the concentration required to be effective on E-ras 20 cells in cell culture, therefore in vivo application of INH2BP appears feasible. The possible use of INH2BP in clinical oncology might be in cases of disseminated metastases, which may be inactivated by mechanisms described in this report. This treatment differs from traditional direct cytocidal methods of chemotherapy, and may fit into the category of biological response modification, a prediction made on the basis of theoretical considerations of cell cycle control in cancer (63).

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KW - 5-iodo-6-amino-1

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