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H Joenje

Publications and source records attributed to H Joenje.

At least 91 records · Page 5Linked to original sources
PubMed1990

Non-P-glycoprotein mediated mechanism for multidrug resistance precedes P-glycoprotein expression during in vitro selection for doxorubicin resistance in a human lung cancer cell line.

Two different mechanisms that contribute to multidrug resistance (MDR) were found in derivatives of the human squamous lung cancer cell line SW-1573. The parental cell line has a low amount of mdr1 P-glycoprotein mRNA. In three independent selections for doxorubicin resistance, MDR variants arose in which mdr1 P-glycoprotein mRNA and protein was not detectable. Selection on higher doxorubicin concentrations gave rise to variants containing high levels of mdr1 mRNA, due to transcriptional activation of the mdr1 gene. Upon continued selection for higher levels of doxorubicin resistance, the mdr1 gene became amplified, resulting in an additional increase in the level of mdr1 mRNA. The cross-resistance pattern of the sublines that lack mdr1 P-glycoprotein expression is different from that seen in the mdr1 overexpressing cells. Both types of MDR cell lines are resistant to doxorubicin, daunorubicin, etoposide, colchicine, gramicidin D, and vincristine. However, in the non-P-glycoprotein-mediated MDR cell lines, resistance levels are lower and a preferential resistance for etoposide is seen.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Respiratory failure and stimulation of glycolysis in Chinese hamster ovary cells exposed to normobaric hyperoxia.

Continuous exposure of Chinese hamster ovary (CHO) cells to an atmosphere of 98% O2, 2% CO2 (normobaric hyperoxia) leads within a period of several days to cytostasis and clonogenic cell death. Here we report respiratory failure as an important early symptom of oxygen intoxication in CHO cells, resulting in a more than 80% inhibition of oxygen consumption within 3 days of hyperoxic exposure. This inhibition appeared to be correlated with selective inactivation of three mitochondrial key enzymes, NADH dehydrogenase, succinate dehydrogenase, and alpha-ketoglutarate dehydrogenase. The latter enzyme controls the influx of glutamate into the Krebs cycle and is particularly critical for oxidative ATP generation in most cultured cells, which depends on exogenous glutamine rather than glucose as a carbon source. As expected, the inactivation of alpha-ketoglutarate dehydrogenase was correlated with a fall in cellular glutamine utilization, which became apparent from the first day of hyperoxic exposure. Thereafter, glucose utilization and lactate excretion started to increase, up to 3-fold, indicating a cellular response to respiratory failure aimed at increased ATP generation from glycolysis. However, in spite of this response, the cellular ATP level progressively decreased, up to 2.5-fold. Thus, killing of CHO cells by normobaric hyperoxia seems to be due to a severe disturbance of mitochondrial metabolism eventually leading to a depletion of cellular ATP pools.

Adenosine Triphosphate↗
PubMed1990

Doxorubicin (adriamycin): a critical review of free radical-dependent mechanisms of cytotoxicity.

The antineoplastic drug doxorubicin is capable of generating a variety of free radical species in subcellular systems and this capacity has been considered critical for its antitumor action. However, for most tumor cell lines this mechanism of cytotoxicity does not appear to play a major role. Free radical-independent cytotoxicity mechanisms, taking place in the nuclear compartment of the cell, may more likely be involved in the antitumor effect of doxorubicin.

Animals↗
PubMed1989

Correlation of multidrug resistance with decreased drug accumulation, altered subcellular drug distribution, and increased P-glycoprotein expression in cultured SW-1573 human lung tumor cells.

Four multidrug-resistant variants of the human squamous lung cancer cell line SW-1573 with levels of doxorubicin resistance ranging from 10- to 2000-fold were characterized with respect to drug accumulation and efflux, subcellular drug distribution pattern, antioxidant defenses, and P-glycoprotein expression. For all these parameters except the antioxidant defenses a correlation was observed with the level of doxorubicin resistance; with increasing drug resistance cellular drug accumulation capacity (as measured for doxorubicin) progressively decreased, initial drug efflux rates (as measured for daunorubicin) progressively increased, while the subcellular doxorubicin distribution (as measured by fluorescence microscopy) gradually shifted from a "mainly nuclear" to a "mainly cytoplasmic" pattern. Our data suggest that in the present set of cell lines the same mechanism of resistance is operating at all levels of doxorubicin resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Increased cytosolic pH in multidrug-resistant human lung tumor cells: effect of verapamil.

In a set of four increasingly multidrug-resistant variants of SW-1573 human lung tumor cells, the pHi (i.e., steady-state cytosolic pH) increased up to 0.44 U as a function of the level of doxorubicin resistance. The elevated pHi in the most resistant (2,000-fold) variant dropped to the control level upon addition of verapamil, a known inhibitor of P-glycoprotein activity. These data suggest that, in the absence of xenobiotic substrates, P-glycoprotein activity can affect cellular pHi. This finding may be important for the elucidation of the physiological function of this protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Effect of N-acetylcysteine on the antiproliferative action of X-rays or bleomycin in cultured human lung tumor cells.

N-Acetylcysteine is currently being considered as a possible selective protector against pulmonary toxicity resulting from X-rays or chemotherapeutic treatment, but its clinical application awaits evidence that it does not interfere with the efficient killing of tumor cells. The capacity of N-acetylcysteine to protect against the antitumor activity of X-rays and of bleomycin was evaluated in a clonogenic cell-survival assay using SW-1573 human squamous lung carcinoma cells as a tumor model. Using the highest non-toxic dose of N-acetylcysteine (incubation for 2 days in the continuous presence of 10 mM) no effect on clonogenic cell killing by X-rays or bleomycin treatment could be detected, even though a twofold enhancement of endogenous glutathione was effectuated. Our data thus indicate that clinically relevant concentrations of N-acetylcysteine are incapable of protecting tumor cells against clonogenic killing by X-rays and by bleomycin.

Acetylcysteine↗
PubMed1989

Effects of lethal exposure to hyperoxia and to hydrogen peroxide on NAD(H) and ATP pools in Chinese hamster ovary cells.

Cell death by oxidative stress has been proposed to be based on suicidal NAD depletion, typically followed by ATP depletion, caused by the NAD-consuming enzyme poly(ADP)ribose polymerase, which becomes activated by the presence of excessive DNA-strand breaks. In this study NAD+, NADH and ATP levels as well as DNA-strand breaks (assayed by alkaline elution) were determined in Chinese hamster ovary (CHO) cells treated with either H2O2 or hyperoxia to a level of more than 80% clonogenic cell killing. With H2O2 extensive DNA damage and NAD depletion were observed, while at a higher H2O2 dosage ATP also became depleted. In agreement with results of others, the poly(ADP)ribose polymerase inhibitor 3-aminobenzamide completely prevented NAD depletion. However, both H2O2-induced ATP depletion and cell killing were unaffected by the inhibitor, suggesting that ATP depletion may be a more critical factor than NAD depletion in H2O2-induced killing of CHO cells. With hyperoxia, only moderate DNA damage (2 X background) and no NAD depletion were observed, whereas ATP became largely (70%) depleted. We conclude that (1) there is no direct relation between ATP and NAD depletion in CHO cells subjected to toxic doses of H2O2 or hyperoxia; (2) H2O2-induced NAD depletion is not by itself sufficient to kill CHO cells; (3) killing of CHO cells by hyperoxia is not due to NAD depletion, but may be due to depletion of ATP.

Adenosine Triphosphate↗
PubMed1989

Effect of artificial electron acceptors on the cytotoxicity of mitomycin C and doxorubicin in human lung tumor cells.

The cytotoxicities of mitomycin C (MMC) and doxorubicin (DOX) have been proposed to depend on intracellular reduction by reduced flavoproteins. We investigated whether MMC- and DOX-induced cytotoxicity could be inhibited by competing for electrons from reduced flavoproteins by the artificial electron acceptors phenazine methosulfate (PMS), menadione (MEN) and methylene blue (MB). In intact SW-1573 human lung tumor cells these compounds proved to be excellent electron acceptors, as judged from their capacity to induce high levels of cyanide-resistant respiration. In addition, PMS, MEN and MB were found to decrease the cytotoxicity of MMC, by 90, 63 and 29%, respectively, at concentrations that were themselves completely nontoxic. In contrast, DOX cytotoxicity was not detectably affected. These results suggest that in SW-1573 cells flavoprotein-mediated bioreduction is required for the cytotoxic effect of MMC, but not for that of DOX.

Carcinoma, Squamous Cell↗
PubMed1989

Chromosomal instability and progressive loss of chromosomes in HeLa cells during adaptation to hyperoxic growth conditions.

By sequential selection for resistance to stepwise increased levels of atmospheric O2, a genetic variant of HeLa cells was obtained capable of stable proliferation under an atmosphere containing 80% O2 (HeLa-80). This cell strain has previously been characterized in terms of growth characteristics, morphology and antioxidant status (Joenje et al., 1985). In an attempt to find cytogenetic clues possibly related to the O2-tolerant character, metaphases of HeLa-80 cells were analyzed and compared to the parental (HeLa-20) strain. Numerical analysis revealed a progressive decrease in the number of chromosomes per cell during selection for O2 resistance, from a modal number of 112 in HeLa-20 cells to 84 in HeLa-80 cells. Cytogenetic endpoints for genetic damage revealed increased frequencies in HeLa-80 cells of both chromosomal aberrations (29.7 versus 6.9% aberrant cells) and sister-chromatid exchanges (0.46 +/- 0.13 versus 0.31 +/- 0.10 SCE/chromosome). G-banded metaphases failed to reveal cytogenetic evidence of gene amplification (homogeneously staining regions, double minutes) in the karyotype of HeLa-80 cells.

Cell Division↗
PubMed1989

Chromosomal instability in an oxygen-tolerant variant of Chinese hamster ovary cells.

Background levels of chromosomal aberrations and sister-chromatid exchanges (SCEs) were determined in CHO-99 cells, an oxygen-tolerant variant substrain of Chinese hamster ovary (CHO-20) cells capable of stable proliferation under an atmosphere of 99% O2/1% CO2, a level of hyperoxia at which cultured mammalian cells normally cannot survive. The mean chromosomal aberration frequency in CHO-99 cells was as high as 1 aberration per cell (mainly chromatid and chromosome gaps and breaks) versus 0.05 aberration/cell in CHO-20 cells, while the SCE frequency was 1.7- to 2.1-fold increased. While most aberrations were apparently distributed at random over the chromosomes, up to 31% of the aberrations appeared to be involved in site-specific fragility at a homologous site in chromosomes Z3 and Z4. Immediately upon shifting CHO-99 cells to air-equilibrated conditions their SCE frequency decreased to the control level, whereas the aberration rate persisted at a still elevated level of 0.16-0.31 aberration per cell, even after a culture period of 14 weeks under normoxia. This indicates that at least part of the chromosomal instability is a constitutional property of the variant cells, i.e., not directly dependent upon hyperoxic stress. In CHO-99 X CHO-20 hybrids the occurrence of chromatid-type aberrations and fragile site but not that of chromosome-type aberrations was suppressed under normoxic conditions, suggesting that chromatid-type aberrations and fragile site expression on the one hand and chromosome-type aberrations on the other hand are mediated by different constitutional defects in CHO-99 cells. No gross alterations in (deoxy)ribonucleoside triphosphate pools were detected in CHO-99 cells that could be held responsible for their chromosomal instability. In addition, no increased level of DNA damage was detected by the technique of alkaline elution. The excessive chromosomal instability in CHO-99 cells, as observed under hyperoxic conditions, may originate from reactive intermediates giving rise to DNA double-strand breaks and/or a type of DNA lesion that is resistant to the conditions of the alkaline elution technique. However, alternative mechanisms based upon reactive species interfering with DNA replication/repair processes cannot be excluded.

Animals↗
PubMed1988

Genes amplified and overexpressed in human multidrug-resistant cell lines.

Multidrug resistance (MDR) is associated with overproduction of Mr 170,000 membrane proteins (P-glycoproteins) caused by either gene amplification, transcriptional activation, or both. In rodents the amplified domain comprises genes that encode P-glycoproteins and at least five unrelated genes, one of which encodes the calcium-binding protein sorcin. The amplification and increased expression of these genes always includes one P-glycoprotein-encoding gene (pgp1 in hamsters, homologous to mdr1 in humans). In human MDR cells only elevated mdr1 expression has been shown thusfar, although another P-glycoprotein encoding gene (mdr3, homologous to hamster pgp3) is closely linked. Here we show that the human homolog of the hamster sorcin gene resides on chromosome 7 like the P-glycoprotein-encoding genes. Furthermore, gene classes designated 4, 5, and 6 are coamplified with mdr1 and mdr3 in the human ovarian carcinoma cell line 2780AD, which strongly suggests that the overall structure of the human MDR domain is the same as in rodents. Class 6 was moderately and mdr1 was highly overexpressed in this cell line. Four other human MDR cell lines also have much higher mdr1 overexpression than expected from the relatively low levels (2- to 30-fold) of gene amplification. This contrasts with the results of previous work with rodent MDR cells, in which the increase in P-glycoprotein mRNA levels usually parallels the increase in gene copy number. Although four of the five human MDR cell lines have coamplified mdr3, its expression was undetectable. Our results confirm the central role of the mdr1 (pgp1) gene in MDR and suggest that different cross-resistance patterns are not due to differential expression of different P-glycoprotein genes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Effect of endogenous glutathione, superoxide dismutases, catalase, and glutathione peroxidase on adriamycin tolerance of Chinese hamster ovary cells.

Based on the concept that activated oxygen species are causally involved in Adriamycin toxicity, endogenous antioxidant defenses are expected to be important determinants of cellular Adriamycin tolerance. We have tested this prediction by making use of an oxygen-resistant variant subline of Chinese hamster ovary cells (CHOr), which is characterized by increased levels of glutathione, copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, and glutathione peroxidase. The levels of antioxidant defenses in wild-type CHO (CHOs) cells were within the range reported for human tumor cell lines, except for catalase, which was comparatively high. Oxygen-tolerant CHOr cells, which contained 4.3-fold more catalase activity than CHOs cells, were proportionally more resistant to H2O2, indicating that catalase activity in wild-type CHOs cells was still limiting H2O2 tolerance. The Adriamycin sensitivity of CHOs cells was compared to that of CHOr cells by clonogenic cell survival. After correcting for differential drug uptake in CHOs and CHOr cells, no significant difference in Adriamycin sensitivity could be detected. Furthermore, drug-induced cyanide-resistant oxygen consumption and electron spin resonance data indicated that both cell strains were equally efficient in reducing Adriamycin to its semiquinone radical and in generating activated oxygen species through oxidation-reduction cycling. These results indicate that Adriamycin tolerance of wild-type CHO cells, as determined by clonogenic cell survival, is not limited by endogenous glutathione, copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, or glutathione peroxidase.

Animals↗
PubMed1988

Effect of normobaric hyperoxia on antioxidant defenses of HeLa and CHO cells.

The effect of increased intracellular oxygen activation on cellular antioxidant defenses in CHO and HeLa cells was studied. In both cell types, hyperoxic exposure (up to 4 days, 600-700 mm Hg O2) and in CHO cells menadione (up to 3 days, 15 microM) failed to affect the enzymatic antioxidant defenses Mn-containing superoxide dismutase (Mn-SOD), CuZn-SOD, catalase and glutathione peroxidase. The markedly increased antioxidant enzyme activities observed in a recently obtained oxygen-tolerant CHO variant persisted under normoxia. These data suggest that the synthesis of antioxidant enzymes is constitutive. Glutathione levels of HeLa cells did not respond to hyperoxia whereas in CHO cells hyperoxia and menadione exposure resulted in a 2- and 7-fold increase in glutathione contents, respectively. However, considering the large variations in glutathione contents observed under normal culture conditions, it is uncertain whether this increase is to be considered as a true adaptive response.

Animals↗
PubMed1988

Characterization of oxygen-tolerant Chinese hamster ovary cells. II. Energy metabolism and antioxidant status.

Further characteristics of an oxygen-tolerant variant of Chinese hamster ovary cells (CHO-99) capable of stable proliferation at 99% O2/1% CO2, and O2 level that is lethal to the parental line (CHO-20), are described. Previous work has revealed that CHO-99 cells have 2- to 4-fold increased activities of superoxide dismutases, catalase and glutathione peroxidase, and substantially increased relative volumes of mitochondria and peroxisomes. To document possible additional mechanisms of O2 tolerance we compared CHO-20 cells growing at 20% O2 (normoxia) and CHO-99 cells at 99% O2 (normobaric hyperoxia). We show the following: (1) the estimated total (oxidative and glycolytic) ATP production in CHO-99 cells was 36% decreased. ATP production through oxidative phosphorylation was 52% lower in CHO-99 cells, while the relative contribution from glycolysis was increased from 6% to 30%. The ATP content was 29% lower in CHO-99 cells, the adenylate energy charge being also significantly decreased, indicating that energy production through oxidative phosphorylation is compromised in CHO-99 cells. Cyanide-resistant respiration was 4-fold higher in CHO-99 cells, probably reflecting, at least partly, the increased peroxisomal activity in these cells. (2) The level of reduced glutathione was several fold increased in CHO-99 cells, oxidized glutathione being unaltered; (NADPH + NADP+) levels were elevated 2.7-fold, while the ratio of NADPH to NADP+ was increased almost two-fold. These changes were associated with a 50% increased metabolism of glucose through the hexose monophosphate pathway. (3) No evidence was obtained for an increased steady-state level of endogenous lipid peroxidation in CHO-99 cells, in spite of a 50% increased content of polyunsaturated fatty acids in the phospholipid fraction.

Animals↗
PubMed1987

Antioxidant status of Fanconi anemia fibroblasts.

Several observations in the recent literature have indicated that Fanconi anemia (FA) cells may be primarily deficient in the detoxification of activated oxygen species. To evaluate the antioxidant status of FA fibroblasts, we measured Mn-containing superoxide dismutase (Mn-SOD), CuZn-containing superoxide dismutase (CuZn-SOD), catalase, and glutathione peroxidase activities, as well as cellular glutathione contents and total nonenzymatic antioxidant potential in FA and control fibroblasts at multiple time points during a single passage. All parameters exhibited a characteristic pattern of changes during a period of 19 days following trypsinization. Unlike FA erythrocytes, which are known to be deficient in CuZn-SOD, FA fibroblasts exhibited normal CuZn-SOD activities. Also, the nonenzymatic "antioxidant potential" as well as glutathione levels were similar in FA and control fibroblasts. However, Mn-SOD, catalase, and glutathione peroxidase activities were consistently higher in FA fibroblasts. We hypothesize that the elevation of these enzyme activities might reflect a cellular "prooxidant" state in FA resulting from an increased formation of endogenous oxidizing molecular species that trigger enhanced synthesis of certain enzymatic antioxidant defenses.

Anemia, Aplastic↗
PubMed1987

Cytogenetic toxicity of paraquat and streptonigrin in Fanconi's anemia.

We reported earlier that the frequency of chromosomal aberrations observed in Fanconi's anemia lymphocyte cultures depends on the oxygen tension during growth of the cultures, suggesting that "activated oxygen species" (superoxide, O2-; hydrogen peroxide, H2O2; hydoxyl radical, OH; and singlet oxygen, 1O2) or other reactive products generated during oxygen metabolism may be involved in the production of chromosomal damage in this syndrome. Paraquat and streptonigrin, agents that have been proposed as model compounds exerting cellular toxicity through overproduction of superoxide, were tested for their clastogenic potency in lymphocyte cultures from healthy controls and patients with Fanconi's anemia. Paraquat, at concentrations that severely affected mitotic activity (100-200 micrograms/ml), appeared to be a weak clastogen in human lymphocytes, whereas a clastogenic effect of streptonigrin was demonstrable already at a concentration as low as 0.005 microgram/ml. The results indicate that Fanconi's anemia lymphocytes fail to exhibit an increased sensitivity to the antimitotic and clastogenic effects of paraquat and streptonigrin. This suggests that intracellular superoxide is not critically involved in the generation of spontaneous chromosomal aberrations in Fanconi's anemia. We infer from these and previous data that singlet oxygen (1O2) may be a critical contributor to the chromosomal breakage in this disorder.

Adolescent↗
PubMed1987

Oxygen toxicity and chromosomal breakage in ataxia telangiectasia.

Toxic effects of ionizing radiation and elevated O2 levels (hyperoxia) are both thought to be mediated by oxidizing free radicals. In view of the reported hypersensitivity of ataxia telangiectasia (A-T) cells to the clastogenic effect of ionizing radiation, the chromosomal sensitivity of A-T cells to hyperoxic culture conditions was investigated in unirradiated and G0-irradiated A-T lymphocyte cultures. Unlike Fanconi's anaemia lymphocytes, which tend to respond to oxygen, especially after treatment with mitomycin C, both non-irradiated and G0-irradiated A-T lymphocytes failed to show an effect. We conclude that the excessive spontaneous and radiation-induced chromosomal breakage in A-T does not result from a deficiency in the cellular defences against the clastogenic effect of hyperoxia.

Ataxia Telangiectasia↗