Phosphorylation of p53 protein in A549 human pulmonary epithelial cells exposed to asbestos fibers. (Research).We examined effects of asbestos exposure on the phosphorylation phosphorylation, chemical process in which a phosphate group is added to an organic molecule. In living cells phosphorylation is associated with respiration, which takes place in the cell's mitochondria, and photosynthesis, which takes place in the chloroplasts. of p53 protein in human pulmonary epithelial type II cells (A549), which express wild-type p53. In cells exposed to two different types of asbestos, chrysotile chrysotile: see serpentine.
Fibrous variety of the magnesium silicate mineral serpentine; it is the most important asbestos mineral. Individual fibres are white and silky, but the aggregate in veins is usually green or yellowish. (~1-6% iron content) and crocidolite crocidolite
or blue asbestos
Gray-blue to green, highly fibrous (asbestiform) form of the amphibole mineral riebeckite. It has higher tensile strength than chrysotile asbestos. (~27% iron content) fibers, at the doses of 1, 5, and 10 [micro]g/[cm.sup.2] for 24 hr, the levels of p53 phosphorylated at Ser15 and p53 protein were correlated with the dose. On a per-weight basis, chrysotile was more potent in inducing Ser15 phosphorylation and accumulation of p53 protein than was crocidolite. After exposure to 10 [micro]g/[cm.sup.2] chrysotile, the levels of p53 phosphorylated at Ser15 and of p53 protein increased after 18 hr. Among serines in p53 protein immunoprecipitated from A549 cells treated with chrysotile, only Ser15 was markedly phosphorylated. In contrast, no dear phosphorylation was observed at Ser6, Ser9, Ser20, Ser37, Ser46, or Ser392. Blocking of the extracellular signal-regulated protein kinase pathway with U0126 or inhibition of p38 activity with SB203580 did not suppress chrysotile-induced Ser15 phosphorylation. On the other hand, treatment with wortmannin, an inhibitor of DNA-activated protein kinase and ataxia--telangiectasia mutated, suppressed both chrysotile-induced Ser15 phosphorylation and accumulation of p53 protein. Treatment with either catalase catalase /cat·a·lase/ (kat´ah-las) a hemoprotein enzyme that catalyzes the decomposition of hydrogen peroxide to water and oxygen, protecting cells. or N-acetyicysteine failed to suppress chrysotile-induced Ser15 phosphorylation, suggesting that reactive oxygen species reactive oxygen species,
n molecules and ions of oxygen that have an unpaired electron, thus rendering them extremely reactive. Many cellular structures are susceptible to attack by ROS contributing to cancer, heart disease, and cerebrovascular disease. do not play a major role in the phosphorylation of p53 protein. The present results show that asbestos, particularly chrysotile, induces phosphorylation of p53 protein at Ser15 in A549 cells depending on a DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. damage--signaling pathway. Key words: A549 cells, chrysotile, crocidolite, p53, phosphorylation, reactive oxygen species, SB203580, Ser15, U0126, wortmannin.
Asbestos is a family of crystalline-hydrated silicates with a fibrous geometry, including chrysotile [[Mg.sub.6][Si.sub.4][O.sub.10][(OH).sub.8]], the curly serpentine-type, and crocidolite [[Na.sub.2][([Fe.sup.3+]).sub.2] [([Fe.sup.2+]).sub.3][Si.sub.8][O.sub.22][(OH).sub.2]], the rodlike amphibole-type asbestos (Mossman et al. 1990; Mossman and Gee 1989). Clinical and epidemiologic studies have established that asbestos fibers are associated with the development of pulmonary interstitial fibrosis, lung cancer, and malignant mesothelioma (Mossman et al. 1990; Mossman and Gee 1989). Asbestos exposure induces diverse cellular events related to lung injury (Jaurand 1997; Kamp and Weitzman 1999; Manning et al. 2002). However, the molecular mechanisms of asbestos-induced fibrogenesis and carcinogenesis and of repair of lung injury are not fully understood.
The p53 tumor suppressor protein plays an important role in the control of genomic integrity or the elimination of damaged or tumorigenic tu·mor·i·gen·ic
Capable of causing tumors. cells (Bargonetti and Manfredi 2002; Levine 1997; Vousden 2000). The mutational inactivation inactivation /in·ac·ti·va·tion/ (in-ak?ti-va´shun) the destruction of biological activity, as of a virus, by the action of heat or other agent. of p53 protein is one of the most common genetic events that occur in human cancers (Hupp et al. 2000). It has been reported that the frequency of p53 protein accumulation is increased in lung carcinomas of patients with clinical or histologic asbestos exposure (Nuorva et al. 1994). Treatment with crocidolite increased the number of p53 protein--expressing cells in A549 human pulmonary epithelial cells (Johnson et al. 1997; Johnson and Jaramillo 1997), and treatment with chrysotile induced the elevation of p53 protein level in rat pleural Pleural
Pleural refers to the pleura or membrane that enfolds the lungs.
Mentioned in: Pneumothorax
emanating from or pertaining to the pleura. mesothelial mesothelial
pertaining to the mesothelium.
cover all serous membranes and normally found in fluid samples aspirated from the pleural or peritoneal cavities. cells (Levresse et al. 1997). In addition, inhaled chrysotile induced the expression of p53 protein at fiber deposition sites (bronchiolar--alveolar duct bifurcations) in rat lungs (Mishra et al. 1997). These findings suggest a possible association between asbestos exposure and accumulation of p53 protein in the pulmonary tissues or cells.
The p53 protein is phosphorylated on multiple residues in both the amino- and carboxy-terminal domains by several different protein kinases (Giaccia and Kastan 1998; Lakin and Jackson 1999; Meek 1998). Among serine serine (sĕr`ēn), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein. residues, phosphorylation at position 15 has been shown to play an important role in the stabilization and subsequent induction and transactivation Transactivation is an increased rate of gene expression triggered either by endogenous cellular or viral proteins - transactivators. These protein factors act in trans (i.e., intermolecularly). function of p53 (Dumaz and Meek 1999; Shieh et al. 1997; Siliciano et al. 1997). Members of the phosphatidylinositol 3-kinase--related kinase (PIKK) family such as DNA-activated protein kinase (DNA-PK DNA-PK DNA-dependent Protein Kinase ) and ataxia-telangiectasia mutated (ATM) have been implicated in the phosphorylation of p53 at Ser15 (Giaccia and Kastan 1998; Lakin and Jackson 1999; Meek 1998). Furthermore, extracellular signal--regulated protein kinase (ERK ERK Extracellular Signal-Regulated Kinase
ERK Electronic Records Keeping
ERK Externally Regulated Kinases ) and p38, the members of mitogen-activated protein kinase Mitogen-activated protein (MAP) kinases (EC 18.104.22.168) are serine/threonine-specific protein kinases that respond to extracellular stimuli (mitogens) and regulate various cellular activities, such as gene expression, mitosis, differentiation, and cell survival/apoptosis. (MAPK MAPK Mitogen-Activated Protein Kinase
MAPK Map Kinase ), have been reported to induce p53 phosphorylation at Ser15 (Bulavin et al. 1999; Kwon et al. 2002; Persons et al. 2000; She et al. 2000, 2001; Shih et al. 2001; Wang and Shi 2001).
In the present study, we examined whether two different types of asbestos, chrysotile and crocidolite, induce the phosphorylation of p53 at Ser15 and other serines in A549 human pulmonary epithelial type II cells, which express wild-type p53 (Jia et al. 1997). Using inhibitors to the members of MAPK and PIKK, we also determined the protein kinases responsible for asbestos-induced p53 phosphorylation. Because reactive oxygen species have been shown to be an important mediator responsible for pulmonary toxicity of asbestos (Kamp et al. 1992; Kamp and Weitzman 1999; Quinlan et al. 1994), effects of antioxidants such as catalase and N-acetyl-cysteine on asbestos-induced p53 phosphorylation were also examined.
Materials and Methods
Preparation of asbestos fibers. Union Internationale Contre le Cancer (UICC UICC Union International Contre le Cancer International Union against Cancer ) standard samples of Rhodesian chrysotile and crocidolite asbestos were used in the present study. The fibers were suspended in distilled water at the concentration of 1 mg/mL. Then the suspensions were passed through a 22-gauge needle eight times and sterilized ster·il·ize
tr.v. ster·il·ized, ster·il·iz·ing, ster·il·iz·es
1. To make free from live bacteria or other microorganisms.
2. by autoclaving. Before the addition to the medium, the fibers were dispersed by sonication sonication /son·i·ca·tion/ (son?i-ka´shun) exposure to sound waves; disruption of bacteria by exposure to high-frequency sound waves.
n. for 10 rain and vortexed.
Cell culture and treatments. A549 cells were obtained from Health Science Research Resources Bank (Japan Health Sciences Foundation, Osaka, Japan) and grown in Earle's minimum essential medium with nonessential amino acids, 10% fetal bovine serum Fetal bovine serum ( or foetal bovine serum) is serum taken from the fetuses of cows. Fetal Bovine Serum (or FBS) is the most widely used serum in the culturing of cells. In some papers the expression foetal calf serum is used. , 100 U/mL penicillin, and 100 [micro]g/mL streptomycin streptomycin (strĕp'tōmī`sĭn), antibiotic produced by soil bacteria of the genus Streptomyces and active against both gram-positive and gram-negative bacteria (see Gram's stain), including species resistant to other (Gibco BRL BRL
In currencies, this is the abbreviation for the Brazilian Real.
The currency market, also known as the Foreign Exchange market, is the largest financial market in the world, with a daily average volume of over US $1 trillion. , Life Technologies, Inc., Rockville, MD, USA) in a humidified atmosphere of 5% C[O.sub.2], 95% air at 37[degrees]C. For each experiment, exponentially growing A549 cells were plated at 1.5 x [10.sup.5] cells/well in 12-well culture plates or 3 x [10.sup.6] cells/dish in 100-mm culture dishes, and cultured for 1 day before the experiments.
A549 cells were incubated with the complete medium containing 1, 5, or 10 [micro]g of fibers/[cm.sup.2] of culture vessel for 24 hr. As a reference, 20 [micro]M cadmium chloride (Cd[Cl.sub.2]; Sigma Chemical Co., St. Louis, MO, USA), which has been shown to induce the phosphorylation of p53 protein at Ser15 (Matsuoka and Igisu 2001), was used. In the time course study, A549 cells were incubated with 10 [micro]g of fibers/[cm.sup.2] for 3-24 hr. Untreated control cells were incubated with the complete medium alone and treated identically to the cells exposed to asbestos.
U0126, SB203580, and wortmannin (Calbiochem, La Jolla, CA, USA) were dissolved in dimethyl sulfoxide (DMSO DMSO dimethyl sulfoxide.
Dimethyl sulfoxide; a colorless hygroscopic liquid obtained from lignin, used as a penetrant to convey medications into the tissues.
n. ). Catalase (Calbiochem) was dissolved in distilled water. N-Acetylcysteine (Sigma) was dissolved in phosphate-buffered saline (PBS PBS
in full Public Broadcasting Service
Private, nonprofit U.S. corporation of public television stations. PBS provides its member stations, which are supported by public funds and private contributions rather than by commercials, with educational, cultural, ) immediately before use, and the pH was adjusted to 7.4 with 2N NaOH. A549 cells were preincubated with the complete medium containing each compound for 30 min (for wortmannin), 1 hr (for U0126, SB203580, and catalase), or 12 hr (for N-acetylcysteine). The control cells were preincubated with the complete medium either alone or containing DMSO at the concentration used in the treated cells (0.05 or 0.08%). Then preincubated and control cells were treated with or without asbestos fibers for 24 hr.
Western immunoblotting immunoblotting,
n the immunologic methods for isolating and quantitatively measuring immunoreactive substances. When used with immune reagents such as monoclonal antibodies, the process is known generically as
Western blot analysis. . After the incubation with asbestos fibers or Cd[Cl.sub.2], cells were washed with PBS and lysed with sodium dodecyl sulfate Sodium dodecyl sulfate (or sulphate) (SDS or NaDS) (C12H25NaO4S),is an anionic surfactant that is used in household products such as toothpastes, shampoos, shaving foams and bubble baths for its thickening effect and its ability to (SDS 1. (company) SDS - Scientific Data Systems.
2. (tool) SDS - Schema Definition Set. )--polyacrylamide gel Laemmli sample buffer. Cell lysates were collected, sonicated, and boiled for 5 min. Aliquots equivalent to 2 x [10.sup.5] cells were subjected to SDS--polyacrylamide gel electrophoresis on a 10% polyacrylamide gel and transferred to a nitrocellulose nitrocellulose, nitric acid ester of cellulose (a glucose polymer). It is usually formed by the action of a mixture of nitric and sulfuric acids on purified cotton or wood pulp. membrane (Hybond-ECL; Amersham Pharmacia Biotech, Buckinghamshire, UK). The membrane was blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 for 2 hr at room temperature. The antibodies used were p53 (Pab 1801) antibody, actin (I-19) antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and phospho-p53 (Ser6, Ser9, Ser15, Ser20, Ser37, Ser46, and Ser392) antibodies (Cell Signaling Technology, Inc., Beverly, MA, USA). The membrane was incubated overnight at 4[degrees]C with the primary antibody diluted 1:100 (for p53 antibody) or 1:1,000 (for phospho-p53 antibodies). Protein was detected with a Phototope-HRP Western blot detection kit (Cell Signaling Technology). After immunodetection, some blots were incubated with Restore Western Blot Stripping Buffer (Pierce Chemical Co., Rockford, IL, USA) for 30 min at room temperature and reprobed with actin antibody diluted 1:500 for 1 hr at room temperature.
Immunoprecipitation--Western immunoblotting. After the incubation with asbestos fibers, cells were washed with PBS and lysed in RIPA RIPA. The bank of a river, or the place beyond which the waters do not in their natural course overflow.
2. An extraordinary overflow does not change the banks of the river. Poth. Pand. lib. 50, h.t. See Banks of rivers; Riparian proprietors; Rivers. buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 0.6 mM phenylmethylsulfonyl fluoride, 30 [micro]L/mL aprotinin aprotinin /apro·ti·nin/ (ap?ro-ti´nin) an inhibitor of proteolytic enzymes used to reduce perioperative blood loss in patients undergoing cardiopulmonary bypass during coronary artery bypass graft. (Sigma A6279), and 1 mM sodium orthovanadate. After sonication, the lysates were stored on ice for 1 hr and centrifuged at 10,000 x g for 10 min at 4[degrees]C. Then, cell lysates equivalent to 6 x [10.sup.6] cells were incubated overnight at 4[degrees]C with 10 [micro]g of p53 (Pab 1801) antibody agarose conjugate (Santa Cruz Biotechnology). The pellet was washed four times with RIPA buffer and suspended in SDS--polyacrylamide gel Laemmli sample buffer. Phosphorylation of p53 protein at Ser6, Ser9, Ser15, Ser20, Ser37, Ser46, and Ser392 in the same samples was analyzed after SDS--polyacrylamide gel electrophoresis and immunoblotting with respective phospho-p53 antibodies. After stripping, the blots were reprobed with p53 antibody.
Accumulation of p53 phosphorylated at Ser15 and p53 protein by asbestos exposure. Exposure to 20 [micro]M Cd[Cl.sub.2] for 24 hr induced a clear phosphorylation of p53 at Ser15 and an accumulation of p53 protein in A549 cells (Figure 1), as has been shown in MCF-7 human breast cancer cells (Matsuoka and Igisu 2001). Similarly, in A549 cells exposed to 1-10 [micro]g/[cm.sup.2] of chrysotile or crocidolite for 24 hr, accumulation of both p53 phosphorylated at Ser15 and p53 protein was found depending on the dose (Figure 1). However, chrysotile exposure induced more marked accumulation of p53 phosphorylated at Ser15 and p53 protein than crocidolite exposure at each dose of 1, 5, and 10 [micro]g/[cm.sup.2]. On the other hand, the levels of actin were not changed by exposure to chrysotile, crocidolite, or Cd[Cl.sub.2] (Figure 1).
[FIGURE 1 OMITTED]
After exposure to 10 [micro]g/[cm.sup.2] chrysotile, the levels of p53 phosphorylated at Ser15 and p53 protein increased after 18 hr, whereas actin levels were not changed after 3-24 hr exposures (Figure 2). In A549 cells exposed to 10 [micro]g/[cm.sup.2] crocidolite, clear Ser15 phosphorylation was not found at 3-18 hr (data not shown). Hereafter, we focused on p53 phosphorylation induced by exposure to 10 [micro]g/[cm.sup.2] chrysotile for 24 hr.
Phosphorylation of serine residues in p53 protein. We examined whether serine residues other than position 15 in p53 protein can be phosphorylated in response to chrysotile exposure. In p53 protein immunoprecipitated from A549 cells exposed to chrysotile, the marked phosphorylation was found only at Ser15 (Figure 3). On the other hand, clear phosphorylation was not observed on Ser6, Ser9, Ser20, Ser37, Ser46, or Ser392 in p53 immunoprecipitated, whereas equal amounts of p53 protein were detected (Figure 3).
[FIGURE 3 OMITTED]
Effects of MAPK inhibitors on Ser15 phosphorylation in p53 protein. Treatment with U0126 (5 and 20 [micro]M), an inhibitor of both activated and nonactivated forms of MAPK/ERK kinase (MEK Noun 1. MEK - a terrorist organization formed in the 1960s by children of Iranian merchants; sought to counter the Shah of Iran's pro-western policies of modernization and opposition to communism; following a philosophy that mixes Marxism and Islam it now attacks the 1/2) (Favata et al. 1998), or with SB203580 (5 and 20 [micro]M), a p38 inhibitor (Cuenda et al. 1995), did not suppress chrysotile-induced Ser15 phosphorylation (Figure 4A, B). Treatment with a higher concentration of U0126 (50 [micro]M) or SB203580 (50 [micro]M) also failed to suppress chrysotile-induced Ser15 phosphorylation, whereas phosphorylated forms of ERK or p38 were not detected in A549 cells treated with each compound (data not shown).
Effects of wortmannin on Ser15 phosphorylation in p53 protein. Treatment with wortmannin (1 and 5 [micro]M), an inhibitor of DNA-PK and ATM (Sarkaria et al. 1998), suppressed chrysotile-induced Ser15 phosphorylation and accumulation of p53 protein depending on the concentration (Figure 5). The levels of actin were not affected by treatment with wortmannin (Figure 5).
[FIGURE 5 OMITTED]
Effects of catalase and N-acetylcysteine on Ser15 phosphorylation in p53 protein. Treatment with catalase (1 and 5 KU/mL) or N-acetylcysteine (1 and 5 mM) did not suppress chrysotile-induced Ser15 phosphorylation (Figure 6A, B). In addition, accumulation of p53 protein in A549 cells exposed to chrysotile was not suppressed by treatment with catalase or N-acetylcysteine (data not shown).
In A549 cells exposed to two different types of asbestos, chrysotile and crocidolite, at the doses of 1-10 [micro]g/[cm.sup.2] for 24 hr, the levels of p53 phosphorylated at Ser15 and p53 protein were found to be elevated. Among serines in p53 protein immunoprecipitated from A549 cells treated with 10 [micro]g/[cm.sup.2] chrysotile for 24 hr, only Ser15 was markedly phosphorylated. In contrast, no clear phosphorylation was observed at other serine residues examined (Ser6, Ser9, Ser20, Ser37, Ser46, and Ser392). Ser15 has been identified as a site on p53 protein phosphorylated in response to DNA-damaging agents such as ionizing radiation (Shieh et al. 1997; Siliciano et al. 1997), ultraviolet radiation (She et al. 2000; Shieh et al. 1997; Siliciano et al. 1997), camptothecin (Shieh et al. 1997), cisplatin (Persons et al. 2000), chromium (Wang and Shi 2001), and cadmium (Matsuoka and Igisu 2001). The present study clearly showed for the first time that asbestos exposure induces phosphorylation of p53 protein at Ser15 in a human pulmonary epithelial cell line. Phosphorylation of p53 at Ser15 was shown to reduce the binding of murine double minute 2 (MDM (Modular Digital Multitrack) An audio recorder that mixes and records multiple tracks of digital audio. The two major MDM technologies are ADAT and DTRS. See ADAT and DTRS. 2) (Shieh et al. 1997), an E3 ligase ligase /li·gase/ (li´gas) (lig´as) any of a class of enzymes that catalyze the joining together of two molecules coupled with the breakdown of a pyrophosphate bond in ATP or a similar triphosphate. that targets both p53 and itself for ubiquitination (Vousden 2000). The level of p53 mRNA as determined using reverse transcriptase--polymerase chain reaction analysis was not elevated in A549 cells treated with chrysotile or crocidolite at 10 [micro]g/[cm.sup.2] for 24 hr (data not shown). Therefore, asbestos-induced Ser15 phosphorylation might be responsible for the accumulation of p53 protein at least in part.
When two types of asbestos fibers were compared in their potency to induce Ser15 phosphorylation and accumulation of p53 protein, chrysotile was more marked than was crocidolite on a per-weight basis. In agreement with our results, treatment with 10 [micro]g/[cm.sup.2] crocidolite for 24 or 48 hr induced less significant increases in p53 protein level than did treatment with chrysotile in rat pleural mesothelial cells (Levresse et al. 1997). When DNA breakage was determined using the single-cell gel (Comet) assay, chrysotile was reported to induce more abnormalities in comet parameters than did crocidolite in rat pleural mesothelial cells (Levresse et al. 2000). If this is the case in human pulmonary epithelial cells as well, a higher genotoxic genotoxic /ge·no·tox·ic/ (je´no-tok?sik) damaging to DNA: pertaining to agents known to damage DNA, thereby causing mutations, which can result in cancer.
adj. potential of chrysotile asbestos might underlie the marked phosphorylation and accumulation of p53 protein observed in the present study.
The MAPKs, including ERK, p38, and c-Jun N[H.sub.2]-terminal kinase (JNK JNK Jun N-terminal Kinase
JNK Junk (File Name Extension) ), are a family of serine/threonine protein kinases that transmit extracellular signals into the nucleus (Schaeffer and Weber 1999). Exposure to chrysotile has been reported to activate ERK in rat pleural mesothelial cells in vitro (Zanella et al. 1996) and mouse pulmonary epithelial cells in vivo (Robledo et al. 2000), although its effects on p38 and JNK are not known. On the other hand, Ser15 phosphorylation induced by various cellular stimuli such as ultraviolet radiation (Bulavin et al. 1999; She et al. 2000), cisplatin (Persons et al. 2000), L-thyroxine (Shih et al. 2001), chromium (Wang and Shi 2001), resveratrol res·ver·a·trol
A natural compound found in grapes, mulberries, peanuts, and other plants or food products, especially red wine, that may protect against cancer and cardiovascular disease by acting as an antioxidant, antimutagen, and (She et al. 2001), and 3-methylcholanthrene (Kwon et al. 2002) has been reported to be mediated by ERK and/or p38. However, in the present study, treatment with neither U0126 nor SB203580 suppressed chrysotile-induced Ser15 phosphorylation, indicating it is unlikely that ERK and p38 are responsible for p53 phosphorylation at Ser15 in A549 cells exposed to chrysotile.
In contrast to MAPK inhibitors, treatment with wortmannin suppressed both chrysotile-induced Ser15 phosphorylation and accumulation of p53 protein. These results suggest that chrysotile-induced Ser15 phosphorylation is dependent on PIKK family such as DNA-PK and ATM, and support the possible role of Ser15 phosphorylation in the accumulation of p53 protein. DNA-PK and ATM are activated after cellular exposure to agents that induce DNA double-strand breaks (DSBs) or other discontinuities in DNA (Canman et al. 1994; Gottlieb and Jackson 1993; Morozov et al. 1994). It has been reported that chrysotile exposure at doses of 8 or 16 [micro]g/[cm.sup.2] for 24 hr induced DNA DSBs in both wild-type and DNA DSB DSB Dispute Settlement Body (World Trade Organization)
DSB Double Strand Break
DSB Defense Science Board (US DoD)
DSB Deep Sand Bed
DSB Deutscher Sportbund repair--deficient mutant Chinese hamster ovary cells (Okayasu et al. 1999). Therefore, DNA damage induced by chrysotile exposure might activate the signaling pathways leading to PIKK activation, and resultant p53 activation might contribute to the protection of cells from fatal genetic injury.
The iron associated with asbestos fibers promotes the formation of hydroxyl radicals via the modified Haber-Weiss (Fenton) reaction (Kamp et al. 1992; Kamp and Weitzman 1999; Quinlan et al. 1994). Although chrysotile contains only ~1-6% iron primarily as a surface contaminant contaminant /con·tam·i·nant/ (kon-tam´in-int) something that causes contamination.
something that causes contamination. (Kamp et al. 1992; Kamp and Weitzman 1999), this asbestos has been reported to generate hydroxyl hydroxyl /hy·drox·yl/ (hi-drok´sil) the univalent radical OH.
The univalent radical or group OH, a characteristic component of bases, certain acids, phenols, alcohols, carboxylic radical-like species and cause DNA strand breaks in human pulmonary epithelial-like WI-26 cells (Kamp et al. 1995). However, treatment with catalase or N-acetylcysteine failed to suppress chrysotile-induced Ser15 phosphorylation in the present study. Furthermore, treatment with deferoxamine (1 and 5 mM), an iron chelator chelator A chemical–eg, EDTA that binds metal ions from solutions. See Chelation therapy. , did not suppress chrysotile-induced Ser15 phosphorylation but induced Ser15 phosphorylation significantly even in the absence of asbestos (data not shown). Consistent with these results, crocidolite asbestos, which has a high iron content (~27%) (Kamp et al. 1992; Kamp and Weitzman 1999), induced less marked Ser15 phosphorylation in A549 cells. Thus, chrysotile-induced p53 phosphorylation at Ser15 might be caused by mechanisms not based on reactive oxygen species, such as the direct physical interaction with cellular components or the generation of reactive nitrogen species (Tanaka et al. 1998).
The p53 protein plays a central role in the control of cell cycle progression or apoptotic cell death (Bargonetti and Manfredi 2002; Levine 1997; Vousden 2000). Therefore, there is a possibility that cell cycle arrest (Levresse et al. 1997) and apoptosis (Aikoh et al. 1998; Broaddus et al. 1996; Dopp et al. 1995; Hamilton et al. 1996; Wu et al. 2000) found in cells exposed to chrysotile might be caused by an activated form of p53 protein. On the other hand, in transgenic mice with reduced and enhanced p53 functions specifically targeted within the pulmonary epithelium, asbestos exposure induced less and more severe fibrogenic lesions in the lung, respectively (Nelson et al. 2001). These findings suggest that p53 protein in cells exposed to asbestos has the fibrosis progressive function as well as the tumor suppressive sup·pres·sive
Tending or serving to suppress.
Adj. 1. suppressive - tending to suppress; "the government used suppressive measures to control the protest" function. In other words Adv. 1. in other words - otherwise stated; "in other words, we are broke"
put differently , activation of p53 by asbestos exposure might intensify its pulmonary toxicity further by forming a positive feedback loop. To clarify the functions of asbestos-induced p53 activation in the pulmonary cells, genes, and/or pathways that are activated after phosphorylation of p53 at Ser15, the critical event for its transactivation (Dumaz and Meek 1999; Shieh et al. 1997; Siliciano et al. 1997), should be investigated.
Aikoh T, Tomokuni A, Matsukii T, Hyodoh F, Ueki H, Otsuki T, et al. 1998. Activation-induced cell death in human peripheral blood lymphocytes Peripheral Blood Lymphocytes (PBL): These are the mature lymphocytes (small white immune cells) that are found circulating in the blood, as opposed to organs, such as the lymph nodes, spleen, thymus, liver or bone marrow. These cells consist of T cells, NK cells and B cells. after stimulation with silicate in vitro. Int J Oncol 12:1355-1359.
Bargonetti J, Manfredi JJ. 2002. Multiple roles of the tumor suppressor p53. Curr Opin Oncol 14:86-91.
Broaddus VC, Yang L, Scavo LM, Ernst JD, Boylan AM. 1996. Asbestos induces apoptosis of human and rabbit pleural mesothelial cells via reactive oxygen species. J Clin Invest 98:2050-2059.
Bulavin DV, Saito S, Hollander MC, Sakaguchi K, Anderson CW, Appella E, et al. 1999. Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation. EMBO J 18:6845-6854.
Canman CE, Wolff AC, Chen C-Y, Fornace AJ Jr, Kastan MB. 1994. The p53-dependent G1 cell cycle checkpoint Cell cycle checkpoints are control mechanisms that ensure the fidelity of cell division in eukaryotic cells. These checkpoints verify whether the processes at each phase of the cell cycle have been accurately completed before progression into the next phase. pathway and ataxia-telangiectasia. Cancer Res 54:5054-5058.
Cuenda A, Rouse J, Doza YN, Meier R, Cohen cohen
(Hebrew: “priest”) Jewish priest descended from Zadok (a descendant of Aaron), priest at the First Temple of Jerusalem. The biblical priesthood was hereditary and male. P, Gallagher TF, et al. 1995. SB 203580 is a specific inhibitor of a MAP kinase homologue homologue /ho·mo·logue/ (hom´ah-log)
1. any homologous organ or part.
2. in chemistry, one of a series of compounds distinguished by addition of a CH2 group in successive members. which is stimulated by cellular stresses and interleukin-1. FEBS FEBS Federation of European Biochemical Societies Lett 364:229-233.
Dopp E, Nebe B, Hahnel C, Papp T, Alonso B, Simko M, et al. 1995. Mineral fibers induce apoptosis in Syrian hamster embryo fibroblasts Fibroblasts
A type of cell found in connective tissue; produces collagen.
Mentioned in: Skin Grafting . Pathobiology pathobiology /patho·bi·ol·o·gy/ (-bi-ol´ah-je) pathology.
The study or practice of pathology with greater emphasis on the biological than on the medical aspects. 63:213-221.
Dumaz N, Meek DW. 1999. Serine15 phosphorylation stimulates p53 transactivation but does not directly influence interaction with HDM HDM - SPECIAL 2. EMBO J 18:7002-7010.
Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, et al. 1998. Identification of a novel inhibitor of mitogen-activated protein kinase kinase Mitogen-activated protein kinase kinase (sic) is a kinase enzyme which phosphorylates mitogen-activated protein kinase. It is also known as MAP2K. It is classified as EC 22.214.171.124. . J Biol Chem 273:18623-18632.
Giaccia AJ, Kastan MB. 1998. The complexity of p53 modulation: emerging patterns from divergent signals. Genes Dev 12:2973-2983.
Gottlieb TM, Jackson SP. 1993. The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell 72:131-142.
Hamilton RF, Li L, Iyer R, Holian A. 1996. Asbestos induces apoptosis in human alveolar macrophages. Am J Physiol Lung Cell Mol Physiol 271:L813-L819.
Hupp TR, Lane DP, Ball KL. 2000. Strategies for manipulating the p53 pathway in the treatment of human cancer. Biochem J 352:1-17.
Jaurand M-C. 1997. Mechanisms of fiber-induced genotoxicity Genotoxic substances are a type of carcinogen, specifically those capable of causing genetic mutation and of contributing to the development of tumors. This includes both certain chemical compounds and certain types of radiation. . Environ Health Perspect 105(suppl 5):1073-1084.
Jia L-Q, Osada M, Ishioka C, Camo M, Ikawa S, Suzuki T, et al. 1997. Screening the p53 status of human cell lines using a yeast functional assay. Mol Carcinog 19:243-253.
Johnson NF, Carpenter TR, Jaramillo R J, Liberati TA. 1997. DNA damage-inducible genes as biomarkers for exposures to environmental agents. Environ Health Perspect 105(suppl 4):913-918.
Johnson NF, Jaramillo RJ. 1997. p53, Cipl, and Gadd153 expression following treatment of A549 cells with natural and man-made vitreous vitreous /vit·re·ous/ (vit´re-us)
1. glasslike or hyaline.
2. vitreous body.
primary persistent hyperplastic vitreous fibers. Environ Health Perspect 105(suppl 5):1143-1145.
Kamp DW, Graceffa P, Pryor WA, Weitzman SA. 1992. The role of free radicals in asbestos-induced diseases. Free Radic Biol Med 12:293-315.
Kamp DW, Israbian VA, Preusen SE, Zhang CX, Weitzman SA. 1995. Asbestos causes DNA strand breaks in cultured pulmonary epithelial cells: role of iron-catalyzed free radicals. Am J Physiol Lung Cell Mol Physiol 268:L471-L480.
Kamp DW, Weitzman SA. 1999. The molecular basis of asbestos induced lung injury. Thorax thorax, body division found in certain animals. In humans and other mammals it lies between the neck and abdomen and is also called the chest. The skeletal frame of the thorax is formed by the sternum (breastbone) and ribs in front and the dorsal vertebrae in back. 54:638-652.
Kwon Y-W Y-W Yule-Walker equation , Ueda S, Ueno M, Yodoi J, Masutani H. 2002. Mechanism of p53-dependent apoptosis induced by 3-methylcholanthrene: involvement of p53 phosphorylation and p38 MAPK. J Biol Chem 277:1837-1844.
Lakin ND, Jackson SP. 1999. Regulation of p53 in response to DNA damage. Oncogene oncogene
Gene that can cause cancer. It is a sequence of DNA that has been altered or mutated from its original form, the proto-oncogene (see mutation). Proto-oncogenes promote the specialization and division of normal cells. 18:7644-7655.
Levine AJ. 1997. p53, the cellular gatekeeper for growth and division. Cell 88:323-331.
Levresse V, Renier A, Fleury-Feith J, Levy F, Moritz S, Vivo C, et al. 1997. Analysis of cell cycle disruptions in cultures of rat pleural mesothelial cells exposed to asbestos fibers. Am J Respir Cell Mol Biol 17:660-671.
Levresse V, Reniar A, Levy F, Broaddus VC, Jaurand M-C. 2000. DNA breakage in asbestos-treated normal and transformed (TSV TSV - tab-separated values 40) rat pleural mesothelial cells. Mutagenesis mutagenesis /mu·ta·gen·e·sis/ (mu?tah-jen´e-sis)
1. the production of change.
2. the induction of genetic mutation.
n. pl. 15:239-244.
Manning CB, Vallyathan V, Mossman BT. 2002. Diseases caused by asbestos: mechanisms of injury and disease development. Int Immunopharmacol 2:191-200.
Matsuoka M, Igisu H. 2001. Cadmium induces phosphorylation of p53 at serine 15 in MCF-7 cells. Biochem Biophys Res Commun 282:1120-1125.
Meek DW. 1998. New developments in the multi-site phosphorylation and integration of stress signalling at p53. Int J Radiat Biol 74:729-737.
Mishra A, Liu J-Y, Brody AR, Morris CF. 1997. Inhaled asbestos fibers induce p53 expression in the rat lung. Am J Respir Cell Mol Biol 16:479-485.
Morozov VE, Falzon M, Anderson CW, Kuff EL. 1994. DNA-dependent protein kinase is activated by nicks and larger single-stranded gaps. J Biol Chem 269:16884-16688.
Mossman BT, Bignon J, Corn M, Seaton A, Gee JBL JBL James Bullough Lansing (audio/speaker engineer)
JBL Journal of Biblical Literature
JBL John Bradshaw Leyfield (wrestler)
JBL Jonathan Bell Lovelace (investment research) . 1990. Asbestos: scientific developments and implications for public policy. Science 247:294-301.
Mossman BT, Gee JBL. 1989. Asbestos-related diseases. N Engl J Med 320:1721-1730.
Nelson A, Mendoza T, Hoyle GW, Brody AR, Fermin C, Morris CF. 2001. Enhancement of fibrogenesis by the p53 tumor suppressor protein in asbestos-exposed rodents. Chest 120 (suppl):33S-34S.
Nuorva K, Makitaro R, Huhti E, Kamel D, Vahakangas K, Bloigu R, et al. 1994. p53 protein accumulation in lung carcinomas of patients exposed to asbestos and tobacco smoke. Am J Respir Grit Care Med 150:528-533.
Okayasu R, Takahashi S, Yamada S, Hei TK, Ullrich RL. 1999. Asbestos and DNA double strand breaks. Cancer Res 59:298-300.
Persons DL, Yazlovitskaya EM, Pelling JC. 2000. Effect of extracellular signal-regulated kinase on p53 accumulation in response to cisplatin. J Biol Chem 275:35778-35785.
Quinlan TR, Marsh JP, Janssen YMW YMW You're Most Welcome
YMW Yahoos Most Wanted (forum)
YMW Yahoo Messenger for the Web , Borm PA, Mossman BT. 1994. Oxygen radicals and asbestos-mediated disease. Environ Health Perspect 102(suppl 10):107-110.
Robledo RF, Buder-Hoffmann SA, Cummins AB, Walsh ES, Taatjes DJ, Mossman BT. 2000. Increased phosphorylated extracellular signal-regulated kinase immunoreactivity associated with proliferative and morphologic lung alterations after chrysotile asbestos inhalation in mice. Am J Pathol 156:1307-1316.
Sarkaria JN, Tibbetts RS, Busby EC, Kennedy AP, Hill DE, Abraham RT. 1998. Inhibition of phosphoinositide 3-kinase related kinases by the radiosensitizing agent wortmannin. Cancer Res 58:4375-4382.
Schaeffer H J, Weber MJ. 1999. Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol Cell Biol 19:2435-2444.
She Q-B, Bode AM, Ma W-Y, Chen N-Y, Dong Z. 2001. Resveratrol-induced activation of p53 and apoptosis is mediated by extracellular-signal-regulated protein kinases and p38 kinase. Cancer Res 61:1604-1610.
She Q-B, Chen N, Dong Z. 2000. ERKs and p38 kinase phosphorylate phos·pho·ryl·ate
tr.v. phos·pho·ryl·at·ed, phos·pho·ryl·at·ing, phos·pho·ryl·ates
To add a phosphate group to (an organic molecule).
phos p53 protein at serine 15 in response to UV radiation. J Biol Chem 275:20444-20449.
Shieh S-Y, Ikeda M, Taya Y, Prives C. 1997. DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 91:325-334.
Shih A, Lin H-Y, Davis FB, Davis PJ. 2001. Thyroid hormone promotes serine phosphorylation of p53 by mitogen-activated protein kinase. Biochemistry 40:2870-2878.
Siliciano JD, Canman CE, Taya Y, Sakaguchi K, Appella E, Kastan MB. 1997. DNA damage induces phosphorylation of the amino terminus of p53. Genes Bev 11:3471-3481.
Tanaka S, Choe N, Hemenway DR, Zhu S, Matalon S, Kagan E. 1998. Asbestos inhalation induces reactive nitrogen species and nitrotyrosine formation in the lungs and pleura pleura (plr`ə), membranous lining of the upper body cavity and covering for the lungs. of the rat. J Clin Invest 102:445-454.
Vousden KH. 2000. p53: death star. Cell 103:691-694.
Wang S, Shi X. 2001. Mechanisms of Cr(VI)-induced p53 activation: the role of phosphorylation, mdm2 and ERK. Carcinogenesis 22:757-762.
Wu J, Liu W, Koenig K, Idell S, Broaddus VC. 2000. Vitronectin adsorption to chrysotile asbestos increases fiber phagocytosis phagocytosis: see endocytosis.
A mechanism by which single cells of the animal kingdom, such as smaller protozoa, engulf and carry particles into the cytoplasm. and toxicity for mesothelial cells. Am J Physiol Lung Cell Mol Physiol 279:L916-L923.
Zanella CL, Posada J, Tritton TR, Mossman BT. 1996. Asbestos causes stimulation of the extracellular signal-regulated kinase 1 mitogen-activated protein kinase cascade after phosphorylation of the epidermal growth factor receptor This article is about a cell suface receptor. For estimated measure of kidney function (eGFR), see Glomerular filtration rate.
The epidermal growth factor receptor . Cancer Res 56:5334-5338.
Masato Matsuoka, (1) Hideki Igisu, (1) and Yasuo Morimoto (2)
Departments of (1) Environmental Toxicology and (2)Occupational Pneumology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Kitakyushu, Japan
Address correspondence to M. Matsuoka, Dept. of Environmental Toxicology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. Telephone: 81-93-6917404. Fax: 81-93-692-4790. E-mail: masatomm@ med.uoeh-u.ac.jp
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Received 19 August 2002; accepted 21 November 2002.