Mutagenic activity of norharman and harman in Chinese hamster lung cells in assays with diphtheria toxin resistance as a marker.
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Alkaloids↗
Biomedical subjects
T Sugimura
Publications and source records attributed to T Sugimura.
A seven-base-pair deletion in an intron of the albumin gene of analbuminemic rats.
Analbuminemic rats, which genetically lack serum albumin, have a mutation affecting albumin mRNA processing. Serum albumin genes were cloned from analbuminemic and normal parental Sprague-Dawley rats. Structural analyses of the two albumin genes showed that the gene from analbuminemic rats had a seven-base-pair deletion in an intron. The deletion extended from base 5 to base 11 from the 5' end of intron HI of the albumin gene. This deletion converted the sequence, G-T-A-G-G-T, which is normally located at the 5' end of intron HI, to G-T-A-G-C-G. RNA blot hybridization of analbuminemic and normal rat liver nuclear RNA using a DNA fragment containing the intron HI as a probe showed that this intron sequence persisted in albumin mRNA precursors of analbuminemic rats.
Animals↗
Presence of 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid, a precursor of a mutagenic nitroso compound, in soy sauce.
After treatment with nitrite, Japanese soy sauce was strongly mutagenic to Salmonella typhimurium TA100 without S9 mixture. Two precursors of the mutagen were isolated from Japanese soy sauce, and these were identified as (-)-(1S,3S)-1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid [(-)-(1S,3S)-MTCA] and its stereoisomer (-)-(1R,3S)-MTCA. After treatment with nitrite, 1-mg samples of these compounds induced 17,400 and 13,000 revertants of TA100, respectively, without S9 mixture. Quantitative analysis of various kinds of soy sauces produced in Japan showed the presence of 82-678 micrograms of MTCA per ml. The mutagenicities of these compounds with nitrite accounted for 16-61% of the total mutagenicity of soy sauce with nitrite. Most soy sauces produced in the United States were less mutagenic than those produced in Japan and little, if any, of these two precursors of the mutagen was found in them. A major reaction product of (-)-(1S,3S)-MTCA and nitrite was a compound having a nitroso substitution at position N-2, but this compound was not mutagenic. Thus, the mutagen(s) formed from (-)-(1S,3S)-MTCA and nitrite was a minor product(s), and its specific mutagenic activity must be very high.
Biotransformation↗
Establishment and characterization of 10 cell lines derived from patients with adult T-cell leukemia.
By using human T-cell growth factor (TCGF), 10 cell lines were established from tissue samples of 10 patients with adult T-cell leukemia (ATL). Three cell lines were adapted to growth in medium lacking TCGF. The surface markers of all cell lines were characteristic of inducer/helper T cells, i.e., OKT3+, OKT4+, OKT6-, OKT8-, OKIa1+, and human Lyt2+ and Lyt3+, except that one cell line was OKT3-. The expression of the viral antigen was examined during establishment of 8 of the 10 cell lines. The viral antigen was not expressed in leukemic cells before cultivation. In 5 lines, the viral antigen was detected by immunofluorescent staining after a short period of cultivation. However, 3 cell lines, ATL-6A, ATL-9Y, and ATL-1K did not express the viral antigen during short-term culture: the ATL-6A and ATL-9Y cell lines became positive for the viral antigen after 5 and 2 months of cultivation, respectively; the ATL-1K cell line remained antigen-negative throughout a culture period of 13 months. Southern blot hybridization assay showed that all of the cell lines, including the viral antigen-negative ATL-1K cell line, contained the viral genome. Thus, the retrovirus was associated with all 10 cell lines established from ATL patients, but there was a heterogeneity in the expression time of the retroviral antigen in leukemic cells maintained in vitro. Our findings suggested that the expression of the viral antigen was not required for maintenance of the leukemic state in vivo and for growth of leukemic cells in vitro.
Antigens, Surface↗
Detection of lymphocytes producing a human retrovirus associated with adult T-cell leukemia by syncytia induction assay.
Recently 10 T-cell lines were established from patients with adult T-cell leukemia (ATL). During establishment of these cell lines, it was found that when T-cell lines expressing the ATL-associated retroviral antigen were cocultivated with 8C cat cells, multinucleated syncytia were formed. Retroviral antigen-negative T-cell lines did not induce syncytia. Peripheral blood lymphocytes obtained from ATL patients did not express the retroviral antigen before cultivation in vitro but became positive for the retroviral antigen after cultivation for a short period; these retroviral antigen-positive lymphocytes, but not retroviral antigen-negative lymphocytes, induced syncytia upon cocultivation with 8C cells. Peripheral blood lymphocytes isolated from patients with chronic lymphocytic leukemia of T-cell origin or Sézary syndrome or from normal adults and lymph node cells from a patient with immunoblastic lymphadenopathy-like T-cell lymphoma did not express the retroviral antigen even after cultivation in vitro and did not induce syncytia upon cocultivation with 8C cells. Thus, there was complete agreement between the presence of the retroviral antigen in established T-cell lines or freshly isolated peripheral blood lymphocytes and their ability to induce syncytia. Syncytia formation was enhanced 5- to 20-fold by the presence of Polybrene and inhibited by addition of plasma of ATL patients to the cocultures. Syncytia were detected within 4 hr on cocultivation of 8C cells with the retroviral antigen-positive T-cells, indicating that most syncytia were formed by early polykaryocytosis. After cocultivation, a clone of 8C cells that harbored the ATL virus genome and had syncytia-inducing activity was isolated. These findings indicate that the retrovirus associated with ATL has syncytia-inducing activity. Syncytia induction assay using 8C cells will be useful for detection and characterization of human retrovirus associated with T-cell malignancies.
Adult↗
Immunoaffinity fractionation of the poly(ADP-ribosyl)ated domains of chromatin.
Antibody to poly(ADP-ribose) has been covalently coupled to Sepharose and utilized to isolate selectively oligonucleosomes undergoing the poly(ADP-ribosyl)ation reaction from the bulk of chromatin. Approximately 12% of the unfractionated oligonucleosomes were bound to the immunoaffinity column and these represented essentially 100% of the original poly(ADP-ribosyl)ated nucleosomal species in the unfractionated chromatin. Poly(ADP-ribosyl)ated chromatin was not bound by preimmune IgG columns. KSCN eluted the modified nucleosomes in the form of nucleoprotein complexes. The eluted chromatin components were shown to contain poly(ADP-ribosyl)ated histones as well as automodified poly(ADP-ribose) polymerase. By using [3H]lysine- and [3H]arginine-labeled chromatin, it was shown that the poly-(ADP-ribosyl)ated histones, attached to stretches of oligonucleosomes bound to the column, had a 6-fold enrichment of the modification compared to histones of the unfractionated chromatin. This indicated that non-poly(ADP-ribosyl)ated nucleosomes, connected and proximal to the modified regions, were copurified by this procedure. This allowed characterization of the oligonucleosomal DNA around poly(ADP-ribosyl)ated chromatin domains to be compared with the unbound bulk chromatin. The data indicated that immunofractionated poly(ADP-ribosyl)ated oligonucleosomal DNA contained significant amounts of internal single-strand breaks compared with bulk chromatin. The bound nucleo-protein complexes were found to be enzymatically active for poly(ADP-ribose) polymerase after elution from the antibody column. In contrast, the unbound nucleosomes, representing 90% of the unfractionated chromatin, were totally inactive in the poly(ADP-ribosyl)ation reaction.
Chromatin↗
Inhibition of specific binding of [3H]phorbol-12,13-dipropionate to an epidermal fraction by certain irritants and irritant promoters of mouse skin.
Specific binding of [3H]phorbol-12,13-dipropionate ([3H]PDPr) to a particulate fraction of mouse skin is demonstrated (KD = 35 nM; Rt = 1.2 pmol/mg protein). A series of compounds of the diterpene ester, indole akaloid and polyacetate types with different degrees of activity as skin tumor promoters and/or irritants have been tested for their capacity to inhibit specific [3H]PDPr binding. Three main categories are found: (i) compounds which exhibit a positive correlation between their potency as irritants and promoters in vivo and their inhibition of specific binding in vitro: 12-O-tetradecanoylphorbol-13-acetate, 3-O-tetradecanoylingenol, pimelea factor P2, 'teleocidin', dihydroteleocidin B, and lyngbyatoxin are active in vivo and in vitro, whereas phorbol and ingenol are inactive and 4-O-methyl-12-O-tetradecanoyl-phorbol-13-acetate is weakly active; (ii) compounds which are strong irritants and inhibitors of binding but are weak or practically non-promoters: mezerein, 12-O-retinoylphorbol-13-acetate and milliamine C; (iii) strong irritants which are weak or marginally active inhibitors of binding: debromoaplysiatoxin and resiniferatoxin. Some consequences of these findings with respect to interpretations of the biochemical mechanism(s) of tumor promotion are discussed.
Animals↗
Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion and ornithine decarboxylase activity by quercetin: possible involvement of lipoxygenase inhibition.
Quercetin (30 mumol/mouse) markedly suppressed the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA, 20 nmol/mouse) on skin tumor formation in the CD-1 mice initiated by 7,12-dimethylbenz[a]anthracene (200 nmol/mouse). TPA (20 nmol/mouse)-induced epidermal ornithine decarboxylase (ODC) activity was also inhibited by quercetin (10-30 mumol/mouse), but it failed to inhibit the stimulation of epidermal DNA synthesis by TPA. In addition, quercetin potently inhibited lipoxygenase from 105 000 g supernatant of epidermal homogenate of mice. The 50% inhibition of lipoxygenase was observed by quercetin at 1.3 microM. These results suggest that the inhibition of lipoxygenase by quercetin is one of the major actions of the above agent to inhibit tumor promotion and TPA-induced ODC activity.
Animals↗
Poly(ADP-ribose) and cancer research.
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Animals↗
Myeloperoxidases of human myeloid leukemia cells HL-60 grown in culture and in nude mice.
Human myeloid leukemia HL-60 cells were grown either in suspension culture or in nude mice. The two types of myeloperoxidase, the small and the large type, in crude extracts of these cells were analyzed by sucrose density gradient centrifugation. The proportions of the small and the large myeloperoxidase varied markedly depending on the growth conditions of cells. In cells in culture, the small myeloperoxidase amounted to 80% of the total myeloperoxidase, whereas in the solid tumors it amounted to only about 30% of the total. Both in cultured cells and solid tumors, 40% of the total myeloperoxidase was found in the soluble fraction and the rest in the granule fraction. However, adult and fetal blood granulocytes contained only the large myeloperoxidase, which was mainly recovered in the granule fraction. Antiserum prepared against purified large myeloperoxidase of HL-60 solid tumors reacted with the small myeloperoxidase as well as the large enzyme of HL-60 cells in culture. The antiserum also precipitated myeloperoxidase of adult and fetal blood granulocytes. An Ouchterlony double immunodiffusion test also revealed their immunological identity.
Animals↗
Molecular mechanism of change in serum alpha-fetoprotein concentration during neonatal development of analbuminemic rats.
Analbuminemic rats, which lack albumin synthesis in the liver, have been shown to have a defect in splicing of albumin mRNA precursors. Change in serum alpha-fetoprotein concentration during the neonatal period in these mutant rats was compared with change of their gene expression of albumin. In analbuminemic rats the serum alpha-fetoprotein concentration at birth was almost the same as that of normal rats, and its concentration gradually decreased to a nondetectable level within 3 to 4 weeks after birth, as in normal rats, without increase in serum albumin concentration. In spite of the absence of increase in serum albumin, increase in transcripts of the albumin gene was observed. The level of "albumin mRNA precursors" in nuclei of the liver was less than 0.05 ng/micrograms of nuclear RNA at birth, but increased to 1 ng/micrograms within 3 weeks after birth in normal rats. Although the extent of increase was less, a similar switch-on of the albumin gene was observed in analbuminemic rats in the developmental period. These data clearly indicated that even in analbuminemic rats, coordinated regulation of the alpha-fetoprotein and albumin genes took place at a transcriptional level during development.
Animals↗
Polyoma virus minichromosomes: poly ADP-ribosylation of associated chromatin proteins.
The host nuclear enzyme poly(ADP-ribose) polymerase has been shown to be associated with the replicative intermediate and mature forms of polyoma virus minichromosomes. Minichromosome-associated histones H2A and H2B as well as several nonhistone proteins were poly ADP-ribosylated by endogenous poly(ADP-ribose) polymerase. In addition, minichromosome fractions catalyzed the formation in vitro of dimers of endogenous histone H1 linked by poly(ADP-ribose). Poly ADP-ribosylated polyoma virus minichromosome chromatin labeled in vivo with [3H]thymidine could be retained and eluted from anti-poly(ADP-ribose) immunoglobulin G-Sepharose. Pulse-labeled replicative intermediate minichromosomes were retained better on the antibody columns than were mature minichromosomes labeled for 2.5 h. The possible role of poly ADP-ribosylation of viral nucleosomes during polyoma replication or transcription is discussed.
Chromatin↗
Biological actions of nitroarenes in short-term tests on Salmonella, cultured mammalian cells and cultured human tracheal tissues: possible basis for regulatory control.
Pure synthetic nitropyrene compounds were subjected to a mutation test using Salmonella typhimurium TA 98 and TA 100 with and without S9 mix, a metabolic activation system. Dinitropyrenes were highly mutagenic. Among them, 1,8-dinitropyrene was the most potent mutagen, producing 940,000 revertants of TA 98/micrograms. 1,3,6-Trinitropyrene and 1,3,6,8-tetranitropyrene were also highly mutagenic, producing 708,000 and 221,000 revertants/micrograms, respectively. 1-Nitropyrene was weakly mutagenic. All nitropyrenes were more mutagenic towards TA 98 than TA 100, and all mutagenic activities were abolished by the presence of S9 mix. Di- and trinitropyrenes were demonstrated to be mutagenic to Chinese hamster lung cells without metabolic activation, by using diphtheria toxin resistancy as a marker. The range of mutagenic potential of nitropyrenes was much narrower with cultured mammalian cells than with Salmonella. 1-Nitropyrene was not mutagenic. 1,6-Dinitropyrene and 1-nitropyrene induced unscheduled DNA synthesis in epithelial cells of in vitro cultured human bronchi, as did diol-epoxides of benzo[a]pyrene, while benzo[a]pyrene itself was inert. 1-Nitropyrene and 3-nitrofluoranthene produced subcutaneous fibrosarcomas at the loci of injections in the backs of rats. Tumors were found in 47% and 40% of animals with total doses of 40 mg of 1-nitropyrene and 30 mg of 3-nitrofluoranthene, respectively. The biomedical significance of nitroarenes is discussed.
Animals↗
New classes of environmental tumor promoters: indole alkaloids and polyacetates.
Tumor promoters are known to induce ODC activity in mouse skin and that this induction can be inhibited by the application of 13-cis-retinoic acid. These two properties of tumor promoters were utilized for screening new tumor promoters in our environment. Two new classes of tumor promoters are presented: indole alkaloids (teleocidin and lyngbyatoxin A) and polyacetates (aplysiatoxin and debromoaplysiatoxin). Teleocidin from streptomyces and lyngbyatoxin A, from the blue-green alga, Lyngbya majuscula, were able to induce ODC activity in mouse skin and showed various biological activities similar to those of TPA. Teleocidin and lyngbyatoxin A are indole alkaloids. Their tumor promoting activities became apparent in the mouse skin through a two-stage carcinogenicity test. The tumor incidence of the group treated with DMBA plus teleocidin was 100% at week 30, which was similar to that of the group given DMBA and TPA. The in vivo carcinogenicity test with lyngbyatoxin A is still underway. The tumor incidence of the group treated with DMBA plus lyngbyatoxin A is 80% at week 21. A second new class of tumor promoter is polyacetate. Aplysiatoxin and debromoaplysiatoxin were isolated from another variety of blue-green alga. Aplysiatoxin and debromoaplysiatoxin induce ODC to the same degree of potency. However, aplysiatoxin induced various membrane effects, such as adhesion of cells and turnover of phospholipid with a similar concentration of TPA, teleocidin and lyngbyatoxin A. On the other hand, debromoaplysiatoxin required an amount almost 100 times greater to achieve the same effects. We are convinced of the possibility that various classes of tumor promoters exist in our environment.
Acetates↗
Enhancement of antitumor activity of bleomycin by benzamide in vitro and in vivo.
The cytotoxic effects of bleomycin on HeLa cells in culture were enhanced by incubation of the cells with benzamide, a potent inhibitor of poly(ADP-ribose) polymerase, at concentrations at which benzamide alone did not show any cytotoxicity. Benzamide plus bleomycin display enhanced therapeutic effects against Ehrlich ascites tumor cells in vivo. On daily treatment with various doses of bleomycin plus benzamide for 10 days, mice with Ehrlich ascites tumors survived longer than mice on treatment with bleomycin alone.
Animals↗
Promoter-induced cellular responses closely correlated with the enhancement of cell transformation.
In order to understand the mechanisms of tumor promotion, the relationship between the early effects induced by promoters and the enhancement of transformation was investigated using transformable, promoter-sensitive mouse clone Balb/3T3 A31-1-1. Emphasis was placed on the measurement of all parameters in the same clonal line under the same conditions. The potentials of various derivatives of phorbol ester, indole alkaloid, and polyacetate for enhancing transformation in 3-methylcholanthrene-initiated cells were, in general, in parallel with their potentials for inhibiting phorbol-12, 13-dibutyrate-binding and for inducing early responses such as the reduction of epidermal growth factor (EGF)-binding to cell surface receptors, the increase in glucose uptake and release of arachidonic acid, and the stimulation of DNA synthesis in cells arrested at G0. There was an exception to this general rule; some agents, such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and debromoaplysiatoxin, showed very strong activities to induce most of the early responses, whereas they showed slight or no activity to enhance cell transformation. The cause of this exception was ascribed to their higher susceptibility to metabolic or physical inactivation. However, we found that the continuous suppression of EGF-binding to cell surface receptors by promoters was well correlated with the enhancement of transformation, without exception. Furthermore, continuous elevation or reduction of the number of EGF receptors by various hormones was associated with the suppression or enhancement of cell transformation. The hypothesis was proposed that a continuous decrease in the number of EGF-receptors on the cell surface, or its underlying mechanisms, plays an important role in the enhancement of transformation. The mechanisms by which activation of protein kinase C leads to the enhancement of transformation were discussed, with emphasis on cytoskeletal alteration.
Animals↗
Nakahara memorial lecture. New classes of tumor promoters: teleocidin, aplysiatoxin, and palytoxin.
Teleocidin and aplysiatoxin, which are structurally different from 12-O-tetradecanoylphorbol-13-acetate (TPA), were found to be potent tumor promoters in two-step mouse skin carcinogenesis. The class of teleocidin includes dihydroteleocidin B, teleocidin, and lyngbyatoxin A. Teleocidin, which is a mixture of 93% teleocidin A and 7% teleocidin B, was isolated from Streptomyces mediocidicus as a strong skin irritant. Teleocidin A consists of C-14S-teleocidin A and C-14R-teleocidin A. One teleocidin A-isomer corresponds to lyngbyatoxin A, which was isolated from the blue-green alga, Lyngbya majuscula. Teleocidin B has four isomers, C-14, C-17-diastereomers. The two teleocidin A-isomers and three of the teleocidin B-isomers (all but one, which was obtained in too low yield) were shown to be biologically active and also potent tumor promoters. Synthetic analogues (indolactams) of teleocidin were obtained and their structure-activity relations were examined by several biological tests. The finding that only (-)-indolactam-V was active showed that the S, S configuration of native teleocidin was necessary for expression of the activity. The class of aplysiatoxin, which was isolated from the blue-green alga, L. majuscula, includes debromoaplysiatoxin, aplysiatoxin, bromoaplysiatoxin, and oscillatoxin A (nordebromoaplysiatoxin). The former three were potent tumor promoters, while oscillatoxin A was a moderate one. Dibromoaplysiatoxin, which is a chemically brominated derivative of debromoaplysiatoxin, in addition to aplysiatoxin and bromoaplysiatoxin, possessed the same promoting activity as that of oscillatoxin A.(ABSTRACT TRUNCATED AT 250 WORDS)
Acrylamides↗
Distinct inhibitory effects of dihydroteleocidin B and the phorbol ester tumor promoters on the adipocyte differentiation of 3T3-L1 cells.
An indole alkaloid tumor promoter, dihydroteleocidin B, was able to modulate a membrane property of 3T3-L1 preadipocytes, showing an almost complete reduction of epidermal growth factor binding capacity. This receptor modulating potency of dihydroteleocidin B, was 10 times that of a phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Dihydroteleocidin B, however, had little effect on the epidermal growth factor receptors of the adipocyte stage of 3T3-L1. Adipocyte differentiation was induced by treating growth-arrested 3T3-L1 cells with dexamethasone and 1-methyl-3-isobutylxanthine for 48 hr. These inducers initiated DNA synthesis, led to one full cycle of cell division, and triggered the adipocyte differentiation program. Dihydroteleocidin B almost completely inhibited this differentiation at concentrations of 1 to 10 ng/ml (10(-9) to 10(-8) M). The inhibition was observed regardless of when the tumor promoter was added: before, during, or after the addition of inducers. Similar inhibition was also observed by TPA, but with over 90% less efficiency than that of dihydroteleocidin B. TPA was most effective when it was added during the inducer treatment. Both dihydroteleocidin B and TPA stimulated DNA synthesis to the same level during the initial 22 hr. The DNA synthesis stimulated by dihydroteleocidin B resulted in extraordinary enhancement of cell proliferation, whereas TPA-treated 3T3-L1 cells did not divide. These findings suggest that dihydroteleocidin B and TPA have distinct potencies in interfering with the mechanisms of adipocyte differentiation and that presumably they are different in action of tumorigenesis.
1-Methyl-3-isobutylxanthine↗