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P Amstad

Publications and source records attributed to P Amstad.

At least 19 recordsLinked to original sources
PubMed2001

Mutability of p53 hotspot codons to benzo(a)pyrene diol epoxide (BPDE) and the frequency of p53 mutations in nontumorous human lung.

p53 mutations are common in lung cancer. In smoking-associated lung cancer,the occurrence of G:C to T:A transversions at hotspot codons, e.g., 157, 248, 249,and 273, has been linked to the presence of carcinogenic chemicalsin tobacco smoke including polycyclic aromatic hydrocarbons suchas benzo(a)pyrene (BP). In the present study, we have used a highly sensitive mutation assay to determine the p53 mutation load in nontumorous human lung and to study the mutability of p53 codons 157, 248, 249, and 250 to benzo(a)pyrene-diol-epoxide (BPDE), an active metabolite of BP in human bronchial epithelial BEAS-2B cells. We determined the p53 mutational load at codons 157, 248, 249, and 250 in nontumorous peripheral lung tissue either from lung cancer cases among smokers or noncancer controls among smokers and nonsmokers. A 5-25-fold higher frequency of GTC(val) to TTC(phe) transversions at codon 157 was found in nontumorous samples (57%) from cancer cases (n = 14) when compared with noncancer controls (n = 8; P < 0.01). Fifty percent (7/14) of the nontumorous samples from lung cancer cases showed a high frequency of codon 249 AGG(arg) to AGT(ser) mutations (P < 0.02). Four of these seven samples with AGT(ser) mutations also showed a high frequency of codon 249 AGG(arg) to ATG(met) mutations, whereas only one sample showed a codon 250 CCC to ACC transversion. Tumor tissue from these lung cancer cases (38%) contained p53 mutations but were different from the above mutations found in the nontumorous pair. Noncancer control samples from smokers or nonsmokers did not contain any detectable mutations at codons 248, 249, or 250. BEAS-2B bronchial epithelial cells exposed to doses of 0.125, 0.5, and 1.0 microM BPDE, showed G:C to T:A transversions at codon 157 at a frequency of 3.5 x 10(-7), 4.4 x 10(-7), and 8.9 x 10(-7), respectively. No mutations at codon 157 were found in the DMSO-treated controls. These doses of BPDE induced higher frequencies, ranging from 4-12-fold, of G:C to T:A transversions at codon 248, G:C to T:A transversions and G:C to A:T transitions at codon 249, and C:G to T:A transitions at codon 250 when compared with the DMSO-treated controls. These data are consistent with the hypothesis that chemical carcinogens such as BP in cigarette smoke cause G:C to T:A transversions at p53 codons 157, 248, and 249 and that nontumorous lung tissues from smokers with lung cancer carry a high p53 mutational load at these codons.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2000

Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation.

Activation of caspases is the key event of apoptosis and new methods are needed to assay this event, particularly in situ, in individual cells. To measure in situ caspases activation in the present study we employed fam-VAD-fmk and fam-VEID-fmk, the fluorochrome (fam)-labeled inhibitors of caspases (FLICA), which through the fluoromethylketone (fmk) moiety bind to active center of the activated enzymes. The peptide moiety of these inhibitors defines their specificity; VAD is generic to most caspases and VEID is caspase-6 specific. The frequencies of cells showing caspases activation were compared with those showing DNA fragmentation (detected by the TUNEL assay) in the same cultures. Apoptosis of HL-60 cells was induced by DNA topoisomerase I inhibitor camptothecin (CPT) or tumor necrosis factor-alpha combined with cycloheximide (TNF-alpha + CHX). The cells that bound FLICA had morphological changes typical of apoptosis. The intensity of their fluorescence was measured by laser scanning cytometry. Maximal rate of activation of the caspases, measured by the increase in frequency of the cells that bound fam-VAD-fmk, occurred between 30 and 90 min after the administration of TNF-alpha + CHX and between 2 and 4 h after the administration of CPT. In the CPT-treated cultures about 30% fewer cells bound fam-VEID-fmk than fam-VAD-fmk which suggests that the activation of caspase-6 was delayed or was not induced in some cells. A strong overall correlation between the cytometric assays of the apoptotic index based on the detection of caspases activation by the FLICA and the TUNEL assay was observed. The data indicate that FLICA offers a rapid and convenient method of assessing caspase's activation in individual cells and can also be used to estimate the frequency of apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Increased p53 mutation load in noncancerous colon tissue from ulcerative colitis: a cancer-prone chronic inflammatory disease.

Ulcerative colitis (UC) is a chronic inflammatory disease that produces reactive oxygen and nitrogen species and increases the risk of colorectal cancer (CRC). The p53 tumor suppressor gene is frequently mutated in UC-associated dysplastic lesions and CRC. We are exploring the hypothesis that p53 mutations in the nontumorous colonic tissue in noncancerous UC cases indicate genetic damage from exposure to exogenous and endogenous carcinogens and may identify individuals at increased cancer risk. We are reporting, for the first time, the frequency of specific p53 mutated alleles in nontumorous colon tissue from donors either with or without UC by using a highly sensitive genotypic mutation assay. Higher p53 mutation frequencies of both G:C to A:T transitions at the CpG site of codon 248 and C:G to T:A transitions at codon 247 were observed in colon from UC cases when compared with normal adult controls (P = 0.001 and P = 0.001, respectively). In the UC cases, higher p53 codon 247 and 248 mutation frequencies were observed in the inflamed lesional regions when compared with the nonlesional regions of their colon (P < 0.001 and P = 0.001). The colonic nitric oxide synthase-2 activity was higher in UC cases than in non-UC adult controls (P = 0.02). Our data are consistent with the hypothesis that a higher frequency of p53 mutant cells can be generated under oxidative stress in people with UC. The increased frequency of specific p53 mutated alleles in noncancerous UC colon tissue may confer susceptibility to the development of CRC in an inflammatory microenvironment.

Adult↗
PubMed1999

Hydroxyl radical production by mouse epidermal cell lines in the presence of quinone anti-cancer compounds.

The effect of quinone anti-cancer compounds, 3,6-diaziridinyl-2, 5-bis(carboethoxyamino)-1,4-benzoquinone (diaziquone or AZQ) and 3, 6-dihydro-2,5-bis(aziridinyl)-1,4-benzoquinone (DZQ), on the production of hydroxyl radical ((*)OH) in a series of mouse epidermal cell lines was examined using a new, highly sensitive method that employs a fluorescamine-derivatized nitroxide probe. Cell lines that were examined included the mouse epidermal cell line, JB6 clone 41, and JB6 cells transfected with the human Cu-Zn superoxide dismutase (SOD) genes (SOD3 and SOD15) and human catalase (CAT) genes (CAT13 and CAT10). Bioreduction of the nitroxide probe by these cell lines was insignificant at the cell densities employed in these experiments, and thus did not interfere with the (*)OH measurements. In the presence of low concentrations of AZQ or DZQ (20-200 microM), (*)OH production rates were highest in the JB6 cells, intermediate in the SOD-transfected cells, and lowest in the CAT-transfected cells, illustrating that both superoxide and hydrogen peroxide are involved in the production of (*)OH in these systems. Further experiments in which the addition of exogenous SOD and CAT was employed, as well as measurements of probe incorporation into the cells, indicated that this probe can cross cell membranes and detect (*)OH generated intracellularly. In the presence of 100 microM diethylenetriaminepentaacetic acid (DTPA), the rate of (*)OH production in the presence of 100 microM AZQ is approximately 4.7 x 10(4) molecules s(-)(1) cell(-)(1); as much as 45% of this production appears to originate within the cells. This new method should be broadly applicable to the rapid screening of compounds or treatments thought to induce oxidative stress in mammalian cells.

Animals↗
PubMed1997

Renal antioxidant enzyme mRNA levels are increased in rats with experimental diabetes mellitus.

Exposure to high glucose concentrations increases the mRNA levels of oxygen radical scavenging enzymes in cultured endothelial cells, suggesting a compensatory response to increased free radical production. To test the hypothesis that this response also occurs in vivo, Cu.Zn-superoxide dismutase (Cu.Zn-SOD) and catalase mRNA levels, were measured in the kidneys of Sprague-Dawley rats 17 days after intravenous injection of streptozotocin (60 mg/kg body weight) and compared with those of control rats. Diabetic rats were either left untreated or given differing insulin regimens (2. 3-8, 6-10 IU/day) in two different experiments that were designed to achieve varying degrees of metabolic control. Cu,Zn-SOD and catalase mRNA levels were measured by Northern blot hybridization and standardized by 28S ribosomal RNA determination. Renal Cu,Zn-SOD and catalase mRNA levels were significantly greater in untreated diabetic and in low-dose (2 IU/day) insulin-treated rats than in controls. Treatment with a moderate dose (3-8 IU/day) of insulin normalized catalase but not Cu,Zn-SOD mRNA levels. The highest insulin regimen (6-10 IU/day), in addition to achieving complete metabolic control as evidenced by normal growth and plasma glucose levels, normalized both catalase and Cu,Zn-SOD mRNA levels. Thus, in rats with streptozotocin-induced diabetes Cu,Zn-SOD and catalase renal mRNA levels are greater than in normal rats. This difference is prevented by sufficient insulin dosage to normalize plasma glucose and might be due to an increased production of free radicals.

Animals↗
PubMed1997

Radon and lung carcinogenesis: mutability of p53 codons 249 and 250 to 238Pu alpha-particles in human bronchial epithelial cells.

Radon-222, a decay product of uranium-238 and a source of high linear energy transfer (LET) alpha-particles, has been implicated in the increased risk of lung cancer in uranium miners as well as non-miners. p53 mutation spectrum studies of radon-associated lung cancer have failed to show any specific mutational hot spot with the exception of a single study in which 31% of squamous cell and large cell lung cancers from uranium miners showed a p53 codon 249 AGGarg --> ATGmet mutation. Although the results of laboratory studies indicate that double-strand breaks and deletions are the principal genetic alterations caused by alpha-particles, uncertainty still prevails in the description of DNA damage in radon-associated human lung cancer. In the present study, we have evaluated the mutability of p53 codons 249 and 250 to alpha-particles in normal human bronchial epithelial (NHBE) cells using a highly sensitive genotypic mutation assay. Exposure of NHBE cells to a total dose of 4 Gy (equivalent to approximately 1460 working level months in uranium mining) of high LET alpha-radiation induced codon 249 AGG --> AAG transitions and codon 250 CCC --> ACC transversions with absolute mutation frequencies of 3.6 x 10(-7) and 3.8 x 10(-7) respectively. This mutation spectrum is consistent with our previous report of radon-associated human lung cancer.

Adolescent↗
PubMed1996

Differential effect of interleukin-4 and transforming growth factor beta 1 on expression of proto-oncogenes and autocrine insulin-like growth factor II in colorectal carcinoma cells.

We have compared the effect of interleukin-4 and transforming growth factor beta 1 on proliferation and gene expression in two colorectal carcinoma cell lines, LS513 and LS1034. Transforming growth factor beta 1 was a potent inhibitor for both cells lines and virtually abolished de novo DNA synthesis. Interleukin-4 inhibited thymidine incorporation up to 60 and 45%, respectively. While both cytokines exerted a comparable cyto-inhibitory activity they displayed differential effects on proto-oncogene expression. Transforming growth factor beta 1 markedly down-regulated c-myc in LS1034 but not in LS513 cells. In contrast, expression of c-fos was induced by interleukin-4 in LS513 but not in LS1034 cells. Interestingly, in agreement with their cyto-inhibitory activity both cytokines suppressed the expression of insulin-like growth factor II in LS1034, which is an autocrine growth factor for these cells.

Cell Division↗
PubMed1996

High glucose induces antioxidant enzymes in human endothelial cells in culture. Evidence linking hyperglycemia and oxidative stress.

It has been suggested that oxidative stress may play an important role in the pathogenesis of diabetic complications. Hyperglycemia may cause increased production of free radicals, and evidence supports a prominent role for these reactive molecules as mediators of endothelial cell dysfunction in diabetes. It has been demonstrated that active oxygen species induce antioxidant enzyme expression in some tissues, and this phenomenon is considered proof of an existing oxygen-dependent toxicity. In this study, human endothelial cells from umbilical vein, immortalized human endothelial cells, and immortalized human endothelial cells transfected to express high glutathione peroxidase levels were grown in normal and high-glucose conditions. High glucose delayed replication after 7 and 14 days of culture of human endothelial cells, both from umbilical vein and immortalized, while transfected cells were not affected. The activity and the mRNA expression of the antioxidant enzymes CuZn-superoxide-dismutase, Mn-superoxide-dismutase, catalase, and glutathione peroxidase were evaluated after 2, 7, and 14 days of culture. High glucose at days 7 and 14 induced an overexpression of CuZn-superoxide-dismutase, catalase, and glutathione peroxidase in both human endothelial cells from umbilical vein and immortalized human endothelial cells, while in transfected cells it did not. This study demonstrates that high glucose induces an increase in antioxidant enzyme levels in human endothelial cells, suggesting that elevated glucose levels may produce an oxidative stress in the cells.

Animals↗
PubMed1995

The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-kappa B.

BACKGROUND: The inducible, higher eukaryotic transcription factor NF-kappa B is activated by a variety of stimuli. Several lines of evidence have suggested that reactive oxygen intermediates (ROIs) serve as messengers for most if not all of these stimuli. To identify the relevant ROI species and to gain more direct evidence for an involvement of ROIs as messengers, we investigated whether changes in the levels of enzymes that control intracellular ROI levels affect the activation of NF-kappa B. RESULTS: Cell lines stably overexpressing the H2O2-degrading enzyme catalase were deficient in activating NF-kappa B in response to tumor necrosis factor alpha (TNF) or okadaic acid. The catalase inhibitor aminotriazol restored NF-kappa B induction. In contrast, stable overexpression of cytoplasmic Cu/Zn-dependent superoxide dismutase (SOD), which enhances the production of H2O2 from superoxide, potentiated NF-kappa B activation. The level of cytoplasmic NF-kappa B-I kappa B complex was unchanged, indicating that synthesis of NF-kappa B was not affected. CONCLUSIONS: Our data show that one ROI species, H2O2 acts as a messenger in the TNF- and okadaic acid-induced post-translational activation of NF-kappa B. Superoxide is only indirectly involved, as a source for H2O2. These data explain the inhibitory effects of many antioxidative compounds on the activation of NF-kappa B and its target genes. H2O2 is overproduced in response to various stimuli, and normal levels of catalase appear insufficient to remove it completely. H2O2 can therefore accumulate and act as an intracellular messenger molecule in the response to pathogens.

Animals↗
PubMed1995

The defence against free radicals protects endothelial cells from hyperglycaemia-induced plasminogen activator inhibitor 1 over-production.

It has been shown that hyperglycaemia may stimulate plasminogen activator inhibitor 1 (PAI-1) over-production in human endothelial cells in culture. At the same time, it has been shown that glucose may enolize, producing free radicals. In this study, the possibility that hyperglycaemia stimulates PAI-1 over-production in human endothelial cells in culture by generating free radicals has been evaluated. For this purpose two experimental models were used: human endothelial cells transfected to express high glutathione peroxidase levels cultured in hyperglycaemic media, and human endothelial cells cultured in hyperglycaemic media with the antioxidant GSH. Cells grown in 20 mM glucose produced higher values of PAI-1 with respect to controls. The production of PAI-1 was not influenced by hyperglycaemia in transfected cells. GSH in the medium reduced hyperglycaemia-induced PAI-1 over-production, but also reduces the basal production of PAI-1 in the cells grown in normal glucose concentration. These data show that antioxidant defences may reduce hyperglycaemia-induced PAI-1 over-production in human endothelial cells in culture. The hypothesis that oxidative stress may play an important role in the pathogenesis of diabetic complications is then supported by this study.

Antioxidants↗
PubMed1995

Interleukin 4 down-regulates expression of c-kit and autocrine stem cell factor in human colorectal carcinoma cells.

Stem cell factor (SCF) is a cytokine which plays an important role in the development of precursor cells. We have investigated the expression of SCF and its receptor, the c-kit proto-oncogene, in human colorectal carcinoma cell lines. Using reverse transcription-PCR, we confirmed the expression of c-kit in two lines (LS174T and LS1034) and of SCF in 9 of 11 cell lines tested. In a Northern blot, a single transcript of 6.6 kb was detected for SCF mRNA. In addition, two lines (LS174T and HT29) synthesized SCF protein, as detected by Western blot analysis. SCF stimulated proliferation and colony formation of LS174T in a dose-dependent manner up to 160%. A half-maximal effect was obtained with about 5.5 ng/ml of SCF under both growth conditions. LS174T cells expressed the M(r) 145,000 c-kit protein on the cell surface and a neutralizing anti-c-kit mAb inhibited colony formation of LS174T by 40%. Interleukin 4 (IL-4) completely inhibited SCF-induced proliferation of LS174T cells. Interestingly, IL-4 induced an almost complete down-regulation of both c-kit and SCF expression in LS174T. Our findings suggest that in LS174T cells, an SCF-mediated autocrine loop is functional and that IL-4 down-regulates the expression of both the receptor and the ligand of this circuit.

Base Sequence↗
PubMed1994

Blockade of the insulin-like growth-factor-I receptor inhibits growth of human colorectal cancer cells: evidence of a functional IGF-II-mediated autocrine loop.

Insulin-like growth factors (IGFs) are potent proliferation stimulators for numerous tumor cells and often function as autocrine growth factors. We have previously shown that exogenous IGF-I and IGF-II enhance proliferation of colorectal carcinoma cells. The biological signal of both factors is transmitted through the IGF-I receptor (IGF-I-R). This receptor was expressed in 12/12 colorectal carcinoma cell lines tested. alpha IR3, a neutralizing monoclonal antibody (MAb) directed against the human IGF-I-R, inhibited proliferation in 7/12 lines (Caco-2, HT-29, LS411N, LS513, LS1034, WiDr and SW620), as reflected by a reduction of MTT conversion (19 to 42%), a decrease in cell number (39 to 72%) and an increase in doubling time (up to 2-fold). In addition, in 4 cell lines (Caco-2, LS513, LS1034, WiDr) alpha IR3 suppressed colony formation in methylcellulose (40 to 84%). Excess of exogenous IGF completely neutralized alpha IR3-mediated inhibitory effects. Northern blot analysis revealed abundant expression of 2 IGF-II transcripts of 5.0 and 4.3 kb in LS1034 cells. In addition, we observed that growth inhibition by alpha IR3 was correlated with a more differentiated phenotype. Our results suggest that growth of many colorectal carcinoma cell lines is regulated by autocrine IGF-II-mediated stimulation of the IGF-I-R.

Antibodies, Monoclonal↗
PubMed1994

Glutathione peroxidase compensates for the hypersensitivity of Cu,Zn-superoxide dismutase overproducers to oxidant stress.

The balance between several components of the antioxidant defenses appears to be important for the cellular resistance to oxidative stress. While Cu,Zn-superoxide dismutase (SOD) transfectants of mouse epidermal cells JB6 clone 41 were sensitized to oxidants produced by xanthine/xanthine oxidase (X/XO) consecutive transfection with catalase corrected their hypersensitivity (Amstad, P., Peskin, A., Shah, G., Mirault, M. E., Moret, R., Zbinden, I., and Cerutti, P. (1991) Biochemistry 30, 9305-9313). We studied the effect of the transfection of bovine selenoglutathione peroxidase (GPx) on the sensitivity of JB6 clone 41 and its SOD transfectants. Sensitivity to DNA strand breakage and killing by X/XO was reversely related to the activity ratios GPx over SOD. A GPx-transfectant of JB6 clone 41 cells with a GPx/SOD ratio of 3.8 was very strongly protected. The hypersensitivity of the SOD clones with GPx/SOD ratios of 0.4 was corrected or overcorrected by secondary transfection with bovine Se-GPx resulting in increased activity ratios GPx/SOD of 1 to 2.4. Our results indicate that small deviations from the physiological activity ratios of GPx/SOD have a dramatic effect on the resistance of cells to oxidant-induced damage to the genome and cell killing.

Animals↗
PubMed1994

Ultraviolet B light-induced mutagenesis of p53 hotspot codons 248 and 249 in human skin fibroblasts.

Mutations in the p53 tumor suppressor gene are detected in approximately half of non-melanoma skin cancers. The type of base-pair changes observed strongly suggests solar radiation as the causative mutagen. Mutations are distributed nonrandomly and form moderate hotspots. We studied the capacity of ultraviolet B light (UVB, 280-320 nm) to induce base-pair changes into the p53 exon 7 sequence extending from nt 14067 to 14075 in human skin fibroblasts. This sequence contains hotspot codon 248. UVB induced mostly C-->A and G-->T transversions. The base-pair change with the highest relative abundance was C-->A in the first position of codon 250 (CCC-->ACC), followed by (in diminishing relative abundance) G-->T in the third position of codon 249 (AGG-->AGT), C-->A in the first position of codon 248 (CGG-->AGG), and C-->A in the third position of codon 247 (AAC-->AAA). The C-->T transition in the third position of codon 247 (AAC-->AAT) occurred with moderate efficiency. These base-pair changes are compatible with pyrimidine photodimers as premutagenic lesions, but they could also form opposite 8-hydroxyguanine, which is the major oxidation product of guanine. No evidence was obtained for the presence of tandem double CC-->TT transitions in the untranscribed strand at codons 247/248 and 250. The relative abundance of mutations induced by UVB in the p53 sequence extending from codon 247 to 250 in human fibroblasts does not correlate with mutations observed in the DNA from non-melanoma skin cancer. This lack of correlation suggests that the mutability of this p53 sequence at the DNA level plays only a minor role in the pathogenesis of non-melanoma skin cancer in humans.

Base Sequence↗
PubMed1994

Differential cytotoxicity in mouse epidermal JB6 cells: a potential mechanism for oxidant tumor promotion.

It has been suggested that superior antioxidant defense systems protect promotion-sensitive (p+t) mouse epidermal JB6 clone 41 cells from excessive deleterious effects of oxidants, allowing their clonal expansion in contrast to that of promotion-resistant (p-) clone 30 cells. In support of this concept, we report that oxidants produced by xanthine/xanthine oxidase cause more cytotoxicity, cellular damage, and cell death in p-cells. Cell surface blebbing, an early morphological consequence of oxidative injury, was detected in cultures grown on glass coverslips. While a rise in cytosolic ionized calcium ([Ca2+]i) preceding bleb formation was observed in both p+ and p- cells by digital imaging fluorescence microscopy, elevated levels of [Ca2+]i were sustained longer in p- cells. This increase was dependent on the levels of extracellular ionized calcium ([Ca2+]e) in p+ but not p- cells. We conclude that the superior antioxidant defense or improved Ca2+ buffering of promotable clone 41 cells protects them from more severe deregulation of [Ca2+]i and, as a consequence, from excessive cytotoxicity after exposure to oxidant promoters.

Animals↗
PubMed1994

The role of the cellular antioxidant defense in oxidant carcinogenesis.

Oxidant carcinogens interact with multiple cellular targets including membranes, proteins, and nucleic acids. They cause structural damage to DNA and have the potential to mutate cancer-related genes. At the same time, oxidants activate signal transduction pathways and alter the expression of growth- and differentiation-related genes. Indeed, the carcinogenic action of oxidants results from the superposition of these genetic and epigenetic effects. All cells possess elaborate antioxidant defense systems that consist of interacting low and high molecular weight components. Among them, superoxide dismutases (SOD), glutathione peroxidases (GPx), and catalase (CAT) play a central role. Our studies with mouse epidermal cells demonstrate that the balance between several antioxidant enzymes rather than the activity of a single component determines the degree of protection. Unexpectedly, increased levels of Cu,Zn-SOD alone in stable transfectants resulted in sensitization to oxidative chromosomal aberrations and DNA strand breaks. However, a concomitant increase in CAT or GPx in double transfectants corrected or overcorrected the hypersensitivity of the SOD clones depending on the ratios of activities CAT/SOD or GPx/SOD. The cellular antioxidant capacity also affected oxidant induction of the growth-related immediate early protooncogene c-fos. Increases in CAT or SOD reduced the accumulation of c-fos message, albeit for different reasons. The cellular antioxidant defense also affects the action of UVB light (290-320 nm) that represents the most potent carcinogenic wavelength range of the solar spectrum. UVB light is known to exert its action in part through oxidative mechanisms. Increases in CAT and GPx protected mouse epidermal cells from UVB-induced DNA breakage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Oxy-radical induced mutagenesis of hotspot codons 248 and 249 of the human p53 gene.

Oxidants are suspected to represent important human carcinogens. They are mutagenic and may participate in the activation of proto-oncogenes and the inactivation of tumor suppressor genes. We have studied the capacity of hydrogen peroxide plus ferric chloride (FeCl3) to induce base pair changes in the hotspot codons 248 and 249 of the p53 tumor suppressor gene in human fibroblasts. In codon 248 (CGG) H2O2/FeCl3 only induced the transversion of G to C in the second position and the transition of G to A in the third position. No evidence was obtained for spontaneous or oxidant-induced deamination of 5-methylcytosine in the CpG dinucleotide of codon 248 since neither C to T transitions in the first position nor G to A transitions in the middle position were observed. H2O2/FeCl3 efficiently induced G to T transversions at both G-residues of codon 249 (AGG) and C to A transversions at the first position of codon 250 (CCC). It is evident that H2O2/FeCl3 possesses essentially the same mutagenic specificity for codons 249 and 250 of p53 as bulky carcinogens such as aflatoxin B1, benzo(a)pyrene or heterocyclic amines. In particular, it is not possible to eliminate oxidants from the list of candidate carcinogens which may be responsible for the high incidence of p53 codon 249 AGT mutations in hepatocellular carcinoma from certain areas of the world.

Cells, Cultured↗