Biomedical subjects

L M Nutter

Publications and source records attributed to L M Nutter.

At least 19 recordsLinked to original sources

PubMed1998

Absence of pyridoxine-5'-phosphate oxidase (PNPO) activity in neoplastic cells: isolation, characterization, and expression of PNPO cDNA.

Major differences in the metabolism of vitamin B6 in various cancers compared to their normal cellular counterparts have been documented. In particular, pyridoxine- 5'-phosphate oxidase (PNPO), the rate-limiting enzyme in pyridoxal 5'-phosphate (PLP) biosynthesis, is absent in liver and neurally-derived tumors. We show that the expression of PNPO is developmentally regulated not only in liver but also in brain. Specifically, PNPO activity in fetal brain tissue is 7.5-fold lower than that found in adult brain tissue. Furthermore, the isolation and characterization of a PNPO cDNA are described. The isolated cDNA was verified to be the authentic PNPO cDNA on the basis of two criteria. First, the translated product from the PNPO cDNA is immunologically reactive to a polyclonal PNPO antibody. Second, PNPO negative hepatoma cell lines stably transfected with the PNPO cDNA express enzymatically active PNPO protein. The availability of these biological reagents will not only facilitate in depth investigations of the reasons for the absence of PNPO in liver and brain malignancies but also aid in an understanding of the biochemical regulation of B6 metabolism in development.

Amino Acid Sequence↗

PubMed1998

Hexahydrocolupulone and its antitumor cell proliferation activity in vitro.

The purpose of this study was to evaluate the ability of hexahydrocolupulone (HHC) to inhibit the growth of tumor cells in vitro and to investigate the potential mechanism(s) involved. HHC was demonstrated to have a wide spectrum of activity against a number of established human tumor cell lines, including some exhibiting drug resistance. Culturing human breast adenocarcinoma (MCF-7) cells in the presence of HHC for 18 hr resulted in a significant decrease in the incorporation of [3H]uridine and [3H]leucine into RNA and protein, respectively. MCF-7 cells cultured in the presence of 1.5 microM HHC for 48 hr demonstrated an increase in the amount of cells detected in G0/G1 and a decrease in the amount of cells detected in S phase. In contrast, treatment with 25 microM HHC decreased the amount of cells detected in G0/G1 and increased the amount of cells detected in S phase. HHC did not cause single-stranded or double-stranded DNA breaks, interfere with topoisomerase function, or generate free radicals. Mice injected intraperitoneally for 5 consecutive days with HHC to a final in vivo blood concentration of 200 microM survived and showed no obvious signs of toxicity. Mass spectroscopy analysis, crystal generation, and structure elucidation confirmed HHC purity. Consequently, all activity observed can be attributed to HHC, a metabolite, and/or a combination thereof. These data suggest that HHC inhibits tumor cell proliferation in vitro via a mechanism(s) that may involve effects on macromolecular synthesis, precursor metabolism/transport, and/or the cell cycle or cell cycle-dependent pathway(s).

Animals↗

PubMed1998

Induction of p53 by the concerted actions of aziridine and quinone moieties of diaziquone.

The biologic functions attributed to the nucleophosphoprotein p53 have been increasing in recent years. Some studies suggested that wild type p53 is responsible for cell cycle arrest brought about as a response to exposure of mammalian cells to DNA-damaging agents. This cell cycle arrest occurs in order for cells to repair the damaged macromolecules. Extensively damaged cells are also thought to undergo apoptosis via the p53-dependent or -independent signal transduction pathways. In this study, we investigated the ability of diaziridinylbenzoquinones to increase p53 levels in the human breast cancer cell line MCF-7. Diaziquone (AZQ), an anticancer agent, and its derivatives, diaziridinequinone (DZQ) and methyldiaziridinequinone (MeDZQ), induced p53 in a dose- and time-dependent manner as measured by the electrophoretic mobility shift assay. Wild type p53 induction by AZQ was suppressed when DT-diaphorase activity was inhibited by pretreating the cells with dicumarol. Aside from their potent alkylating activity, these agents also undergo redox cycling as evidenced by oxygen consumption and the production of reactive oxygen species (ROS). Inhibition of ROS production by the antioxidant enzyme catalase reduced AZQ- and DZQ-mediated p53 induction by about 45%. Thiotepa, a non-quinone aziridine-containing agent, and 1,4-benzoquinone (p-BQ), a redox cycling quinone, increased p53 levels. The nonalkylator oxygen-radical-generating agent menadione (MD) caused p53 induction only when MCF-7 cells were allowed to recover in drug-free media. On the basis of these data, we propose that the bioreductive activation of AZQ is a prerequisite for p53 induction. Moreover, the induction of p53 by AZQ requires both the quinone and the aziridine moieties of the AZQ molecule. Although AZQ and its analogues increased p53 levels in MCF-7 cells, p53 induction in these cells may not be responsible for the apoptosis seen upon treatment of MCF-7 cells with these agents. The uncoupling of p53 induction and apoptosis is evidenced by the generation of nucleosomal DNA laddering in aziridinequinone-treated T47D cells, a breast cancer cell line bearing a p53 mutation.

Adenocarcinoma↗

PubMed1998

Antimicrobial activity of the semisynthetic compound, hexahydrocolupulone.

In this study we demonstrate that hexahydrocolupulone (HHC) more effectively inhibits the growth in vitro of Gram-positive organisms than Mycobacterium tuberculosis or Escherichia coli. Vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, and coagulase-negative staphylococci were inhibited by HHC at concentrations < or = 4.06 mg/L. Growth inhibition profiles varied according to the microorganism evaluated (static for S. aureus and bactericidal for Bacillus subtilis).

Anti-Bacterial Agents↗

PubMed1997

Insensitivity of cultured rat cortical neurons to mitochondrial DNA synthesis inhibitors: evidence for a slow turnover of mitochondrial DNA.

Mitochondrial dysfunction is a major contributor to aging and neurodegeneration. Defects in mitochondrial DNA (mtDNA) have been identified in several neuromuscular diseases. Even though there is a high rate of phenotypic expression of mtDNA mutations in the central nervous system and replication of DNA introduces errors, little is known about the replicative activity of mtDNA in the brain. In this study, we investigated the sensitivity of cultured rat cortical neurons to mtDNA synthesis inhibitors as a means to assess the turnover rate of mtDNA. Four-day treatment with dideoxycytidine (ddC) (0.2 microM) or ethidium bromide (EtB) (0.25 microg/mL) reduced the mtDNA content approximately 80% in the human lymphoblastoid cell line, CEM. Concentrations of ddC ranging from 0.2 to 10 microM did not reduce mtDNA content in primary cultures of rat cortical neurons. Similarly, treatment with EtB (0.1, 0.25, and 0.5 microg/mL) did not affect significantly neuronal mtDNA. EtB (0.25 microg/mL) was effective in reducing mtDNA content in the undifferentiated embryonic carcinoma cell line, P 19. However, once P 19 cells were differentiated into a neuronal phenotype, they became insensitive to inhibition of mtDNA synthesis by EtB. Thus, cultured rat cortical neurons were less sensitive to mtDNA synthesis inhibitors than cell lines, suggesting that the turnover of mtDNA in central neurons is very slow. This may protect central neurons from accumulating mutations during the replication of mtDNA.

Animals↗

PubMed1995

alpha-Ketoacids scavenge H2O2 in vitro and in vivo and reduce menadione-induced DNA injury and cytotoxicity.

We demonstrate that alpha-ketoacids reduce and, in some instances, abrogate menadione-induced DNA damage and cytotoxicity in the human breast cancer cell line, MCF7. We confirm that alpha-ketoacids quench the copious amounts of H2O2 generated by menadione while these alpha-ketoacids undergo nonenzymatic oxidative decarboxylation; our data thus support enhanced H2O2 production as an important pathway for menadione-induced DNA damage and cytotoxicity. We also demonstrate that alpha-ketoacids scavenge H2O2 generated by mitochondria and microsomes when these organelles are exposed to menadione; additionally, alpha-ketoacids protect oxidant-vulnerable enzymes against functional impairment induced by H2O2. Finally, we provide the first in vivo demonstration that acute elevations in concentrations of alpha-ketoacids in rat tissues and urine scavenge H2O2. We conclude that enhanced H2O2 production is a major pathway for menadione-induced DNA damage and cytotoxicity and that the diverse alpha-ketoacids present within the cell must be considered, along with glutathione peroxidase and catalase, as part of the intracellular antioxidant defense mechanisms that regulate the ambient levels of H2O2.

Animals↗

PubMed1994

Status of glutathione and glutathione-metabolizing enzymes in menadione-resistant human cancer cells.

Cloned menadione (MD)-resistant human breast cancer cell lines have been developed and characterized with respect to glutathione (GSH) content and GSH-metabolizing enzymes. Increases in the activities of gamma-glutamyltranspeptidase and glutathione-S-transferase were demonstrated in the absence of alterations in the GSH content of two cloned MD-resistant cell lines. The MD-resistant cells also displayed alterations in their growth kinetics, possessing longer doubling times and increased fractions in the G1/O phase of the cell cycle as compared to parental MD-sensitive cells. The possible mechanisms for the resistance to MD, including an increase in repair of MD-induced DNA damage, are discussed.

Cell Cycle↗

PubMed1994

2',3'-Dideoxycytidine alters calcium buffering in cultured dorsal root ganglion neurons.

Mitochondria play a prominent role in shaping intracellular calcium concentration ([Ca2+]i) transients in dorsal root ganglion neurons. Mitochondrial DNA polymerase is inhibited by antiviral compounds such as 2',3'-dideoxycytidine (ddC). Here, we test the hypothesis that ddC can alter mitochondrially mediated Ca2+ buffering in neurons. Chronic treatment of dorsal root ganglion cultures with ddC (1 microM) lowered mitochondrial DNA levels and decreased the mitochondrially mediated component of depolarization-induced [Ca2+]i transients. The inhibition increased in a time-dependent manner, reaching a maximum at 6 days. ddC did not affect small, action potential-evoked, [Ca2+]i transients that are predominantly buffered by Ca(2+)-ATPases, suggesting that ATP levels were not depleted. The drug did not inhibit whole-cell Ca2+ currents, indicating that the Ca2+ load was not affected. Thus, ddC produces a graded, time-dependent inhibition of mitochondrial function that is reflected, in part, by a decrease in the direct buffering of Ca2+ by mitochondria. This effect may contribute to the peripheral neuropathy that results from ddC treatment. Furthermore, ddC promises to be a useful tool to study the role of mitochondria in [Ca2+]i homeostasis and neurodegenerative processes.

Animals↗

PubMed1994

Heme oxygenase does not protect human cells against oxidant stress.

The induction of heme oxygenase in cells under conditions of oxidative stress has been hypothesized to represent a cellular antioxidant defense mechanism. The objectives of this study were to characterize the induction of heme oxygenase by the oxidant stress-inducing quinone agent menadione (2-methyl-1,4-naphthoquinone) and to elucidate the roles of basal and induced heme oxygenase enzyme activities in menadione-induced DNA damage and growth inhibition in human MCF-7 cells. Time- and dose-dependent inductions of heme oxygenase messenger RNA and enzyme activity in menadione-treated MCF-7 cells were demonstrated. Intracellular and extracellular bilirubin concentrations were less than 100 nmol/L and were not altered when heme oxygenase was induced. The roles of the basal and induced heme oxygenase enzyme activities in menadione-mediated DNA damage were evaluated by means of the heme oxygenase competitive inhibitor tin protoporphyrin. Inhibition of the basal heme oxygenase enzyme activity by tin protoporphyrin resulted in a decrease in the number of menadione-induced DNA breaks and an attenuation of the cellular growth inhibition caused by menadione. Induced heme oxygenase did not protect MCF-7 cells from menadione-induced DNA breaks. Basal heme oxygenase enzyme activities in two cloned menadione-resistant cell lines were significantly less than that measured in a menadione-sensitive parental MCF-7 cell line. Collectively, these data do not support a protective role for basal or induced heme oxygenase enzyme activities against oxidant stress-related DNA strand breakage or cytotoxic effects engendered by menadione in human cells.

Bilirubin↗

PubMed1992

DNA strand scission and free radical production in menadione-treated cells. Correlation with cytotoxicity and role of NADPH quinone acceptor oxidoreductase.

Menadione (MD; 2-methyl-1,4-naphthoquinone), a redox cycling quinone was shown to induce single (ss)- and double (ds)-strand DNA breaks in human MCF-7 cells. This DNA damage was mediated via the hydroxyl radical as evidenced by electron spin resonance spectroscopy (ESR) studies utilizing the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide. The free radical production and DNA damage were shown to play a role in MD cytotoxicity as revealed by the reversal of MD toxicity and inhibition of hydroxyl radical production by exogenously added catalase. The role of NADPH quinone acceptor oxidoreductase in the metabolism of MD was evaluated. Purified quinone acceptor oxidoreductase in combination with MD resulted in the production of significant levels of the hydroxyl radical as measured by ESR. Dicumarol, an inhibitor of quinone acceptor oxidoreductase, decreased the production of the hydroxyl radical and attenuated DNA strand breaks in MCF-7 cells treated with MD.

Catalase↗

PubMed1992

A microassay for heme oxygenase activity using thin-layer chromatography.

A sensitive and facile assay for heme oxygenase (HO) has been developed. The basis of the assay is the detection of [14C]bilirubin formation in a coupled enzyme assay involving HO and biliverdin reductase actions, respectively. Separation of substrate from product is accomplished by thin-layer chromatography with subsequent quantitation by liquid scintillation counting of radioactive material present on chromatograms. As little as 20 micrograms of total cellular protein derived from cells growing in a standard 25-cm2 culture flask is sufficient for detection of HO enzyme activity using this assay. The reaction is inhibited by tin-protoporphyrin (10 microM final concentration), a specific inhibitor of HO. The linearity of the enzyme reaction with respect to incubation time and amount of protein used was established. Comparison of the new HO assay with a spectrophotometric assay was made, and good agreement of the results from both methods was found. The assay described here should facilitate measurements of this important heme-degrading enzyme in tissue culture studies and cases where limited amounts of material are available.

Autoradiography↗

PubMed1991

Menadione-induced DNA damage in a human tumor cell line.

The nature and extent of menadione (MD)-induced DNA damage were explored using the human breast cancer cell line MCF-7. Concentration-dependent single-strand (ss) and double-strand (ds) DNA breaks were detected in MD-treated MCF-7 cells using the alkaline- and neutral-elution techniques, respectively. The repair of ss and ds DNA breaks was extensive but not complete after a 6-hr incubation in drug-free medium. Evidence was found for the production of DNA interstrand cross-links in MCF-7 cells treated with the bifunctional alkylating agent, mitomycin C, but not for cells treated with MD. Exposure of MCF-7 cells to etoposide (VP-16), mitoxantrone and camptothecin resulted in the detection of significant amounts of protein-linked DNA breaks, whereas none were found in MD-treated cells. These results support the proposition that MD-induced DNA damage is not likely to be mediated via topoisomerases, nor do significant amounts of protein-linked DNA form in MD-treated cells. Thus, MD serves as a good model for examination of the role of the quinone moiety in DNA damage in relation to redox cycling. Future studies directed at elucidation of the biochemical determinants mediating formation of reactive oxygen species effecting the MD-induced DNA damage are necessary and underway.

Breast Neoplasms↗

PubMed1991

Characterization of DNA damage induced by 3,4-estrone-o-quinone in human cells.

The DNA damage induced in a human breast cancer cell line treated with 1,5 (10)-estradiene-3,4,17-trione (3,4-estrone-o-quinone; 3,4-EQ) has been measured qualitatively and quantitatively. Single-strand (ss) but not double-strand (ds) DNA breaks were formed in MCF-7 cells treated with 3,4-EQ. The ss DNA breaks formed in MCF-7 cells were partially repaired after incubation of cells in 3,4-EQ-free media for 2 and 4 h (i.e. 33 and 23% repair, respectively, as compared to the ss DNA breaks in cells after a 1-h exposure to 3,4-EQ without a recovery period). The formation of interstrand DNA cross-links was demonstrated in MCF-7 cells exposed to the bifunctional alkylating agent, mitomycin C, but not in those exposed to 3,4-EQ. Protein-linked DNA breaks were detected in MCF-7 cells after exposure to camptothecin and etoposide but not 3,4-EQ, suggesting that the ss DNA breaks induced by 3,4-EQ are unlikely to be mediated via topoisomerases. The induction of ss DNA breaks was detected in the estrogen receptor-negative cell line, BT-20, after exposure to 3,4-EQ. Furthermore, excess estradiol in culture media did not prevent 3,4-EQ-induced ss DNA breaks, suggesting that the DNA damage was not mediated via the estrogen receptor. Evaluation of the newly synthesized quinone analogue, 5,6,7,8-tetrahydro-1-2-naphthoquinone, in the ss DNA breakage assay revealed that the A and B ring moiety of 3,4-EQ is sufficient to produce ss DNA breaks in MCF-7 cells.

Breast Neoplasms↗

PubMed1991

Menadione: spectrum of anticancer activity and effects on nucleotide metabolism in human neoplastic cell lines.

The spectrum of cytotoxicity of menadione (MD) was examined in a panel of human cancer cell lines. MD was equipotent against multidrug-resistant and parental leukemia cell lines with IC50 values of 13.5 +/- 3.6 and 18 +/- 2.4 microM respectively. A cervical carcinoma cell line resistant to the antimetabolite, methotrexate (MTX), was as sensitive to MD as its parental cell line. The interactions of fifteen clinically utilized anticancer drugs with MD were examined in vitro and the majority were found to be additive, with four agents exhibiting synergism and one agent exhibiting antagonism. MD inhibited the incorporation of radioactive thymidine, uridine and amino acids into DNA, RNA and protein, respectively, in three human cancer cell lines. Some possible reasons for the inhibition of DNA synthesis including effects of MD on intracellular deoxyribonucleoside triphosphate pools were examined and ruled out. Although results from previous studies using rat hepatocytes suggested that mitochondria may be a target of MD, no significant effect of this compound on total intracellular adenosine triphosphate (ATP) pools in human cancer cell lines was observed. Collectively, these in vitro results demonstrate that MD possesses a broad spectrum of anticancer activity and suggest the potential utility of this agent in cancer therapy. Future studies directed at elucidation of the mechanism of MD action in human cancer cells are warranted and are under study.

Antineoplastic Agents↗

PubMed1989

Enhancement of herpes simplex virus type 2 (HSV-2) DNA synthesis in infected cells that constitutively express the BglII-N region of the HSV-2 genome.

The BglII-N fragment of the herpes simplex virus type-2 (HSV-2) genome encodes one of two known transforming regions of this DNA virus. In this study, we report the derivation of HeLa S3 cells (2DC4) that stably express the HSV-2 BglII-N region, including the small subunit of HSV-2 ribonucleotide reductase (RR). Superinfection of the 2DC4 cells with wild-type HSV-2 resulted in the efficient induction of HSV-2-encoded ICP10, DNA polymerase, and thymidine kinase. The amount of HSV-2 DNA synthesis in 8-hr HSV-2-infected 2DC4 cells was enhanced 2.6 +/- 0.6-fold relative to infected control cells. Furthermore, the replication kinetics of HSV-2 DNA in 2DC4 cells were accelerated relative to HeLa S3 cells; HSV-2 DNA synthesis was detectable as early as 3 hr postinfection in 2DC4 cells as compared to 6 hr postinfection in HeLa S3 cells. These results suggest that the BglII-N region of HSV-2 encodes function(s) that activate the viral DNA synthesis apparatus and that this activation could relate to the transforming ability of this DNA region.

Base Sequence↗

PubMed1987

Induction of virus enzymes by phorbol esters and n-butyrate in Epstein-Barr virus genome-carrying Raji cells.

Phorbol esters and n-butyrate (SB) together could induce Epstein-Barr virus (EBV) DNA polymerase and DNase activities in Raji cells (virus nonproducer). Neither 12-O-tetradecanoylphorbol-13-acetate (TPA) nor SB alone could induce these EBV enzyme activities, transcription of the EcoRI C-region or other EBV proteins in Raji cells. The enzyme induction caused by exposure of Raji cells to TPA-SB was the result of the synthesis of virus-specified RNA, and the increase of linear EBV DNA content in Raji cells caused by TPA alone was not sufficient for induction of EBV-enzyme activities. Temporal characteristics of the TPA-SB induction process, but not the phorbol 12,13-dibutyrate-SB induction process, in Raji cells were observed; a critical phase (10-24 h) postphorbol ester treatment in phorbol 12,13-dibutyrate-SB-treated Raji cells which was responsible for the synthesis of virus RNA and enzymes was found. Phospholipase C, which increases intracellular diacylglycerols (and subsequently activates protein kinase C) was able to partially substitute for TPA in the TPA-SB induction for EBV polymerase and DNase activities. Sphingosine, a protein kinase C inhibitor, partially prevented the induction of virus enzyme activities in Raji cells treated with phorbol 12,13-dibutyrate and SB. No apparent changes in the methylation state of EBV DNA (EcoRI C region) were observed when Raji cells were treated with SB and TPA, alone or in combination. These results suggest that induction of EBV polymerase and DNase activities by TPA-SB may involve protein kinase C activation and another factor triggered by SB which together increase transcription of EBV DNA.

Burkitt Lymphoma↗

PubMed1987

Study of ribonucleotide reductase in cells infected with six clinical isolates of herpes simplex virus type 2 (HSV-2) with mutations in its larger subunit.

Herpes simplex virus type 2 (HSV-2) induces a novel ribonucleotide reductase (RR) composed of two subunits (140 and 38 kDa) in infected cells. Other investigators have developed a monoclonal antibody, A6, against the 140-kDa subunit of RR and have found, in about 1% of the cases, an inability to detect this protein in cells infected with clinical isolates of HSV-2. We therefore investigated whether in such cases the clinical isolates were capable of inducing viral RR activity and whether the lack of detection of the 140-kDa protein by the monoclonal antibody was due to an alteration in the antigenic site of this protein. Six such isolates were examined and were found to induce RR activity, similar to HSV-2 (strain 333) RR, which did not require ATP for CDP reduction. Western blot analyses using A6 failed to detect the protein. However, R1, a polyclonal antibody raised against viral RR was capable of detecting this subunit. In addition, R1 was also capable of neutralizing RR activity induced by all the isolates and HSV-2 (strain 333). In conclusion, the lack of detection of the large subunit of RR was not due to the lack of induction but was due to an alteration in the antigenic site recognized by A6; this alteration did not appear to affect the properties of the induced RR activity.

Animals↗

PubMed1987

Demonstration of viral thymidine kinase inhibitor and its effect on deoxynucleotide metabolism in cells infected with herpes simplex virus.

The thymidine analog 5'-ethynylthymidine was a potent inhibitor of herpes simplex virus type 1 (strain KOS)-induced thymidine kinase with a Ki value of 0.09 microM. 5'-Ethynylthymidine was less inhibitory against herpes simplex virus type 2 (strain 333)-induced thymidine kinase with a Ki of 0.38 microM and showed no inhibition against human cytosolic thymidine kinase under the conditions tested. The compound was effective against the altered thymidine kinase induced by acyclovir- and bromovinyldeoxyuridine-resistant virus variants. At 100 microM 5'-ethynylthymidine, the cellular pool size of dTTP in herpes simplex virus type 1-infected cells was 5% that of infected cells receiving no drug treatment, while there was no significant effect on the pool sizes of dATP, dGTP, and dCTP. There was a positive correlation between dTTP pools and the intracellular thymidine kinase activity of herpes simplex virus type 1-infected cells. When tested alone, 5'-ethynylthymidine exhibited no antiviral activity, but it antagonized the antiviral efficacy of five compounds which require viral thymidine kinase for their action.

Antiviral Agents↗