Biomedical subjects

R H Schiestl

Publications and source records attributed to R H Schiestl.

At least 19 recordsLinked to original sources

PubMed2002

X radiation causes a persistent induction of reactive oxygen species and a delayed reinduction of TP53 in normal human diploid fibroblasts.

Multiple genetic changes are required for the development of a malignant cell. The frequency of such changes in cancer cells is higher than can be explained through random mutation, and it was proposed that a subpopulation of cells develop a persistent mutator phenotype. Evidence for such a phenotype has been observed in mammalian cells after treatment with ionizing radiation. The mechanism that promotes this effect has not been defined, but proposed explanations include increased levels of reactive oxygen species (ROS) in irradiated cells and their progeny. The tumor suppressor TP53 is of prime importance in coordinating the cellular response to damage, and it has been suggested to have a role in regulating the cellular redox state. We investigated the persistence of induced levels of ROS in normal diploid human cells for 1 month after X-ray exposure and the role of TP53 in this oxidant response. X radiation induced an oxidant response that persisted for 2 weeks after exposure in cells with normal TP53 function. ROS levels in cells with abrogated TP53 function were decreased in magnitude and duration. X radiation caused a primary transient induction of TP53 followed by a reinduction of TP53 5 days after irradiation. This reinduction persisted for at least 2 days and coincided with the largest induction of apoptosis. The persistently elevated levels of ROS and delayed reinduction of TP53 reported here are further evidence of the delayed effects of ionizing radiation and add to the growing number of such observations.

Antimycin A↗

PubMed2001

Restriction enzymes increase efficiencies of illegitimate DNA integration but decrease homologous integration in mammalian cells.

Mammalian cells repair DNA double-strand breaks by illegitimate end-joining or by homologous recombination. We investigated the effects of restriction enzymes on illegitimate and homologous DNA integration in mammalian cells. A plasmid containing the neo(R) expression cassette, which confers G418 resistance, was used to select for illegitimate integration events in CHO wild-type and xrcc5 mutant cells. Co-transfection with the restriction enzymes BamHI, BglII, EcoRI and KpnI increased the efficiency of linearized plasmid integration up to 5-fold in CHO cells. In contrast, the restriction enzymes did not increase the integration efficiency in xrcc5 mutant cells. Effects of restriction enzymes on illegitimate and homologous integration were also studied in mouse embryonic stem (ES) cells using a plasmid containing the neo(R) gene flanked by exon 3 of HPRT: The enzymes BamHI, BglII and EcoRI increased the illegitimate integration efficiency of transforming DNA several-fold, similar to the results for CHO cells. However, all three enzymes decreased the absolute frequency of homologous integration approximately 2-fold, and the percentage of homologous integration decreased >10-fold. This suggests that random DNA breaks attract illegitimate recombination (IR) events that compete with homology search.

Animals↗

PubMed2001

Homologous recombination as a mechanism of carcinogenesis.

Cancer develops when cells no longer follow their normal pattern of controlled growth. In the absence or disregard of such regulation, resulting from changes in their genetic makeup, these errant cells acquire a growth advantage, expanding into pre-cancerous clones. Over the last decade many studies have revealed the relevance of genomic mutation in this process, be it by misreplication, environmental damage or a deficiency in repairing endogenous and exogenous damage. Here we discuss homologous recombination as another mechanism that can result in loss of heterozygosity or genetic rearrangements. Some of these genetic alterations may play a primary role in carcinogenesis, but they are more likely to be involved in secondary and subsequent steps of carcinogenesis by which recessive oncogenic mutations are revealed. Patients whose cells display an increased frequency of recombination also have an elevated frequency of cancer, further supporting the link between recombination and carcinogenesis. In addition, homologous recombination is induced by a wide variety of carcinogens, many of which are classically considered to be efficiently repaired by other repair pathways. Overall, homologous recombination is a process that has been widely overlooked but may be more central to the process of carcinogenesis than previously described.

Animals↗

PubMed2001

Susceptibility of proliferating cells to benzo[a]pyrene-induced homologous recombination in mice.

The pink-eyed unstable mutation, p(un), is the result of a 70 kb tandem duplication within the murine pink-eyed, p, gene. Deletion of one copy of the duplicated region by homologous deletion/recombination occurs spontaneously in embryos and results in pigmented spots in the fur and eye. Such deletion events are inducible by a variety of DNA damaging agents, as we have observed previously with both fur- and eye-spot assays. Here we describe a study of the effect of exposure to benzo[a]pyrene (B[a]P) at different times of development on reversion induction in the eye. Previously we, among others, have reported that the retinal pigment epithelium (RPE) displays a position effect variegation phenotype in the pattern of pink-eyed unstable reversions. Following an acute exposure to B[a]P or X-rays on the tenth day of gestation an increased frequency of reversion events was detected in a distinct region of the adult RPE. Examining exposure at different times of eye development reveals that both B[a]P and X-rays result in an increased frequency of reversion events, though the increase was only significant following B[a]P exposure, similar to our previous report limited to exposure on the tenth day of gestation. Examination of B[a]P-exposed RPE in the present study revealed distinct regions where the induced events lie and that the positions of these regions are found at increasing distances from the optic nerve the later the time of exposure. This position effect directly reflects the previously observed developmental pattern of the RPE, namely that cells in the regions most distal from the optic nerve are proliferating most vigorously. The numbers and positions of RPE cells displaying the transformed (pigmented) phenotype strongly advocate the proposal that dividing cells are at highest risk to deletions induced by carcinogens.

Animals↗

PubMed2001

Cytotoxic and genotoxic consequences of heat stress are dependent on the presence of oxygen in Saccharomyces cerevisiae.

Lethal heat stress generates oxidative stress in Saccharomyces cerevisiae, and anaerobic cells are several orders of magnitude more resistant than aerobic cells to a 50 degrees C heat shock. Here we characterize the oxidative effects of this heat stress. The thermoprotective effect in anaerobic cells was not due to expression of HSP104 or any other heat shock gene, raising the possibility that the toxicity of lethal heat shock is due mainly to oxidative stress. Aerobic but not anaerobic heat stress caused elevated frequencies of forward mutations and interchromosomal DNA recombination. Oxidative DNA repair glycosylase-deficient strains under aerobic conditions showed a powerful induction of forward mutation frequencies compared to wild-type cells, which was completely abolished under anaerobiosis. We also investigated potential causes for this oxygen-dependent heat shock-induced genetic instability. Levels of sulfhydryl groups, dominated mainly by the high levels of the antioxidant glutathione (reduced form) and levels of vitamin E, decreased after aerobic heat stress but not after anaerobic heat stress. Aerobic heat stress also led to an increase in mitochondrial membrane disruption of several hundredfold, which was 100-fold reduced under anaerobic conditions.

Anaerobiosis↗

PubMed2001

Mitochondrial respiratory electron carriers are involved in oxidative stress during heat stress in Saccharomyces cerevisiae.

In the present study we sought to determine the source of heat-induced oxidative stress. We investigated the involvement of mitochondrial respiratory electron transport in post-diauxic-phase cells under conditions of lethal heat shock. Petite cells were thermosensitive, had increased nuclear mutation frequencies, and experienced elevated levels of oxidation of an intracellular probe following exposure to a temperature of 50 degrees C. Cells with a deletion in COQ7 leading to a deficiency in coenzyme Q had a much more severe thermosensitivity phenotype for these oxidative endpoints following heat stress compared to that of petite cells. In contrast, deletion of the external NADH dehydrogenases NDE1 and NDE2, which feed electrons from NADH into the electron transport chain, abrogated the levels of heat-induced intracellular fluorescence and nuclear mutation frequency. Mitochondria isolated from COQ7-deficient cells secreted more than 30 times as much H(2)O(2) at 42 as at 30 degrees C, while mitochondria isolated from cells simultaneously deficient in NDE1 and NDE2 secreted no H(2)O(2). We conclude that heat stress causes nuclear mutations via oxidative stress originating from the respiratory electron transport chains of mitochondria.

Cell Nucleus↗

PubMed2001

Ozone causes lipid peroxidation but little antioxidant depletion in exercising and nonexercising hamsters.

Ozone (O(3)), a major component of urban air pollution, is a strong oxidizing agent that can cause lung injury and inflammation. In the present study, we investigated the effect of inhalation of O(3) on levels of F(2)-isoprostanes in bronchoalveolar lavage fluid (BALF) and on levels of antioxidants in the BALF and plasma of hamsters. Because antioxidants, including urate, ascorbate, GSH, and vitamin E, defend the lungs by reacting with oxidizing agents, we expected to find a decrease in antioxidant levels after O(3) exposure. Similarly, we expected an increase in the levels of F(2)-isoprostanes, which are lipid peroxidation products. Exposure to 1.0 or 3.0 parts/million (ppm) O(3) for 6 h resulted in an increase in BALF neutrophil numbers, an indicator of acute inflammation, as well as elevation of BALF F(2)-isoprostanes. The higher dose of O(3) caused an increase in the BALF level of urate and a decrease in the plasma level of ascorbate, but 1.0 ppm O(3) had no effect on BALF or plasma antioxidant levels. Exposure to 0.12 ppm O(3) had no effect on BALF neutrophils or F(2)-isoprostanes nor on BALF and plasma antioxidants. We also investigated the effect of O(3) exposure of hamsters during exercise on F(2)-isoprostane and antioxidant levels. We found that exposure to 1.0 ppm O(3) during 1 h of exercise on a laddermill increased BALF levels of F(2)-isoprostanes but had no effect on BALF neutrophils or on BALF and plasma antioxidants. These results indicate that O(3) induces inflammation and biomolecule oxidation in the lungs, whereas extracellular antioxidant levels are relatively unchanged.

Animals↗

PubMed2001

Persistent genomic instability in the yeast Saccharomyces cerevisiae induced by ionizing radiation and DNA-damaging agents.

A "hypermutable" genome is a common characteristic of cancer cells, and it may contribute to the progressive accumulation of mutations required for the development of cancer. It has been reported that mammalian cells surviving exposure to gamma radiation display several highly persistent genomic instability phenotypes which may reflect a hypermutability similar to that seen in cancer. These phenotypes include an increased mutation frequency and a decreased plating efficiency, and they continue to be observed many generations after the radiation exposure. The underlying causes of this genomic instability have not been fully determined. We show here that exposure to gamma radiation and other DNA-damaging treatments induces a similar genomic instability in the yeast Saccharomyces cerevisiae. A dose-dependent increase in intrachromosomal recombination was observed in cultures derived from cells surviving gamma irradiation as many as 50 generations after the exposure. Increased forward mutation frequencies and low colony-forming efficiencies were also observed. Persistently elevated recombination frequencies in haploid cells were dominant after these cells were mated to nonirradiated partners, and the elevated recombination phenotype was also observed after treatment with the DNA-damaging agents ultraviolet light, hydrogen peroxide, and ethyl methanesulfonate. Radiation-induced genomic instability in yeast may represent a convenient model for the hypermutability observed in cancer cells.

DNA Damage↗

PubMed2000

Benzo(a)pyrene and X-rays induce reversions of the pink-eyed unstable mutation in the retinal pigment epithelium of mice.

The pink-eyed unstable (p(un)) mutation is the result of a 70kb tandem duplication within the murine p gene. Homologous deletion/recombination of the locus to wild-type occurs spontaneously in embryos and results in pigmented spots in the fur and eye that persist for life. Such deletion events are also inducible by a variety of DNA damaging agents, as we have observed previously with the fur spot assay. Here, we describe the use of the retinal pigment epithelium (RPE) of the eye to detect reversion events induced with two differently acting agents. Benzo(a)pyrene (B(a)P) induces a high frequency, and X-ray exposure a more modest increase, of p(un) reversion in both the fur and the eye. The eye-spot assay requires fewer mice for significant results than the fur spot assay. Previous work had elucidated the cell proliferation pattern in the RPE and a position effect variegation phenotype in the pattern of p(un) reversions, which we have confirmed. Acute exposure to B(a)P or X-rays resulted in an increased frequency of reversion events. The majority of the spontaneous reversions lie toward the periphery of the RPE whereas induced events are found more centrally, closer to the optic nerve head. The induced distribution corresponds to the major sites of cell proliferation in the RPE at the time of exposure, and further advocates the proposal that dividing cells are at highest risk to develop deletions.

Animals↗

PubMed2000

Simultaneous measurement of the frequencies of intrachromosomal recombination and chromosome gain using the yeast DEL assay.

The yeast DEL assay measures the frequency of intrachromosomal recombination between two partially-deleted his3 alleles on chromosome XV. The his3Delta alleles share approximately 400bp of overlapping homology, and are separated by an intervening LEU2 sequence. Homologous recombination between the his3Delta alleles results in deletion of the intervening LEU2 sequence (DEL), and reversion to histidine prototrophy. In this study we have attempted to further extend the use of the yeast DEL assay to measure the frequency of chromosome XV gain events. Reversion to His(+)Leu(+) in the haploid yeast DEL tester strain RSY6 occurs upon non-disjunction of chromosome XV sister chromatids, coupled with a subsequent DEL event. Here we have tested the ability of the yeast DEL assay to accurately predict the aneugenic potential of the diversely-acting, known or suspected aneugens actinomycin D, benomyl, chloral hydrate, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and methotrexate. Actinomycin D and benomyl strongly induced aneuploidy. EMS and methotrexate modestly induced aneuploidy, while chloral hydrate and MMS failed to illicit any significant induction. In addition, by FACS-analysis of DNA content it was shown that the majority of both spontaneous- and chemically-induced His(+)Leu(+) revertants were heterodiploid. Thus, our results indicate endoreduplication of almost entire chromosome sets as a major mechanism of aneuploidy induction in haploid Saccharomyces cerevisiae.

Aneuploidy↗

PubMed2000

Atm deficiency causes an increased frequency of intrachromosomal homologous recombination in mice.

Ataxia telangiectasia (AT) patients have inactivating mutations in both copies of the ATM gene. The ATM protein that the gene encodes is involved in DNA double-strand break (DSB) recognition; in its absence, p53 response to DSBs is delayed and reduced. In addition, AT patients have a high propensity for cancer, and cells from these patients show chromosomal instability. Here, using an in vivo mouse model system with the pink-eyed unstable mutation, we demonstrate that the absence of functional Atm results in a significantly elevated frequency of intrachromosomal recombination resulting in deletion events (wild-type 17.73%, heterozygous Atm 15.72%, and mutant Atm 30.33%). No such increase was observed in mice heterozygous for Atm. These results further advocate the role of ATM in maintaining genomic integrity after the onset of endogenous damage. This system relies on the initiation of events during a relatively short time frame to produce an observable deletion product. AT patients have a lifelong exposure to endogenous damage and perhaps similarly acting external agents. Because 25% of our genome consists of repeated elements, genomic instability due to an increased level of homologous recombination between such repeats, as observed here, may contribute to carcinogenesis in AT patients.

Animals↗

PubMed2000

Mis-targeting of multiple gene disruption constructs containing hisG.

Gene targeting by homologous recombination occurs in Saccharomyces cerevisiae efficiently when there are as few as 30 base pairs of sequence homology at both ends of the targeting construct. Multiple gene disruptions within a single cell are possible using the hisG cassette, which allows recovery of the marker but leaves a single hisG sequence imbedded in the disrupted gene(s). We use an integration hisG construct, which has limited homology to the target at one end, to show that a single genomic copy of hisG decreases the percentage of integration at the target locus from 44% to 4.5% and two genomic hisG copies decrease it to less than 1%. Enlarging the homology at the disruption construct abolishes this effect. Thus competition between endogenous hisG sequences and successive hisG cassette transformations occurs if there is limited homology at one end of the targeting construct. Therefore, methods using limited homology, such as PCR-mediated gene targeting, are inefficient when significant internal homology exists.

Base Sequence↗

PubMed2000

Homologous recombination as a mechanism for genome rearrangements: environmental and genetic effects.

Novel findings over the last 2 years have led to an increased emphasis on homologous recombination (HR) as both a pathway for DNA repair and a cause for genomic rearrangements. Indeed, environmental carcinogens increase the frequency of HR, as can be observed when two copies of a duplicated sequence recombine to delete the intervening sequences. Such HR events between dispersed homologous sequences may result in not only deletions, but also gene duplications or translocations. These types of genomic rearrangement have been observed to be the cause of several different genetic diseases, including cancer. In reflection of this, several genes have been identified that, when mutant, predispose an individual to an increased frequency of cancer. These genes have been shown to be either directly or indirectly involved in HR. In addition, HR is induced by a wide variety of carcinogens, preferentially in proliferating cells. This fits the most current models of recombination and its involvement in reinitiating stalled replication forks. Thus, 'correct' HR repair may act with high fidelity, an important issue for proliferating cells, but in the context of alternative homologous partner sequences, 'aberrant' HR can cause genomic rearrangements with dire consequences.

Animals↗

PubMed2000

DNA integration by Ty integrase in yku70 mutant Saccharomyces cerevisiae cells.

In the present work we examined nonhomologous integration of plasmid DNA in a yku70 mutant. Ten of 14 plasmids integrated as composite elements, including Ty sequences probably originating from erroneous strand-switching and/or priming events. Three additional plasmids integrated via Ty integrase without cointegrating Ty sequences, as inferred from 5-bp target site duplication and integration site preferences. Ty integrase-mediated integration of non-Ty DNA has never been observed in wild-type cells, although purified integrase is capable of using non-Ty DNA as a substrate in vitro. Hence our data implicate yKu70 as the cellular function preventing integrase from accepting non-Ty DNA as a substrate.

Antigens, Nuclear↗

PubMed1999

The aromatic amine carcinogens o-toluidine and o-anisidine induce free radicals and intrachromosomal recombination in Saccharomyces cerevisiae.

Aniline-based aromatic amine carcinogens are poorly detected in short-term mutagenicity assays such as the Salmonella reverse mutation (Ames) assay. More information on the mechanism of toxicity of such Salmonella-negative carcinogens is needed. Aniline and o-toluidine are negative in the Ames assay, but induce deletions (DEL) due to intrachromosomal recombination in Saccharomyces cerevisiae with an apparent threshold. We show here that the DEL assay also detects the genotoxic activity of another aromatic amine carcinogen, o-anisidine, which is also negative in the Salmonella assay. We also show that the DEL assay distinguishes between o-anisidine and its non-carcinogenic structural analog 2, 4-dimethoxyaniline. We have investigated whether the ability of the DEL assay to detect the carcinogens and to distinguish between the carcinogen/non-carcinogen pair is linked to rises in intracellular free radical species following exposure to the carcinogens. Toxicity induced by all three compounds was reduced in the presence of the free radical scavenger and antioxidant N-acetyl cysteine, recombination induced by o-anisidine and o-toluidine was also reduced by N-acetyl cysteine. All three compounds induced oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate. Superoxide dismutase-deficient strains, however, were hypersensitive to cytotoxicity induced by o-toluidine and o-anisidine but not by the non-carcinogen 2,4-dimethoxyaniline, indicating a different potential for generating superoxide radical between the carcinogens and the non-carcinogen analog. The results indicate that the yeast DEL assay is a useful tool for investigating the genotoxic activity of aromatic amine carcinogens.

Aniline Compounds↗

PubMed1999

Cell division transforms mutagenic lesions into deletion-recombinagenic lesions in yeast cells.

Cell proliferation has been recognized as an important factor in human and experimental carcinogenesis. Point mutations as well as larger chromosomal rearrangements are involved in the initiation of cancer. In this paper we compared the relative potencies of radiation and chemical carcinogens for inducing point mutations vs. deletions in cell cycle arrested with dividing cells of Saccharomyces cerevisiae. Point mutation substrates and deletion (DEL) recombination substrates were constructed with the genes CDC28 and TUB2 that are required for cell cycle progression through G1 and G2, respectively. The carcinogens ionizing radiation, UV, MMS, EMS and 4-NQO induced point mutations in G1 and in G2 arrested as well as in dividing cells. UV, MMS, EMS and 4-NQO caused very weak if any increases in DEL recombination in G1 or G2 arrested cells, but large increases in dividing cells. When cells treated with carcinogen either in G1 or G2 were allowed to progress through the cell cycle, a time-dependent increase in DEL recombination was seen. Ionizing radiation and the site-specific endonuclease I-SceI, which both directly create double-strand breaks, induced DEL recombination in G1 as well as in G2 arrested cells. In conclusion, UV-, MMS-, EMS- and 4-NQO-induced DNA damage was converted during DNA replication to a lesion capable of inducing DEL recombination which is probably a DNA strand break. Thus, cell proliferation is not necessary to turn DNA alkylation or UV damage into a mutagenic lesion but to convert the damage into a lesion that induces DNA deletions. These results are discussed with respect to mechanisms of carcinogenesis.

4-Nitroquinoline-1-oxide↗

PubMed1999

Involvement of p53 in X-ray induced intrachromosomal recombination in mice.

The tumor suppressor gene Trp53 (also known as p53) is the most frequently mutated gene in human cancers. p53 is induced in response to DNA damage and effects a G(1) cell cycle arrest. It is believed that p53 plays a key role in maintaining genomic integrity following exposure to DNA-damaging agents. We determined the frequency of spontaneous and DNA damage-induced homologous intrachromosomal recombination in p53-deficient mouse embryos. Homologous intrachromosomal recombination events resulting in deletions at the pink eyed unstable (p(un)) locus result in reversion to the p gene. Reversions occurring in embryonic premelanocytes give rise to black spots on the gray fur of the offspring. Pregnant C57BL/6J p(un)/p(un) p53(+/-) mice were exposed to X-rays (1 Gy) or administered benzo¿apyrene (B¿aP; 30 or 150 mg/kg i.p.) 10 days after conception. Frequencies of spontaneous p(un) reversions in p53(-/-) and p53(+/-) animals were not significantly different compared with their wild-type littermates. X-ray treatment increased the recombination frequency in wild-type and p53(+/-), but surprisingly not in p53(-/-) offspring. In contrast, B¿aP treatment caused a dose-dependent increase in p(un) reversion frequencies in all three genotypes. Western blot analysis of embryos indicated that p53 protein levels increased approximately 3-fold following X-ray treatment, while B¿aP had no effect on p53 expression. These results are in agreement with the proposal that p53 is involved in the DNA damage response following X-ray exposure and suggest that X-ray-induced double-strand breaks are processed differently in p53(-/-) animals.

Animals↗

PubMed1999

Effect of ionizing radiation on transgenerational appearance of p(un) reversions in mice.

Multiple genetic changes are required for the development of a malignant tumor cell and many environmentally induced cancers show a delayed onset of > 20 years following exposure. In fact, the frequency of genetic changes in cancer cells is higher than can be explained by random mutation. A high level of genetic instability in a subpopulation of cells may be caused by a mutator phenotype transmitted through many cell divisions. We have determined the effects of irradiation of parental male mice on the frequency and characteristics of mitotically occurring DNA deletion events at the p(un) locus in the offspring. Reversion of the p(un) marker in mouse embryos is due to deletion of 70 kb of DNA resulting in fur spots in the offspring. We found that irradiation of male mice caused a significantly higher frequency of large spots in the offspring, indicative of the induction of DNA deletions early in embryo development. These deletion events occurred, however, many cell divisions after irradiation. The present data indicate that exposure of the germline to ionizing radiation results in induction of delayed DNA deletions in offspring mice.

Animals↗