Toxicological Sciences 55, 60-68 (2000)
Copyright © 2000 by the Society of Toxicology
Carcinogenicity |
Apoptosis and P53 Induction in Human Lung Fibroblasts Exposed to Chromium (VI): Effect of Ascorbate and Tocopherol
,§,1
* Departments of Pharmacology and
Pathology, and Programs in
Molecular and Cellular Oncology and
§ Genetics, The George Washington University Medical Center, Washington, DC 20037
Received December 17, 1999; accepted January 10, 2000
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Some forms of hexavalent chromium [Cr(VI)] are known to cause damage to respiratory tract tissue, and are thought to be human lung carcinogens. Because Cr(VI) is mutagenic and carcinogenic at doses that evoke cell toxicity, the objective of these experiments was to examine the effect of Cr(VI) on the growth, survival, and mode of cell death in normal human lung fibroblasts (HLF cells). DNA adduct formation was monitored as a marker for bioavailability of genotoxic chromium. We also examined the modulation of these endpoints by vitamins C and E. Long-term Cr(VI) exposures were employed, which decreased clonogenic cell survival by 25% to 95% in a dose-dependent manner. The predominant cellular response to Cr(VI) was growth arrest. We found that Cr(VI) caused up to 20% of HLF cells to undergo apoptosis, and documented apoptotic morphology and the phagocytosis of apoptotic bodies by neighboring cells. P53 levels increased 4- to 6-fold in chromium-treated cells. In contrast with previous studies using CHO cells, the present study using HLFs found that pretreatment with either vitamin C or E did not exhibit a significant effect on Cr-induced apoptosis or clonogenic survival. In addition, pretreatment with vitamin C did not affect the p53 induction observed after chromium treatment. Neither vitamin had any effect on Cr-DNA adduct formation. These data indicate that although pretreatment with vitamin C or E alters the spectrum of cellular and/or genetic lesions induced by chromium(VI), neither vitamin altered the initiation or progression of apoptosis in diploid human lung cells.
Key Words: genotoxicity; carcinogenesis; ascorbate; tocopherol; LL-24 cells; clonogenic survival; electron microscopy.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Some forms of chromium(VI) [(Cr(VI)] are human occupational respiratory tract irritants and carcinogens. They are known to cause nasal polyps, atrophy of the nasal septum, and lung and nasal cancer (reviewed in (IARC, 1990)). The deposition of chromium waste in landfills and waterways by chromate industries has also raised concerns about the potential of chromium as an environmental hazard (Burke et al., 1991
). Although these compounds have been recognized as irritants and carcinogens for over 100 years, the mechanisms by which they have these effects are not fully understood. Studies have shown that the insoluble forms of Cr(VI), such as zinc and lead chromate, are the most potent carcinogens, probably due to their persistence within the lungs (Bencko, 1985; Gibb and Chen, 1989; Mancuso, 1997). The more soluble forms of chromium (VI) do not persist in exposed tissue and are rapidly reduced by extracellular body fluids to non-toxic and non-carcinogenic chromium(III) (De Flora et al., 1997). Recent studies have shown that particulate Cr(VI), in the form of lead chromate, undergoes enhanced dissolution at the cell surface where the chromate oxyanion is released (Wise, J. P., Sr. et al., 1994). These ions enter cells through the sulfate ion transporter, are rapidly reduced by glutathione and ascorbate into several reactive intermediate species, and finally to chromium(III) (Aiyar et al., 1992; Standeven and Wetterhahn, 1992, 1991a, b; Stearns and Wetterhahn, 1994). During this reduction process, these reactive intermediates, which may include chromium(V), chromium(IV), and reactive oxygen, bind both protein and DNA, and cause many types of DNA damage (Manning et al., 1994, 1992; Sugiyama et al., 1986; Wise et al., 1992; Xu et al., 1992), as reviewed by Singh (1998). Cell culture experiments have shown that long (24 h) exposures to soluble sodium chromate can mimic many of the effects of lead chromate in vitro (Blankenship et al., 1997; Wise, J.P., Sr. et al., 1994 DNA damage and cellular toxicity. These two events can be linked by the p53 gene, in some cases. P53 has been shown to induce growth arrest and apoptosis after treatment of some cells with DNA-damaging agents, and may be a mechanism by which damaged cells are removed from a population (Wyllie et al, 1994). The object of the current study was to examine the mechanism and potential modulation of chromium-induced toxicity in a relevant human cell culture model. Although most lung cancers are epithelial in nature, lung fibroblasts are potential targets for toxicity caused by hexavalent chromium, and may indirectly contribute to tumor formation through stromal-epithelial interactions. In addition, lung fibroblasts may be a direct target of chromium-induced toxicity, and the death of these cells may directly contribute to the toxicities of acute chromium exposure. The HLF cells used in this study were normal, diploid human lung fibroblasts. To examine the effect of Cr(VI) on this cell type, we tested the hypotheses that sodium chromate inhibits growth of HLF cells, that the mode of cell death seen after chromium treatment is apoptosis, and that this these effects can be modulated by the antioxidants ascorbate and alpha-tocopherol.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials
All chemicals, unless otherwise noted, were purchased from Sigma (St. Louis, MO). Sodium chromate solutions were prepared fresh in deionized water, and sterile-filtered before use. ApoAlert Apoptosis Kit was purchased from Clonetics (Palo Alto, CA). All cell culture materials were purchased from Fisher Scientific (Pittsburgh, PA). Cell counting was done using a Coulter Cell Counter (Multisizer II, Luton, UK). Fluorescence microscopy was performed using an Opelco (Sterling, VA) AX70 microscope with green wide range filter suitable for FITC analysis.
Cell Culture
HLF cells (LL-24 cell line, ATCC #151-CCL) are normal diploid human lung fibroblasts which were isolated from a 5-year-old male. They were used before passage 17, and were not put through more than 2 freeze-thaw cycles in order to ensure consistent cellular responses. They were grown in F12K medium (GIBCO, Grand Island, NY) with 15% FBS (Hyclone, Logan, UT), at 5% CO2, 37°C.
Cell Growth and Survival
Cells were seeded at 500,000 cells per T25 flask with 5 ml complete medium. After 48 h, sodium chromate was added to the medium, with final concentrations from 3–9 µM. To determine cell growth, after 24 h, the medium was aspirated, the cells rinsed with phosphate-buffered saline (PBS) twice, and the medium replaced. At the time points indicated after exposure, cells were removed from the flasks with 0.25% trypsin and counted twice using a Coulter Cell Counter, 3 flasks per time point. To determine survival, after 24 h, the medium was aspirated, the cells were rinsed with PBS, and were removed from the flasks with trypsin. Each sample was then reseeded at 1000 cells per 60-mm dish with 5-ml complete medium, in triplicate. After 14 days, the colonies that formed were stained with crystal violet, counted, and survival, relative to untreated control, was determined.
Electron Microscopy
Cells were seeded at 500,000 cells per 10-cm dish and treated with sodium chromate for 2 h. After treatment, cells were washed with cold PBS, then the dish filled with 10 ml cold 0.1 M sodium cacodylate-buffered 2.5% glutaraldehyde (pH 7.2), and sealed with parafilm. Cells were then observed and images captured with an electron microscope, as previously described (Blankenship et al., 1994).
Phosphatidyl Serine Assay
Cells were seeded at 80,000 cells per 60-mm plate in 5-ml medium. After 24 h, sodium chromate, at a final concentration of 6 µM, was added to the medium. After a 24-h treatment at 6 µM, the medium was aspirated, cells rinsed with PBS, and 5 ml of fresh medium added to plates. At various times after treatment, plates were taken for analysis. To determine the extent of apoptosis, the medium was aspirated, cells rinsed with PBS, and detached from plates with trypsin. The cells were centrifuged at 1000 rpm, 4°C for 5 min, the supernatant aspirated, the pellet resuspended in PBS, counted, centrifuged as previously, and resuspended in 100 µl binding buffer (provided with kit). Five liters of Annexin(V)-FITC was then added to each sample and incubated in the dark for 5–15 min before it was placed on a slide with coverslip, for microscopic observation. For quantitation, the number of FITC-stained cells were compared to the total number of cells present in the same field.
Western Blotting
Cells were seeded at 106 cells per 10-cm culture dish. After 24 h, cells were pretreated with 1 mM ascorbate then treated with 9 µM sodium chromate for 24 h, 200 M sodium chromate for 2 h, or 20 J of ultraviolet irradiation. UV irradiation was done with a germicidal lamp, with the dose calculated before exposure using a spectroradiometer (Optronics Laboratory, Inc., model 742). Two h after the sodium chromate treatments, or 18 h after UV irradiation, cells were scraped from the dishes into RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing 0.1 mg/ml PMSF, 30 µl aprotinin/ml RIPA, and 1 mM sodium orthovanidate. Cells were incubated in RIPA plus the inhibitors for 20 min, then microfuged at 12,000 rpm for 20 min at 4°C. The supernatant was collected and total cell protein was quantitated from whole cell lysates using Pierce BCA protein assay. Forty g of each sample was electrophoresed on 10% SDS–polyacrylamide gel and electroblotted onto PVDF membrane (Biorad, Hercules, CA). Blots were incubated overnight with 5% nonfat dry milk (Biorad, Hercules, CA) in TBS-T buffer (0.1% Tween-20 (Biorad, Hercules, CA), 2.7 mM KCl, 138 mM Nace, 20 mM Tris Base pH 7.4), then probed for 1 h with 10 µl p53 Ab6 (Calbiochem, Cambridge, MA) in 10 ml TBS-T with 5% milk, and washed with TBS-T. Goat antimouse IgG-linked horseradish peroxidase secondary antibody (1:10,000 dilution) (Amersham, Heights, IL) in TBS-T with 5% milk added, and incubated for 1 h, then washed with TBS-T. Secondary antibody was detected using enhanced chemiluminescence followed by autoradiography.
Measurement of Cr-DNA adduct formation.
Cells were seeded at 300,000 cells per T75 flask, and incubated. After 24 h, 1 mM ascorbate or 20 µM tocopherol was added for 24 h. Flasks were then rinsed with PBS and media replaced with 75 µM Na251CrO4 per flask (specific activity 6 mCi/mg) in complete medium. After 2 h, cells were put on ice, rigorously rinsed 2–3 times in order to remove unbound or loosely bound chromium, and scraped from the flasks. DNA adducts were then quantitated by isolating the DNA and scintillation counting, as described previously (Xu et al., 1996).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We determined the effect of hexavalent chromium on growth and clonogenic survival of HLF cells. Figure 1
shows the effect of 9 µM sodium chromate for 24 h on the growth of these same cells. We find that the major effect of this treatment was complete growth arrest for at least 3 days. Over time, the treatment also caused a decrease in cell number, which is indicative of cell death.
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In order to confirm cell death and to determine the mode by which this took place, we examined sodium chromate-treated HLF cells by electron microscopy. Figure 2A
shows the morphology of normal, untreated HLF cells in culture, while Figure 2B shows HLF cells that have been treated with sodium chromate. The mode of cell death of treated cells is apoptosis, characterized by cell shrinkage, intact organelles and membranes, and chromatin condensation. Necrotic cells were rarely observed and may derive from late state apoptosis. In addition, Figure 2B shows an apoptotic body that has been phagocytized by a neighboring cell, as would occur physiologically.
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This toxicity was confirmed in Figure 3
, which shows the results of clonogenic survival assays. These assays indicate the ability of the treated cells not only to survive, but also to replicate and form colonies. Figure 3 shows that a 24-h sodium chromate treatment causes a similar decrease in clonogenic survival, from 100% to 3% as the sodium chromate dose increases from 0–9 µM. Clonogenic survival indicates that these cells exhibited a decreased ability to survive and replicate to form colonies after treatment with sodium chromate, probably due to both the severe, prolonged growth arrest indicated in Figure 1
and because of the apoptosis indicated in Figure 2.
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Because vitamins C and E have been shown to alter the speciation of DNA damage after Cr(VI) treatment without altering uptake, we tested the hypothesis that pretreatment with antioxidant vitamins C or E would protect HLF cells against Cr(VI)-induced apoptosis and thereby increase clonogenic survival. Figure 4
shows that, following a 24-h sodium chromate treatment, there appeared to be a consistent trend towards increased cloning ability of pretreated cells; however, this slight difference was not significant in either paired or unpaired statistical tests at any dose. Likewise, vitamin C pretreatment does not have a statistically significant effect on apoptosis after a 24-h treatment (Fig. 5). The effect of vitamin E pretreatment on clonogenic survival and apoptosis induced by sodium chromate is show in Figures 6 and 7, respectively. Figure 6 shows that vitamin E had no protective effect on clonogenic survival after a 24-h sodium-chromate treatment and Figure 7 shows that vitamin E had no effect on apoptosis. Overall, Figures 4–7 indicate that the antioxidant vitamins C and E did not significantly protect against sodium chromate-induced apoptosis, nor did they change clonogenic survival. The lack of protection from clonogenic and apoptotic death is consistent with our finding that neither vitamin had any effect on chromium-DNA adduct levels (Fig. 8). This also indicates that neither vitamin had any effect on the uptake of sodium chromate by the cells.
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We have previously shown that pretreatments with vitamin C but not E markedly increased clonogenic survival and decreased chromosomal damage in the CHO cell line (Blankenship et al., 1997
). The lack of a statistically significant effect using these vitamins in the present study caused us to seek to evaluate a molecular marker for cell damage and response. Increased intracellular p53 levels signify genotoxic stress and p53 has been implicated in both growth arrest and apoptosis. Figures 9 and 10 show that p53 levels increase 4–6-fold in chromium-treated HLF cells after either a 2- or a 24-h exposure to sodium chromate. A treatment of 20 J of ultraviolet radiation was included as a postive control of p53 induction (Fig. 9). Pretreatment with vitamin C did not have a significant effect on the increase of p53 levels induced by chromium (Fig. 10).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At doses that overwhelm both extracellular and intracellular reducing capacity, chromate oxyanions and their reduction products cause many types of DNA damage including single-strand breaks, double-strand breaks, DNA adducts, DNA–DNA cross-links, DNA–protein cross-links and clastogenesis (Manning et al., 1994
, 1992; Singh et al., 1998). Some of these types of DNA damage, such as clastogenesis, can be decreased by pretreating cells with the antioxidant vitamins C and E, which suggests that oxidative damage might have a role in causing these types of DNA damage within the cell (Blankenship et al., 1997; Manning et al., 1992; Singh et al., 1998; Wise J.P., Sr. et al., 1994. Other types of damage, such as chromium-DNA adducts and DNA-Cr-DNA cross-links cannot be prevented by vitamin pretreatments, suggesting that their formation is not dependent on free radical formation (Singh et al., 1998). We have previously shown that in CHO cells (AA8 strain) exposure to Cr(VI) initiated apoptosis, which was the predominant mode of death in these cells (Blankenship et al., 1994). It was found that pretreatment with vitamin C, but not vitamin E, significantly increased clonogenic survival after hexavalent chromium treatment, whereas clastogenesis was markedly decreased by both vitamins, and adducts were not decreased by either vitamin (Blankenship et al., 1997). This lack of correlation suggested that Cr(VI)-induced apoptosis in CHO cells was not initiated by cellular or genetic lesions, which are affected by vitamins C or E.
These previous studies provided important mechanistic data into hexavalent chromium-induced toxicity, but did not utilize human respiratory cells. Studying the regulation of apoptosis and survival in the most relevant cell types available is particularly important, because the regulation of apoptosis is cell-type specific and modulation of apoptosis may not necessarily lead to equivalent alterations in long-term survival in different cell types (Wyllie et al., 1980). One specific concern in this study was the effect of p53 status in CHO cells, since p53 is an important regulator of apoptosis and CHO cells have mutant p53 (Lee et al., 1997) whereas HLF cells are primary human lung fibroblasts, which are diploid and presumably have wild type p53. The studies described here characterized the interaction of chromium(VI) with diploid human lung fibroblasts (HLF), which are likely targets for chromium toxicity in vivo, since they come in contact with chromium following exposure. In addition, these cells may contribute to tumor formation through cell–cell interactions with epithelial cells, as in the model proposed for some colorectal neoplasms, where the presence of mutations in stromal cells precedes cancer of epithelial cells (Kinzler and Vogelstein, 1998). It is important to note that the HLF cells in these studies were not used past passage 16, and that they were not subjected to more than two freeze-thaw cycles. We have found that HLF cells become resistant to chromium-induced apoptosis after passage 16, or even sooner if subjected to more than 2 freeze-thaw cycles. Later-passage cells would appear frozen, unable to either divide or die after chromium treatment (data not shown). Recently it was reported that older (passage 15 and older) HLF cells may not undergo apoptosis, but instead go into a state of premature permanent growth arrest (Gadbois and Lehnert, 1997). Thus, one explanation for our observations may be that, as the diploid cells progress towards senescence, they become resistant to apoptosis. Alternatively, the stressful conditions of passaging and freeze-thaw cycles may gradually select for HLF cells that are abnormally resistant to cell death. In either case, this presents an interesting model to study both the genotypic and phenotypic parameters that distinguish the apoptosis-permissive and apoptosis-resistant states.
We have found that the predominant response of normal human lung fibroblasts to Cr(VI) is an immediate population-wide growth arrest. Even at moderately cytotoxic doses, this growth arrest is protracted for several days, during which time up to 25% of the cells die apoptotically. We have previously demonstrated that Cr(VI) arrests cells in S-phase of the cell cycle and that Cr-induced DNA–DNA cross-links caused guanine-specific arrest of replication by DNA polymerase (Bridgewater et al., 1994; Manning et al., 1992; Singh et al., 1998; Xu et al., 1996). Thus, one aspect of Cr-induced growth arrest may be through direct obstruction of DNA replication.
An alternative mechanism for Cr-induced growth arrest may be related to our demonstration that p53 is activated following hexavalent chromium exposure at the same doses at which DNA damage and growth inhibition take place. p53 is thought to be upregulated at the protein level by preventing the binding of MDM-2, which targets p53 for degradation. MDM-2 is prevented from interacting with p53 either by phosphorylation of MDM-2 by DNA-PK (Mayo et al., 1997), or phosphorylation of p53 by JNK (Jun/SAP kinase) (Fuchs et al., 1998). Increased p53 levels caused by the attenuation of degradation has been demonstrated to stop progression of the cell cycle in response to the exposure of a cell to DNA damaging agents (Dulic et al., 1994). The mechanism for this p53-dependent cell cycle block includes transcriptional activation of p21waf1/cip1, which inhibits cyclin-dependent kinases to block entry into S-phase at the G1 cell cycle checkpoint (Agarwal et al., 1995; Kastan et al., 1991; Kuerbitz et al., 1992; Stewart et al., 1995). One possible trigger for this type of growth inhibition by hexavalent chromium may be related to our finding of premature arrest of transcriptional elongation by RNA polymerase (Xu et al., 1996). This may trigger the accumulation of p53, as has been suggested for ultraviolet radiation by Ljungman and Zhang (1996).
We have found that the sodium-chromate exposure caused a decrease in cloning efficiency of these cells, indicative of a decrease in survival and/or replicative competence. Because chromium (VI) may be both toxic and carcinogenic to humans, we further investigated the mode by which these human cells die, using electron microscopy. We found that these cells die predominantly by apoptosis and that apoptotic cells can be actively phagocytized by neighboring cells. The accumulation of p53 may be important here as well because although p53 can stimulate transcription of p21waf1/cip1, leading to growth arrest, it can also initiate signaling pathways that cause apoptosis (Wyllie et al., 1994) either by stimulating transcription of bax (Miyashita and Reed, 1995) or by directly interacting with the Fas apoptotic pathway (Bennett et al., 1998).
Both of these pathways may ultimately converge on the mitochondria, which have been implicated as playing a central role in the regulation of apoptosis. The release of cytochrome c from the mitochondria activates a cascade of proteases, the caspases, which are responsible for many of the morphological features of apoptosis (Green and Kroemer, 1998; Liu et al., 1996; Wyllie, 1997). In this case, cytochrome c may be released from the mitochondria in response to signaling molecules. Alternatively, hexavalent chromium is taken up by the mitochondria where it damages mitochondrial DNA, proteins, and lipids, and inhibits respiration (Alexander et al., 1982; Ryberg and Alexander, 1990). These effects of chromium(VI) may directly damage the integrity of the mitochondria and cause the release of cytochrome c, thereby initiating apoptosis.
The modulation of chromium-induced apoptosis in human lung fibroblasts with vitamins C and E was also investigated. We had previously shown that pretreatment of CHO cells with vitamin C increased clonogenic survival after treatment with Cr(VI) but vitamin E had no effect on survival (Blankenship et al., 1997). Recently though, it has become apparent that interventions designed to prevent apoptosis do not necessarily result in parallel increases in clonogenic survival (Carlisle et al., 1999). Furthermore, different cell types may respond differently to cellular stress depending on their genetic status and intra- and extracellular milieu. Therefore, we explored whether vitamins C and E would alter apoptosis and clonogenic survival in normal human lung cells. We found that HLF cells were more sensitive to sodium chromate exposure than were CHO cells, exhibiting decreased survival and increased apoptosis at slightly lower exposure levels. This study shows that pretreatment with vitamin C did not significantly increase clonogenic survival after exposure in HLF cells, as it did in CHO cells (Blankenship et al., 1997), and it did not significantly protect against sodium chromate-induced apoptosis. This result was surprising because Stearns et al. have shown that changing the ratio of vitamin C to chromium(VI) in vitro changes the reduction kinetics and alters the spectra of the reactive intermediates formed (Stearns et al., 1994, 1995), and we have shown, both in vitro (Bridgewater et al., 1994a) and in living cells (Blankenship et al., 1997; Wise et al., 1993) that vitamin C markedly alters the spectrum of resulting DNA damage, as have others (Capellmann et al., 1995; Sugiyama et al., 1991). For example, although vitamin C may protect cells from the formation of some types of DNA damage such as strand breakage and clastogenesis (Blankenship et al., 1997), it does not protect cells from the formation of chromium-DNA adducts and chromium-induced DNA crosslinks (Blankenship et al., 1997; Capellmann et al., 1995; Sugiyama, 1992). Our results suggest that in HLF cells, the trigger for apoptotic cell death is for the most part unresponsive to vitamin C-sensitive lesions. This is further supported by our observation that pretreatment with vitamin C had no effect on p53 induction after Cr(VI) exposure.
An important difference between HLF cells and CHO cells is their p53 status. HLF cells express wild type p53, whereas CHO cells express mutant p53. As previously discussed, p53 has an important role in the cellular response to DNA damage, having its activity modulated by kinases with DNA-binding domains that recognize unrepaired DNA damage. The lack of a functional p53 response in CHO cells probably decreases the efficiency of the damage-response pathway and may contribute to the relative resistance of these cells to the effect of chromium on clonogenicity. Moreoever, the lack of an intact damage-response mechanism may also make CHO cells more sensitive to the direct effects of structural DNA damage, and thus more sensitive to the altered spectrum of genotoxicity caused by pretreatment with vitamin C, which resulted in a statistically significant increase in survival (Blankenship et al., 1997). In contrast, HLF cells, with an intact p53-mediated damage-response mechanism, are more sensitive to lower levels of chromium but are relatively unaffected by the altered genotoxic spectrum caused by pretreatment with vitamin C. In this case the DNA damage that is insensitive to vitamin C may be sufficient to induce p53, growth arrest, and apoptosis. This is supported by the observation that pretreatment of HLF cells with vitamin C did not affect the upregulation of p53 after sodium chromate exposure.
Chromium-induced growth arrest and apoptosis are at the molecular decision point between chromium toxicity and chromium carcinogenesis. When normal cells of the respiratory tract come in contact with carcinogenic forms of chromium, they may respond by undergoing growth arrest, apoptosis, or necrosis. There may also emerge a population of genetically damaged cells that exhibits either intrinsic or induced resistance to apoptosis. Such cells may be predisposed to neoplasia as result of their altered growth/death ratio, disrupted cell cycle control, or genomic instability. These concepts require further exploration because they raise the question of whether interventions designed to decrease chromium toxicity may actually increase the incidence of cancer by allowing the inappropriate survival of genetically damaged cells.
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ACKNOWLEDGMENTS |
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We would like to express our appreciation to L.E. Dye for his assistance with electron microscopy. This work was done in partial fulfillment of the Ph.D. requirements of the Molecular and Cellular Oncology program by DLC.
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NOTES |
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1 To whom correspondence should be addressed at The George Washington University Medical Center, Department of Pharmacology, 2300 Eye Street, NW, Washington, DC 20037. Fax: (202) 994-2870. E-mail: phmsrp{at}gwumc.edu.
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