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Biomedical subjects

A M Rauth

Publications and source records attributed to A M Rauth.

At least 37 records · Page 2Linked to original sources
PubMed1996

Differential cell death in immortalized and non-immortalized cells at confluency.

Rat embryo fibroblast cells that have been immortalized by viral transfection have an altered cell cycle control. These cells have been observed to die via an apoptotic death when grown to confluency which, in at least one of the cell line pairs studied, was independent of the presence of wildtype or mutant p53. This confluency-dependent apoptotic cell death was observed in thirteen of fourteen rodent and human cell lines tested. In contrast, primary rodent and human cell strains (fibroblasts) entered a quiescent G1/G0 state at confluency. Two weeks later, these non-immortalized cells underwent necrosis, not apoptosis. As the medium was not replenished during this two week period, cell necrosis was probably due to deprivation of nutrients and growth factors. These results indicate that mechanisms of cell death may differ between transformed and non-transformed cells under physiological stressed situations, such as high cell density. It may be possible to exploit these differences to increase the efficacy of chemotherapeutic compounds towards neoplastic cells.

Animals↗
PubMed1996

In vitro and in vivo evaluation of a technetium-99m-labeled 2-nitroimidazole (BMS181321) as a marker of tumor hypoxia.

UNLABELLED: The 99mTc-labeled 2-nitroimidazole derivative BMS181321, previously studied in experimental models of myocardial and cerebral ischemia, has been evaluated in single-cell and tumor models. METHODS: Accumulation of BMS181321 was studied in aerobic and hypoxic (<10 PPM O2) suspension cultures of Chinese hamster ovary (CHO) cells at 37 degrees C and the oxygen dependency and stability of the accumulated radioactivity determined. Biodistribution studies of the tracer after intravenous injection in C3H mice bearing three different murine solid tumors were performed noninvasively using a gamma camera, as well as invasively by determining blood and tissue levels of radioactivity from 10 min to 24 hr after injection. RESULTS: Accumulation of BMS181321 in aerobic cells in vitro equilibrated within 5 min at a approximately 10-fold level over the external medium. Hypoxic cells showed a linear increase in radioactivity up to 4 hr for cell densities < or = 1 x 10(6)/ml. At higher cell densities (2-4 x 10(6)/ml) there was substantial depletion of radioactivity from the growth medium and increased alteration in the chemical state of the tracer that remained. Low O2 levels (approximately 40 ppm) inhibited the maximal accumulation rate by 50%. Approximately 30% of radioactivity accumulated under hypoxic conditions remained cell-associated after 24 hr. Following intravenous injection, the tracer rapidly distributed throughout the mouse and was predominately cleared through the hepatobiliary system. Blood levels of radioactivity cleared quickly and plateaued at approximately 4% of the total dose from 2-24 hr. Absolute uptake in the tumors was highest 10 min after injection, and the tumor-to-muscle activity ratios increased and plateaued from 4-8 hr at values of 3.5-4.0. Two drugs which affect blood flow and increase hypoxic cell fraction in these tumors, hydralizine and nitro-L-arginine, significantly increased levels of BMS181321 radioactivity over control levels with minimal effects on normal tissue retention. CONCLUSION: These results suggest BMS181321 or an analog of it will be a useful agent to investigate the status of hypoxia in solid tumors experimentally and potentially in the clinic.

Animals↗
PubMed1996

Immortalisation of a human diploid fibroblast cell strain: a DT-diaphorase paradox.

Transfection of a normal human diploid fibroblast cell strain, GM38, with a simian virus 40 (SV40) large T antigen containing plasmid, yielded an immortal cell line, G38-8X, which had a similar sensitivity as the parental cell strain to the quinone-containing chemotherapeutic agent mitomycin C (MMC), under both aerobic and hypoxic exposure conditions. The activity level of DT-diaphorase was similar in both the parental GM38 and G38-8X cells. Although DT-diaphorase could be detected by Western blot analysis, using two mouse anti-human monoclonal antibodies, in GM38 cells, it was not detected in the G38-8X cells. G38-8X cells have a slightly increased P450R activity (2-fold), and have elevated P-glycoprotein levels compared with the parental GM38 cell strain. The immortal G38-8X cell line is 2-fold more resistant to ionising radiation than the parental GM38 cell strain (D10 approximately 5 Gy). Although these SV40 large T antigen immortalised human diploid fibroblasts behaved similarly to their parental cell strain in terms of MMC sensitivity and DT-diaphorase activity, careful characterisation revealed that these cells had enhanced P-glycoprotein activity and had a decreased sensitivity to ionising radiation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

BMS181321 accumulation in rodent and human cells: the role of P-glycoprotein.

A 2-nitroimidazole with a side chain that contains technetium-99m as a chelate, BMS181321, is undergoing evaluation as an imaging agent for myocardial and cerebral ischaemia, as well as a diagnostic probe for hypoxic cells in solid tumours. Its accumulation in hypoxic and aerobic populations of three lines of Chinese hamster ovary cells of differing P-glycoprotein status, as well as one rat and two human cell lines has been determined. There was selective accumulation of BMS181321 in hypoxic vs aerobic cells. P-glycoprotein level was not a factor in this accumulation and hypoxic human cells accumulated BMS181321 more rapidly than the rodent cells. These results indicate P-glycoprotein levels in tumour cells will not confound the use of BMS181321 as a hypoxic cell marker.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Isolation and characterization of a cell line resistant to 5-[3-(2-nitro-1-imidazoyl)-propyl]-phenanthridinium bromide (2-NLP-3), a DNA-intercalating hypoxic cell radiosensitizer and cytotoxin.

A DNA-targeted hypoxic cell radiosensitizer and cytotoxin, 5-[3-(2-nitro-1-imidazoyl)-propyl]-phenanthridinium bromide (2-NLP-3), has been shown previously to have increased efficacy over untargeted analogues in vitro. To further study the mechanism of action of this compound, a cell line, CHO-1000, derived from Chinese hamster ovary (CHO) AA8-4 cells was isolated. This cell line is capable of continuously growing in a concentration of 2-NLP-3 approximately 10-fold greater than that tolerated by wild-type CHO cells. The resistance of CHO-1000 to 2-NLP-3 was compared with that of the P-glycoprotein overexpressing, multidrug resistant Chinese hamster cell line CHR-C5 (C5). The resistance of CHO-1000 cells to the acute toxic effects of 2-NLP-3 under both hypoxic and aerobic exposure conditions was intermediate to that of the sensitive CHO wild-type cells and the resistant C5 cells. A similar pattern was seen for the hypoxic cell radiosensitizing ability of 2-NLP-3. 2-NLP-3 produced significant depletion of glutathione under both hypoxic and aerobic conditions in all three cell lines studied, and the degree of depletion was correlated with drug toxicity. CHO-1000 and C5 cells were significantly more resistant to colchicine and doxorubicin compared with wild-type cells. The toxicity pattern of 2-NLP-3 and its comparison phenanthridinium ion, P3, was not the same for CHO-1000 cells compared with C5 cells. Verapamil was an effective agent for reversing the hypoxic resistance to 2-NLP-3 in both CHO-1000 and C5 cells, but only a partial reversal of aerobic resistance was observed in CHO-1000 cells. These results indicate that the resistant phenotype of CHO-1000 is mediated to some degree by P-glycoprotein expression, but that other as yet unidentified factors are also involved.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Presence of a heterozygous substitution and its relationship to DT-diaphorase activity.

A point mutation in the mRNA of NADP(H): quinone oxidoreductase 1 (NQO1, DT-diaphorase) is believed to be responsible for reduced enzyme activity in the adenocarcinoma BE cell line. The present study examined nine cultured human non-cancerous fibroblast cell strains, five of which were from members of a single cancer-prone family, which demonstrated widely varying activity levels of DT-diaphorase (41 - 3462 nmol min-1 mg-1 protein), to determine if genetic alteration of the NQO1 or NOQ2 gene was involved in determining enzyme activity. All cell strains expressed NQO1 and NQO2 mRNA as measured by a quantitative polymerase chain reaction amplification technique. No relationship was found between the level of mRNA expressed and the enzyme activity in the cells. Sequencing of the entire complementary DNA from the cell strains revealed only a single base substitution at nucleotide 609 in one allele encoding NQO1 in every cell strain from members of the cancer-prone family, except for one cell strain which expressed only the T at nucleotide 609 in both alleles. Subsequent examination of genomic DNA from 44 individuals revealed that this base substitution is present in approximately 50% of the population. The presence of the T at nucleotide 609 in the NQO1 locus does not appear to be directly causal for altered DT-diaphorase activity.

Adenocarcinoma↗
PubMed1994

Constitutive expression of P-glycoprotein as a determinant of loading with fluorescent calcium probes.

Determination of intracellular calcium levels in Chinese hamster ovary (CHO) cells using the fluorescent calcium probe indo-1AM was hindered by the low level of accumulation of indo-1 in these cells. CHO cells are known to express basal levels of the multidrug resistance efflux pump P-glycoprotein (P-gp). Rhodamine-123, which is a known substrate of P-gp, was used to confirm the presence of P-gp in CHO cells. Verapamil and cyclosporin (CsA), both inhibitors of P-gp, enhanced accumulation of indo-1 in these cells and therefore allowed for improved intracellular calcium measurements. P-gp overexpressing colchicine-resistant CHO cells (CHRC5) also displayed enhanced indo-1AM loading with P-gp inhibitors. Nondetectable levels of P-gp activity were found in wild-type CEM-CCRF cells (human T lymphoblasts), and these cells did not show any difference in indo-1AM loading in the presence or absence of P-gp inhibitors. Loading of a second calcium fluorescent probe fluo-3AM was improved in CHO cells by P-gp inhibition, whereas the structurally related pH probe BCECF-AM was minimally affected. Because low levels of P-gp may be expressed by a range of cell lines and normal tissues, it is suggested that this be considered if difficulties are encountered in loading fluorescent calcium probes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Mechanism of the selective hypoxic cytotoxicity of 1-methyl-2-nitroimidazole.

2-Nitroimidazoles were introduced into radiation therapy to test their ability to radiosensitize hypoxic cells in solid human tumours. In addition, they are selectively reduced in hypoxic cells to form reactive metabolites that may be effective cytotoxins. 1-Methyl-2-nitroimidazole (INO2) was investigated as a model compound to study the mechanism of selective bioreduction in hypoxic cells. Results demonstrated that INO2 was toxic under hypoxic conditions (tested via colony-forming assay) at concentrations where no toxicity was observed for aerobic cells. This selective hypoxic toxicity was a function of both concentration and time. The depletion of both glutathione and protein thiols occurred under hypoxic conditions and preceded a rise in intracellular calcium levels. Previous work with INO, the nitroso intermediate of INO2 reduction, also showed concentration-dependent cytotoxicity, and glutathione and protein thiol depletion, which was followed by an increase in intracellular calcium levels. The kinetics of cytotoxicity and cellular reactions were slower for the parent compound, INO2, as compared with the 2e- reductive metabolite, INO, reflecting the limited enzymatic production of the reactive intermediate in the INO2 experiments. Zeiosis (membrane blebbing) and chromatin condensation occurred shortly after treatment of cells with equitoxic concentrations of both INO2 (under hypoxic conditions) and INO (under aerobic conditions), suggesting that an apoptotic-like death mechanism may be involved. However, analysis of DNA isolated from both INO2- and INO-treated cells, up to 2 hr after treatment, did not reveal any nucleosomal fragmentation, another characteristic feature of cells undergoing apoptosis. The toxicity of high INO2 concentrations toward CHO cells is consistent with the production of an INO intermediate and has several features characteristic of an apoptotic mechanism of cell death.

Animals↗
PubMed1994

DNA-targeted 2-nitroimidazoles: in vitro and in vivo studies.

A series of compounds in which a 2-nitroimidazole is linked to a DNA intercalating phenanthridine moiety has been synthesised. Previously, three such compounds, termed nitroimidazole-linked phenanthridines or NLPs, were tested in vitro and showed a greatly enhanced molar efficiency as hypoxic cell radiosensitisers and cytotoxins compared with the untargeted 2-nitroimidazole, misonidazole. Since the cytoxicity of these compounds was shown to be inversely proportional to linker chain length while radiosensitising ability was dependent of it, compounds with five and six carbons in the chain were synthesised in an attempt to lower the toxicity of the drugs while increasing their ability to 'scan' DNA for target radicals. These compounds and a comparison series of n-alkylated phenathridinium ions have been characterised and evaluated in vitro using Chinese hamster ovary and V79 cells and their effects compared with misonidazole. Based on in vitro results, one member of the series was selected and evaluated in vitro using a V79 spheroid tumour model and in vivo using an SCCVII transplantable tumour system. These studies have demonstrated the potential utility of this class of compound.

Animals↗
PubMed1994

Radiosensitizing and cytotoxic properties of DNA targeted phenanthridine-linked nitroheterocycles of varying electron affinities.

2-Nitroimidazoles targeted to DNA via intercalation have previously been shown to be as much as 10-100 times more efficient on a molar basis than the untargeted nitroimidazole, misonidazole, in vitro as hypoxic cell selective radiosensitizers and cytotoxins based on extracellular concentrations. In this work the effect of varying the nitroaromatic group has been examined through the preparation of a DNA-targeted 4-nitroimidazole (4-MeNLP-3), a 5-nitroimidazole (5-NLP-3) and a 5-nitrofuran (FEP-2) linked to phenanthridinium ions. With the previously synthesized 2-nitroimidazoles, this provides a series of DNA targeted compounds of varying electron affinity as well as structure at the nitroaromatic position. The present series of compounds was tested for partition coefficient, DNA binding ability, reduction potentials and in vitro radiosensitizing and cytotoxic abilities. The 4-nitro compound displays good radiosensitizing ability, with an SER of approximately 2, despite its low reduction potential, -551 mV. However, hypoxia selective toxicity is lost at the lower reduction potentials presumably due to redox-independent mechanisms. The results obtained indicate that targeting such compounds to DNA diminishes the dependency of radiosensitizing and cytotoxic properties on reduction potential and may allow significant uncoupling of toxicity from radiosensitizing ability.

Animals↗
PubMed1993

Cellular approaches to bioreductive drug mechanisms.

Mitomycin C is being used as an adjunct to ionizing radiation in the treatment of some solid tumors. A rationale for this is that radioresistant hypoxic cells in solid tumors will have enhanced sensitivity to this bioreductively activated drug, compared to aerobic cells. The role of oxygen concentration and enzymatic drug reduction in bioreductive drug activation have been investigated. Techniques are reviewed for the in vitro determination of the oxygen concentration dependency of bioreductive drug activation. One of these techniques, an open cell suspension system using Chinese hamster ovary cells, is described. Results are shown that indicate that the oxygen concentration dependency of toxicity of mitomycin C and one of its analogues profiromycin, though qualitatively complementing the oxygen dependency of ionizing radiation toxicity, are not quantitatively optimal. Using a mitomycin C resistant human cell strain (3437T) from a cancer prone family, a possible role for DT-diaphorase, an oxygen insensitive 2-electron transfer enzyme, is suggested. A correlation between a low level of DT-diaphorase in 3437T cells and mitomycin C resistance under aerobic exposure conditions is seen. Under hypoxic exposure conditions this resistance is lost, suggesting 1-electron transfer enzymes control hypoxic cell bioreductive activation. An activation role for DT-diaphorase in mitomycin C toxicity in the treatment of solid tumors is contrasted to a potential detoxification role for the enzyme with other xenobiotics in the cancer prone family phenotype.

Animals↗
PubMed1993

Hyperresistance to 4-nitroquinoline 1-oxide cytotoxicity and reduced DNA damage formation in dermal fibroblast strains derived from five members of a cancer-prone family.

Dermal fibroblasts cultured from members of a family presenting multiple polyps and sarcomas were compared with fibroblast strains from unrelated healthy donors for sensitivity to killing by four genotoxic agents. Cells from the sister of the male proband (strain 3437T), mother (strain 3703T), two of his paternal aunts (3701T and 3704T) and one paternal uncle (3702T) displayed marked resistance (1.8 to 4.3 times greater than the normal mean) to 4-nitroquinoline 1-oxide (4NQO), a procarcinogen whose DNA-damaging properties encompass those of both far (254 nm) ultraviolet (UV) light and ionising radiation. These same 4NQO-resistant cells, however, responded normally to reproductive inactivation by UV light, 60Co gamma radiation or the alkylating agent methylnitrosourea, signifying that the abnormal resistance of these cells to 4NQO is not associated with aberrant DNA metabolism. In keeping with this conclusion, exposure to a given dose of 4NQO produced decreased amounts of DNA damage and stimulated lower levels of repair DNA synthesis in all five 4NQO-resistant strains than in normal controls. Moreover, exogenous radiolabelled 4NQO accumulated to a lesser extent in the 4NQO-resistant than in the normal fibroblasts. Cell sonicates of strains 3437T, 3701T and 3702T exhibited reduced capacities (40-60% of normal) to catalise the conversion of 4NQO to the proximate carcinogen 4-hydroxyaminoquinoline 1-oxide. However, the 4NQO-resistant strains 3703T and 3704T carried out 4NQO bioreduction at normal rates. Our data therefore indicate that enhanced resistance to 4NQO cytotoxicity in 3437T, 3701T and 3702T is a consequence of anomalies in both intracellular accumulation and enzymatic reduction of 4NQO, whereas 4NQO resistance in 3703T and 3704T appears to result solely from reduced intracellular drug accumulation.

4-Nitroquinoline-1-oxide↗
PubMed1993

Characterization of a set of Chinese hamster ovary variant cell lines demonstrating differing sensitivity to mitomycin C.

Three related Chinese hamster ovary (CHO) cell lines derived from CHO-K1R cells (MMC3-A2, 21-1 and G1B) previously shown to differ in their sensitivity to mitomycin C (MMC), were investigated in more detail to determine the factors controlling this sensitivity. A separately maintained wild type cell line (CHO-K1TOR) was included in this study for comparison. Continuous (chronic) exposure of the five cell lines to MMC during the 10-day colony forming assay demonstrated a 15-fold range in MMC sensitivity between the most sensitive cell line (MMC3-A2) and the most resistant cell line (G1B) with CHO-K1R, 21-1 and CHO-K1TOR falling at intermediate levels. Acute aerobic exposure (0-5 h) to MMC resulted in a reduced fivefold range of sensitivities, which was further reduced to a three-fold range under hypoxic exposure conditions. These results were suggestive of differences in the aerobic enzymatic activation of MMC as a possible mechanism contributing to the varying sensitivities. There was no correlation between the one-electron reducing enzyme NADPH:cytochrome P-450 oxidoreductase (P450R) activity and cellular sensitivity to MMC. The five cell lines had similar levels of reduced glutathione (GSH), suggesting that oxygen homeostasis was not correlated with the cells, differing sensitivity to MMC. A correlation did exist between NAD(P)H:quinone oxidoreductase (DT-diaphorase) activity and cellular sensitivity to MMC under chronic exposure conditions for the cell lines. High DT-diaphorase levels were also correlated with a reduced ability of oxygen to modulate MMC toxicity. Levels of P450R and DT-diaphorase were not altered significantly during five-hour aerobic or hypoxic exposures of control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Effect of hypoxia on the accumulation of technetium-99m-glucarate and technetium-99m-gluconate by Chinese hamster ovary cells in vitro.

The accumulation of 99mTc-glucarate, an agent recently reported to localize in acutely infarcted myocardium, zones of acute cerebral injury and tumors, has been compared with 99mTc-gluconate in an in vitro system of cultured Chinese hamster ovary fibroblasts. The effects on accumulation of hypoxia and competition with fructose have been studied. Both labeled glucose analogs showed a two- to threefold enhanced accumulation in hypoxic cells relative to aerobic cells. No such enhanced accumulation under hypoxia was observed for the nonsugar tracers pertechnetate and 99mTc-DTPA. The presence of 20 mM fructose reduced the accumulation of 99mTc-glucarate by 30% (p = 0.067) and 99mTc-gluconate by 40% (p < 0.05) in hypoxic cells, but had no significant effect in aerobic cells. These results suggest that both compounds at least partially share a common mechanism of uptake and/or accumulation with fructose.

Animals↗
PubMed1992

Depletion of intracellular glutathione by 1-methyl-2-nitrosoimidazole.

In addition to their ability to radiosensitize, nitroimidazoles are selectively toxic toward hypoxic cells. Reduction of the nitro group is required to observe cytotoxicity. One of the reduction products believed to play a role in this cytotoxicity is the nitroso-derivative. One-methyl-2-nitrosoimidazole (INO), chemically synthesized from one-methyl-2-nitroimidazole (INO2), has been used as a model to study the reactivity of 2-nitrosoimidazoles. The ability of INO to react rapidly with glutathione in Chinese hamster ovary cells treated with a sub-toxic and toxic level of the drug has been measured. The kinetics of GSH loss as well as oxidized GSH (GSSG) formation and loss were assessed at short times (0-15 min) after INO exposure using a high pressure liquid chromatography (HPLC) assay for GSH and GSSG. The results obtained were consistent with a model, based on previous chemical studies of the reaction of INO with GSH, whereby GSH reduces INO forming GSSG and as well reacts with a reduced form of INO to form an adduct (I-SG). These results suggest possible strategies for modifying the toxicity of reduction products of one-substituted-2-nitroimidazoles.

Animals↗
PubMed1992

Mode of interaction of 5-fluorouracil, radiation, and mitomycin C: in vitro studies.

An examination of the effects of radiation combined with either 5-fluorouracil, Mitomycin C, or both drugs in vitro has been made using a mouse squamous tumor cell line SCC VIITo and cell viability as an endpoint. Depending on how the survival endpoint was calculated, the interaction of 5-fluorouracil, Mitomycin C, or 5-fluorouracil plus Mitomycin C with radiation was greater than additive (plating efficiency) or only additive (viable cells per flask). These results suggest that the cytostatic effect of these drugs may be an important aspect of their action clinically.

Animals↗
PubMed1992

Mechanistic studies of enhanced in vitro radiosensitization and hypoxic cell cytotoxicity by targeting radiosensitizers to DNA via intercalation.

In an effort to increase the molar efficiency of 2-nitroimidazoles as hypoxic cell radiosensitizers and cytotoxins, they have been linked to a DNA intercalating group. The lead compound in this series, NLP-1, is a 2-nitroimidazole, linked at the one position to a phenanthridine ring system via a three carbon chain. Studies of the hypoxic cell specific radiosensitizing properties and hypoxic cell selective toxicity of NLP-1 toward CHO AA8-4 cells show the drug does have enhanced efficiency compared to the untargeted 2-nitroimidazole, misonidazole, based on external drug concentrations. To see if this enhanced efficiency was due to the proposed mechanism, targeting to DNA, or to a general increase in the intracellular concentration of the drug, its uptake and accumulation intracellularly were determined. Radioactive NLP-1 was synthesized labelled with 14C at the 2 position of the imidazole ring. Cells were incubated with the radioactive drug under aerobic and hypoxic exposure conditions, and intracellular levels of the drug were determined by a spin-through-oil technique. Results indicated that, at a drug concentration of 0.5 mM, there was no net accumulation of the drug over the external drug levels after aerobic exposure. Under hypoxic conditions, the drug did accumulate intracellularly, presumably because of hypoxia specific drug metabolism. Experiments with radioactive misonidazole labelled with 14C in the 2 position of the imidazole ring were run as controls. These results suggest that, under the conditions used, NLP-1 has an increased molar efficiency as a hypoxic cell radiosensitizer and cytotoxin, compared to misonidazole, based on intracellular drug concentrations.

Animals↗