Biomedical subjects

A M Rauth

Publications and source records attributed to A M Rauth.

At least 19 recordsLinked to original sources

PubMed2003

Accumulation of technetium-99m glucarate: in vitro cell cultures and in vivo tumour models.

99mTc-glucarate is an investigational radiopharmaceutical which has been shown to accumulate in acute cerebral and myocardial injuries and in some tumours. In the present work, a survey of possible factors affecting the cellular accumulation of 99mTc-glucarate was carried out in cell lines and strains in vitro and in murine tumours in vivo. Accumulation was enhanced under hypoxic conditions in 12 of the 16 human and murine cell lines and strains studied, and inhibited in the presence of nitroimidazoles. At temperatures lower than 37 degrees C, accumulation was reduced, but a hypoxic/aerobic differential was maintained. Aerobic accumulation of 99mTc-glucarate was enhanced by cyanide. In transplanted tumours in mice, 99mTc-glucarate showed high tumour/muscle and tumour/blood ratios at early times after injection. Pharmacological enhancement of the extent of hypoxia by the administration of hydralazine or nitro-L-arginine resulted in significantly increased accumulation of 99mTc-glucarate in the tumour. The in vitro and in vivo properties of 99mTc-glucarate suggest that it may be useful for tumour imaging in the clinic, although the exact mechanism(s) by which it localizes in tumours remains unknown.

Animals↗

PubMed2002

Expression of the prodrug-activating enzyme DT-diaphorase via Ad5 delivery to human colon carcinoma cells in vitro.

Intratumoral injection of recombinant adenoviral type 5 (Ad5) vectors that carry prodrug-activating enzymes like DT-diaphorase (DTD) could be used to selectively target tumor cells for chemotherapy. To demonstrate the feasibility of this approach, Ad5 vectors were constructed, which express human DTD minigenes for both wild-type and mutant (C-to-T change in nucleotide 609 in DTD cDNA) DTD under the control of the cytomegalovirus (CMV) promoter. HT29 human colon carcinoma cells express wild-type DTD, whereas BE human colon carcinoma cells express mutant DTD, have low to undetectable DTD activity, and are 4- to 6-fold more resistant to mitomycin C (MMC) than HT29 cells. A test of the ability of Ad5 to infect these cells (using a beta-galactosidase CMV-driven minigene) indicated that 90-100% of BE cells were infected at a multiplicity of infection (MOI) of 100, whereas only 15-40% of HT29 cells were infected at this MOI. Infection of BE cells in vitro with recombinant Ad5 carrying a minigene for wild-type DTD at MOIs of 3-100 resulted in a progressive increase in DTD activity and a maximal 8-fold increase in sensitivity to MMC as measured by a colony-forming assay. HT29 cells were sensitized 2- to 3-fold following treatment with Ad5.DTD at an MOI of 100. These results indicate that adenovirus-mediated gene transfer and expression of wild-type DTD can sensitize resistant tumor cells to MMC and that this therapeutic strategy may exert a significant bystander effect.

Adenoviridae↗

PubMed2001

A study of doxorubicin loading onto and release from sulfopropyl dextran ion-exchange microspheres.

The objective of this study was to investigate various factors that influence doxorubicin (Dox) loading onto and release from sulfopropyl dextran ion-exchange microspheres (MS), and to evaluate the anticancer activity of the released drug in vitro. Dox was incorporated into the MS by incubating the MS with aqueous solutions of Dox at room temperature. The drug release was carried out at 37 degrees C in aqueous solutions containing NaCl with or without CaCl2. The kinetics of drug absorption and release, the amount of Dox released, and the stability of Dox after loading, freeze-drying, and release were determined by spectrophotometry. The cytotoxicity of Dox (the original drug or that released from MS) against murine EMT6 breast cancer cells was assessed using a clonogenic assay. An increase in the MS to drug ratio resulted in a higher absorption rate and a higher fraction of the drug extracted from the solution. The release rate and the equilibrium fraction of Dox released increased with a decrease in the initial amount of Dox loaded or an increase in the salt concentration. The addition of divalent ions (Ca2+) promoted drug release compared to NaCl alone. The percent loss of colony forming ability of the cells, a measure of cytotoxicity of the released Dox, was the same as parent Dox solutions, indicating that the drug bioactivity was fully preserved after the drug loading and release cycle. This work demonstrated that various drug release rates were achieved by varying the drug loading and that the MS-delivered Dox was effective against the cancer cells in vitro.

Animals↗

PubMed2001

Cellular accumulation and retention of the technetium-99m-labelled hypoxia markers BRU59-21 and butylene amine oxime.

BRU59-21 and 99mTc-butylene amine oxime (BnAO, HL91) are being evaluated for imaging hypoxia in tumors. Both tracers: 1) rapidly reached a plateau in aerobic Chinese hamster ovary cells in vitro but continuously accumulated in hypoxic cells; 2) ceased to accumulate when hypoxic cells were exposed to air; 3) showed approximately 40% retention upon washing the cells; 4) showed selective hypoxic accumulation only at 37 degrees C; 5) accumulation could be modulated by addition of electron-affinic compounds; and 6) exhibited higher accumulation in cells which overexpress cytochrome P450 reductase. Both BRU59-21 and 99mTc-BnAO share properties making them suitable for hypoxia imaging.

Animals↗

PubMed2000

Role of NADPH:cytochrome P450 reductase in the hypoxic accumulation and metabolism of BRU59-21, a technetium-99m-nitroimidazole for imaging tumor hypoxia.

Nitroimidazoles labeled with technetium-99m are being investigated as non-invasive markers of tumor hypoxia. They are bioreductive compounds that require enzymatic reduction for retention in hypoxic cells, but little is known about the cellular factors affecting their accumulation in hypoxic cells. If the absolute accumulation of hypoxia markers is affected by enzyme levels, an inaccurate assessment of the hypoxic cell fraction in tumors may occur. BRU59-21, (99m)Tc-oxo[[3,3,9, 9-tetramethyl-6-[(2-nitro-1H-imidazol-1-yl)methyl]5-oxa-4, 8-diazadioximato]-(3-)-N,N',N",N"'] technetium (V), a technetium-99m-nitroimidazole that is being studied as a potential marker of tumor hypoxia, was used in the present study to evaluate the effect of NADPH:cytochrome P450 reductase (EC 1.6.2.4) levels on BRU59-21 accumulation and metabolism. Metabolism of BRU59-21 in hypoxic cellular lysates derived from Chinese hamster ovary cells overexpressing NADPH:cytochrome P450 reductase was 8-fold greater than in control cells. This effect required the presence of exogenous NADPH. The increased metabolism of BRU59-21 in lysates overexpressing NADPH:cytochrome P450 reductase was inhibited at 4 degrees and by the addition of NADPH:cytochrome P450 reductase inhibitors. The addition of inhibitors of other nitroreductase enzymes had no effect on BRU59-21 metabolism in these lysates. When the accumulation and metabolism of BRU59-21 were studied in stirred suspension cultures, it was found that cells overexpressing NADPH:cytochrome P450 reductase exhibited about a 3-fold increase in both the hypoxic metabolism and the accumulation of BRU59-21. These findings suggest that NADPH:cytochrome P450 reductase is an important enzyme in BRU59-21 metabolism in model systems of tumor hypoxia.

Animals↗

PubMed2000

Comparison of the accumulation and efflux kinetics of technetium-99m sestamibi and technetium-99m tetrofosmin in an MRP-expressing tumour cell line.

The potential clinical use of technetium-99m labeled sestamibi (Tc-MIBI) and tetrofosmin (Tc-Tfos) to image tumours is currently being evaluated. In this study. the accumulation and efflux of Tc-MIBI and Tc-Tfos in the nasopharyngeal carcinoma cell line CNE-1 were examined in the presence or absence of various inhibitors of P-glycoprotein (PGP) and/or multidrug resistance associated protein (MRP) activity [GG918, PSC833, verapamil (Vrp), cyclosporin A (CsA) and buthionine sulfoximine (BSO)]. Reverse-transcriptase polymerase chain reaction analysis and immunodetection of the CNE-1 cells detected expression of MRP, MRPI and MRP2 but not PGP. Tc-MIBI and Tc-Tfos accumulation was increased (P < 0.0001) and efflux decreased (P < 0.05) in the presence of BSO, CsA, Vrp and PSC833 but not GG918, which is a specific inhibitor of PGP. The absolute accumulation of Tc-MIBI was approximately twofold higher than that seen with Tc-Tfos, whereas the addition of inhibitors caused a much greater suppression of Tc-Tfos transport (>2 times greater than for Tc-MIBI). However, no qualitative differences in inhibitors were seen between Tc-MIBI and Tc-Tfos. These results suggest that both Tc-MIBI and Tc-Tfos are substrates for the MRP transporter and that PSC833, Vrp, CsA and BSO but not GG918 can inhibit MRP activity. These results indicate that Tc-MIBI and Tc-Tfos may be suitable imaging agents for detecting MRP-mediated drug resistance in human cancers.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2000

Assessment of the relationship between genotypic status of a DT-diaphorase point mutation and enzymatic activity.

DT-diaphorase, a cytosolic reductase, has been implicated as an activator of chemotherapeutic prodrugs and a detoxifier of certain potentially carcinogenic xenobiotics. A common C to T nucleotide 609 substitution in DT-diaphorase cDNA has been associated with protein instability and reduced catalytic activity. The degree to which the allelic status of the substitution correlates with enzymatic activity was assessed in 45 normal human skin fibroblast strains using a PCR-RFLP assay. Included in this study was the 3437T strain, which is unique in that it is heterozygous for the polymorphism yet contains undetectable enzymatic activity. An allele-specific RT-PCR-RFLP technique attributed this phenomenon to exclusive DT-diaphorase mRNA expression from the variant allele. Overlap in activities was observed between individual strains homozygous for the wild-type allele and heterozygotes, but the former group displayed enzymatic activity that was on average 2-fold higher. Western blot analysis of the two strains in this panel that are homozygous for the variant allele revealed that they express relatively low amounts of DT-diaphorase protein, consistent with the role of the substitution in protein instability. This work confirms that genotypic status is a reliable initial estimate of DT-diaphorase activity.

Adolescent↗

PubMed2000

BRU59-21, a second-generation 99mTc-labeled 2-nitroimidazole for imaging hypoxia in tumors.

UNLABELLED: Hypoxia in tumors is believed to be an important cause of local failure of radiotherapy in certain types of cancer. BRU59-21 (BMS194796) is a second-generation 99mTc-labeled 2-nitroimidazole that has been shown to offer improved characteristics for imaging myocardial ischemia. It has now been evaluated in models of tumor hypoxia. METHODS: Accumulation of BRU59-21 was compared with that of BMS181321 in Chinese hamster ovary cells incubated under aerobic or hypoxic conditions. The effects of competition with unlabeled nitroimidazoles and oxygen were studied. Biodistribution studies were performed in mice bearing transplanted KHT-C tumors in the leg. RESULTS: Within 5 min, BRU59-21 partitioned into aerobic cells in vitro at a level 10 times higher than external medium with no further increase over time. In hypoxic cells this initial partitioning was followed by selective accumulation to levels 5 times higher than in aerobic cells by 4 h. Low levels of oxygen (approximately 40 ppm) inhibited the maximal accumulation rate by 50%. Unlabeled misonidazole, a 2-nitroimidazole, inhibited accumulation of radioactivity, whereas tinidazole, a 5-nitroimidazole, enhanced accumulation; similar effects had been reported with BMS181321. Biodistribution studies in mice showed rapid clearance of radioactivity from the blood, resulting in enhanced tumor-to-blood ratios compared with BMS181321. Increasing the hypoxic fraction in the tumor by injection of nitro-L-arginine resulted in increased retention of tracer in the tumor without affecting other tissues. CONCLUSION: These results suggest that BRU59-21 warrants further investigation as an agent for imaging tumor hypoxia in the clinic.

Animals↗

PubMed1998

Studies of 99mTc-BnAO (HL-91): a non-nitroaromatic compound for hypoxic cell detection.

PURPOSE: Solid tumours of similar type and stage can vary widely in their hypoxic cell fraction. Such cells may be prognostic for aggressive, metastatic, and radiation-resistant disease. A 99mtechnetium (99mTc)-labelled non-nitroaromatic agent, butyleneamine oxime (99mTc-BnAO) or HL-91 (Amersham International, Inc., Amersham, UK) has been evaluated both in vitro and in vivo for its possible efficacy as a noninvasive marker for the clinical detection of hypoxic cells in solid tumours. MATERIALS AND METHODS: Suspension cultures of Chinese hamster ovary (CHO) cells under controlled levels of oxygen were used to measure the oxygen dependency of 99mTc-BnAO accumulation. V79 cells grown as multilayers on a semipermeable membrane served as an in vitro model for drug penetration through the extravascular space of the tumour. C3H mice bearing KHT-C leg tumours were the in vivo models for selective drug accumulation as a function of time after i.v. administration of 99mTc-BnAO. RESULTS: 99mTc accumulated selectively in hypoxic vs. aerobic cells, resulting in a 9 +/- 2-fold differential in radioactivity per cell at 4 h. The k(m) for this selective accumulation was 20 ppm of oxygen. The labelled drug was equally effective in penetrating the cellular multilayer under aerobic or hypoxic conditions. In vivo measurements indicated favourable labelling of solid tumours containing hypoxic cells with 1% of the total activity per g of tumour, a tumour-to-blood ratio of 1.2, and a tumour-to-muscle ratio of 4.6 at 4 to 6 h after drug administration. In contrast to more lipophilic 99mTc- labelled compounds, excretion was primarily via the urinary tract. Nitro-L-arginine selectively increased solid tumour labelling over normal tissue. CONCLUSIONS: 99mTc-BnAO or HL-91 is a promising agent for clinical studies of tumour hypoxia, although the mechanism of its selective hypoxic cell accumulation remains unexplained.

Animals↗

PubMed1998

Bioreductive therapies: an overview of drugs and their mechanisms of action.

PURPOSE: Bioreductively activated drugs have been used as antimicrobials, chemotherapeutic agents, and radiation sensitizers. The present paper is an overview of their mechanism of action and application in the treatment of cancer. MATERIALS AND METHODS: Drugs such as nitroimidazoles, mitomycins, and benzotriazine di-N-oxides were a focus of this research. Studies have ranged from the chemistry of the reductive process of activation to in vitro and in vivo studies in rodent and human cells, through to clinical testing. The variety of techniques and test systems brought to bear on these compounds is a strength of this field of research. RESULTS: A detailed chemical understanding of the mechanism of action of a variety of bioreductives is now available. The enzymatic processes by which these drugs are activated and the cofactors involved in this activation are becoming well understood. Recent advances have been made in the design and use of dual-function bioreductives, bioreductive triggers of drug activation, and DNA-targeted bioreductives. Significant success has been demonstrated clinically with bioreductive drugs, used in combination with radiation and front-line chemotherapeutic agents. The areas of antibody-directed enzyme prodrug therapy (ADEPT) and gene-directed enzyme prodrug therapy (GDEPT) are identified as new directions for bioreductive therapy. CONCLUSION: The use of bioreductively-activated drugs for the treatment of cancer has made steady progress. The success obtained clinically and the new molecular approaches currently being implemented promise significant advances in the future.

Animals↗

PubMed1998

Role of a DT-diaphorase mutation in the response of anal canal carcinoma to radiation, 5-fluorouracil, and mitomycin C.

PURPOSE: To determine, retrospectively, the status of the bp 609 mutation in the DT-diaphorase gene in anal canal carcinoma patients who have undergone radical radiotherapy with concurrent 5-fluorouracil (5-FU) and mitomycin C (MMC), to determine the relationship of the mutant form of the gene to treatment outcomes. METHODS AND MATERIALS: Paraffin blocks of pretreatment tumor biopsies were obtained on 49 patients who underwent treatment with curative intent using radiation, infusional 5-FU and bolus MMC from January 1991 to December 1993. DNA was extracted and subjected to polymerase chain reaction (PCR) analysis using primers that encompassed the bp 609 C to T mutation. Restriction endonuclease cleavage with Hinf 1 and gel electrophoresis were used to determine the polymorphism status of each patient. RESULTS: DNA of 46 patients was successfully amplified. The 46 patients were distributed as follows: 26 (56.5%) C/C-homozygous wildtype, 18 (39%) T/C-heterozygous, and 2 (4.5%) T/T-homozygous mutant. Eleven of 46 patients had suffered treatment failure. The status of the bp 609 polymorphism in this group was 5 (45.5%) C/C, 5 (45.5%) C/T, and 1 (9%) T/T. CONCLUSION: In this series, there was not an overrepresentation of the mutant allele in patients with treatment failure, suggesting that the bp 609 alteration is not a strong determinant of treatment outcome.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1998

Oxidative stress and 1-methyl-2-nitroimidazole cytotoxicity.

The 2-nitroimidazoles have been used clinically to radiosensitize resistant hypoxic cells, but a dose-limiting peripheral neuropathy has restricted their therapeutic effectiveness. A model compound, 1-methyl-2-nitroimidazole (INO2), was used to investigate the possible role of oxidative stress in this normal tissue toxicity. Chinese hamster ovary (CHO) cells were 10-15 times more resistant to 20 mM INO2 under aerobic than hypoxic conditions. In comparison, a pair of transformed rat embryo fibroblasts (ER17-1wtp53 and ER12L5mtpP53), differing in their p53 genotype, were approximately 3- to 4-fold more sensitive than Chinese hamster ovary cells to INO2 under aerobic conditions, but had the same sensitivity as Chinese hamster ovary cells under hypoxic conditions. These results are consistent with an earlier hypothesis that the mechanism of aerobic toxicity is different from that of hypoxic toxicity (nitroreduction) and show that neither toxicity is dependent on cellular p53 status. There was an increase in the production of reactive oxygen intermediates and a decrease in the antioxidant glutathione following aerobic exposure to INO2, which correlated with cell survival in all three cell lines. No evidence of reductive adducts of the 2-nitroimidazole 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetam ide (EF5) was found by immunofluorescent techniques in aerobic cells. Differing activities of the antioxidant enzymes superoxide dismutase and catalase could be correlated with INO2 aerobic cytotoxicity. DNA strand breaks, as measured by the comet assay, paralleled the appearance of INO2 aerobic cytotoxicity in all three cell lines. Taken together, these results strongly support the conclusion that the aerobic toxicity of IN02 is due to active oxygen species created by futile redox cycling of the parent compound.

Aerobiosis↗

PubMed1998

Prostate-specific antigen induction by a steroid hormone in T47D cells growing in SCID mice.

Previous studies revealed that prostate-specific antigen (PSA) is present in > 30% of human breast tumor cytosols. Survival analysis showed that patients with PSA-producing tumors have a reduced risk for relapse, suggesting PSA to be an independent favorable prognostic marker for a large subset of breast cancer patients. The present investigation established an in vivo model for the induction of PSA in human breast cancer tumors growing as xenografts in severe combined immunodeficient (SCID) mice. The human mammary cancer cell-line T47D was grown i.m. in female mice. When the tumor and leg diameter reached 10 mm, the mice were stimulated daily with norgestrel for either 5 or 7 days to produce PSA, and sacrificed on day 8. The prostate cancer cell-line LNCaP was grown in male mice and functioned as a positive control for PSA production. After T47D and LNCaP mice were sacrificed, a highly sensitive immunofluorometric assay was used to analyze the PSA concentration in the tumor, muscle, liver, and kidney cytosols. Norgestrel-stimulated T47D mice showed significantly more PSA in the tumors compared to tumors of the control mice. However, PSA levels in tumors of the stimulated mice were significantly lower than those in the LNCaP xenografts. No PSA levels above background were present in the blood and normal tissue of the norgestrel-stimulated or control T47D xenografts. This mouse model will be a valuable tool for investigating and screening new therapies for a subgroup of breast cancer patients who have significant PSA concentrations in their tumors.

Animals↗

PubMed1998

Transfection of COS-1 cells with DT-diaphorase cDNA: role of a base change at position 609.

DT-diaphorase, a homodimeric flavoenzyme, can provide for a defence mechanism against carcinogenesis mediated by dietary or environmental quinones as well as bioactivate quinone-containing chemotherapeutic drugs. Human cell lines and strains have been identified with very low or undetectable enzymatic activity and a C to T transition at nucleotide 609 of the DT-diaphorase cDNA. This single base change is predicted to result in a proline to serine change in amino acid 187. Human cells homozygous for this base transition fail to exhibit Western blot reactivity for DT-diaphorase, suggesting that this substitution results in protein instability. To directly test whether this base change affects DT-diaphorase enzymatic activity and/or protein stability in vivo, mammalian expression vectors containing DT-diaphorase cDNA with or without the nucleotide 609 base transition were transiently transfected in COS-1 cells. Co-transfection with a human growth hormone expression vector allowed normalization for transfection efficiency. COS-1 transfectants expressing the C to T base change displayed at least a tenfold reduction in DT-diaphorase activity (P < 0.001) and a two- to threefold reduction in protein levels compared with wild-type transfectants. These results are the first to detect the presence of DT-diaphorase protein coded for by the 609 base transition in mammalian cells and confirm its predicted reduced enzymatic activity.

Animals↗

PubMed1997

Modifying the in vitro accumulation of BMS181321, a technetium-99m-nitroimidazole, with unlabelled nitroaromatics.

BMS181321, [99mTc]oxo[[3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazol-1-yl)-4,8-diaz aundecane-2,10-dione dioximato]-(3)-N,N',N",N"']technetium, is a 99mTc-nitroimidazole that is being investigated as a hypoxic marker in tumors. Due to the high specific activity of 99mTc, the concentration of BMS181321 used in its applications is very low. Metabolic depletion and non-specific binding of the drug may limit its ability to fully map out hypoxic regions. An attempt has been made to modify the in vitro accumulation of BMS181321 in hypoxic Chinese hamster ovary (CHO) cells with unlabelled nitroaromatics. The 2-nitroimidazole etanidazole (0.08 to 8 mM) caused a concentration-dependent decrease in BMS181321 accumulation to 70-28% and metabolism to 70-40% of the control level in hypoxic cells at 4 hr. In contrast, the 5-nitroimidazole tinidazole (0.09 to 9 mM) caused a concentration-dependent increase in BMS181321 accumulation to 110-170% and metabolism to 100-150% of the control level in hypoxic cells at 4 hr. Nitroaromatics with an electron affinity similar to or greater than that of BMS181321 inhibited its accumulation and metabolism, and 5-nitroimidazoles, which have an electron affinity lower than that of BMS181321, enhanced its accumulation and metabolism. The enhanced accumulation with the addition of metronidazole was not observed in the presence of low oxygen levels or of a nitrofuran of higher electron affinity than BMS181321. These results suggest that a competition for reducing equivalents and/or for the BMS181321 radical anion itself can occur in cells, leading to the inhibition of BMS181321 reduction in the presence of nitroaromatics of similar or greater electron affinity. A transfer of electrons from the radical anion form of the reduced 5-nitroimidazole to the more electron affinic BMS181321 compound may occur, causing increased hypoxic accumulation of BMS181321.

Animals↗

PubMed1997

Mechanism of ultrasound enhanced porphyrin cytotoxicity. Part I: A search for free radical effects.

Intense ultrasound beams may have the potential to treat malignant tumours when combined with sensitizers, often called sonodynamic agents. Some of these agents, e.g., the porphyrins, are currently used for photodynamic therapy. However, the experimental evidence for ultrasound activation of sensitizers is inconsistent. This paper attempts to discover whether they yield of free radicals such as .OH and .H, which are produced by transient cavitation, could explain the killing of Chinese hamster ovary (CHO) cells in vitro with and without sonodynamic agents. CHO cells were irradiated with ultrasound beams in phosphate-buffered saline or in growth medium, and the immediate cell lysis and loss of cell colony forming ability were measured. Under our specific conditions, in which the standing wave patterns were minimized, a general correlation was observed between the transient cavitation, free radical production, and cytotoxicity. However, the yield of free radicals was much too small to explain the cell killing observed. We conclude that cytotoxicity is not linked to attack from free radicals formed outside the cells. In our experiments, immediate cell lysis is closely linked to the transient cavitation, which is known to produce shear forces that disrupt cellular membranes. We hypothesize that the loss of cell colony forming ability is also linked to damage of cellular membranes.

Animals↗

PubMed1997

Apoptosis and 1-methyl-2-nitroimidazole toxicity in CHO cells.

The time course and characteristics of the selective hypoxic cytotoxicity of the 2-nitroimidazole model compound 1-methyl-2-nitroimidazole (INO2) were analysed during prolonged time periods (up to 5 days post treatment). When control populations were seeded at the same cell density as drug-treated cells, they entered confluency at day 3 and underwent apoptosis at day 5, which appeared to be mediated by an autocrine mechanism. In subsequent studies of drug-treated cells, the seeding density of treated cells was adjusted to avoid this cell confluency effect. Treatment with a low INO2 concentration (2.5 mM) resulted in apoptotic DNA fragmentation (ladders), which was observed 4-5 days after an acute 6-h hypoxic drug exposure. In contrast, at a high INO2 concentration (40 mM) for 2 h, which was equitoxic to the low concentration, no characteristic DNA ladders were observed. Fluorescence microscopy revealed apoptotic bodies and pyknotic nuclei 5 days following hypoxic 2.5 mM INO2 exposure, whereas 40 mM INO2 hypoxic treatment produced cellular ghosts devoid of DNA 5 days after exposure, consistent with the DNA ladder results. However, characteristic apoptotic morphology was previously observed immediately after the acute hypoxic exposure of 40 mM INO2. Cell cycle analysis and DNA fragmentation as measured by the TdT assay suggested that dose-dependent differences in the apoptotic response occur post exposure after an equitoxic acute hypoxic exposure to either the low or the high INO2 concentration. This dose-dependent differential in response may be attributed to the degree of initial DNA damage as measured by the comet assay.

Animals↗

PubMed1997

DT-diaphorase: possible roles in cancer chemotherapy and carcinogenesis.

DT-diaphorase [also called NAD(P)H:menadione oxidoreductase; NAD(P)H:(quinone acceptor) oxidoreductase; quinone reductase, azo-dye reductase] (EC.1.6.99.2) has been shown to have a role in activation of quinone-containing cancer chemotherapeutic prodrugs to their active form, as well as inactivation of a variety of xenobiotics involved in carcinogenesis. To illustrate the basis of this dichotomy, a brief review of the historical and recent background of studies on DT-diaphorase is given. Recent data from the laboratory of the authors on the nature of the protein coded for by a recently identified human polymorphism, a C to T transition at nucleotide 609 of DT-diaphorase cDNA, and the frequency of this mutation in anal canal carcinoma patients is presented. Finally, a series of questions about the role of DT-diaphorase in both normal and malignant cells is raised based on the results of others that have been published in the last 2-3 years.

Amino Acid Sequence↗