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[Cancer Research 62, 1443-1449, March 1, 2002]
© 2002 American Association for Cancer Research

Experimental Therapeutics

Transformed and Tumor-derived Human Cells Exhibit Preferential Sensitivity to the Thiol Antioxidants, N-Acetyl Cysteine and Penicillamine

Pamela A. Havre, Sandra O’Reilly, J. Justin McCormick and Douglas E. Brash1

Departments of Therapeutic Radiology [P. A. H., D. E. B.] and Genetics [D. E. B.], and Yale Comprehensive Cancer Center [P. A. H., D. E. B.], Yale University School of Medicine, New Haven, Connecticut 06520-8040, and Departments of Microbiology & Molecular Genetics and Biochemistry & Molecular Biology, Carcinogenesis Laboratory, Food Safety and Toxicology Building, Michigan State University, East Lansing, Michigan 48824-1316 [S. O., J. J. M.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Thiol antioxidants, typified by N-acetyl cysteine, are known to induce p53-dependent apoptosis in transformed mouse embryo fibroblasts but not in normal mouse embryo fibroblasts. We now report that this is also the case for human cells. First, we used an isogenic fibroblast cell lineage exhibiting progressive stages of transformation, from primary derived cells to v-MYC immortalized to tumorigenic. At the immortalization stage, cells became 12- and 480-fold more sensitive to the thiol antioxidants N-acetyl cysteine (NAC) and penicillamine (PEN), respectively. Although immortalization of these cells was associated with v-MYC expression, overexpression of MYC was not sufficient for sensitizing these cells to antioxidants. To test whether sensitivity to antioxidants is a general property of immortalized human cells, including fully transformed cells, 12 tumor-derived cell lines were treated with PEN, the more potent of the two antioxidants. Ten of 11 caspase-proficient tumor cell lines underwent apoptosis after treatment, whereas primary fibroblasts and keratinocytes were resistant. The difference between normal and transformed cells was apparent whether the assay used measured caspase 3 activation, Annexin V binding, or cell viability. Tumor cell lines containing wild-type p53 were more sensitive than p53-null cell lines. The requirement for p53 was tested using the p53 inhibitor, pifithrin-{alpha}, or using stable transfectants of a v-MYC-immortalized, telomerase-positive cell line that expresses HPV16 E6 to bind and degrade p53. In the latter case, >=80% of the PEN-induced apoptosis was dependent on the presence of wild-type p53. These studies suggest that treatment with thiol-containing antioxidants, such as PEN, may offer a useful approach for preferential induction of apoptosis in preneoplastic and neoplastic cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antioxidants have been heralded as cancer-preventive compounds, generally because of their ability to neutralize reactive oxygen species. Reactive oxygen species can cause damage to DNA, protein, and lipids, and overproduction can be toxic to the cell (1 , 2) . In fact, hydrogen peroxide is often used as a tool to induce apoptosis in vitro. Consequently, it is counterintuitive that antioxidants might be used to initiate apoptosis. However, several laboratories have reported that antioxidants can induce apoptosis in cells (3, 4, 5, 6) . Although thiol compounds are probably most closely associated with radical quenching, one of their most important functions is to act as cellular redox buffers by regulating protein thiol/disulfide composition (7) . It is known that many transcription factors are active only when their sulfhydryl groups are in the reduced state. Two of the best studied of these are AP-1 and NF-{kappa}B (2) . Reduced cysteine groups are important for the activity of p53, as well, potentiating its participation in apoptosis (8 , 9) . All caspases (10) , in addition to many other enzymes, including several src-related phosphokinases, contain cysteines in their active sites and require a reduced environment for optimal activity (7 , 11) .

Previous results obtained in our laboratory using MEFs2 established that thiol-containing antioxidants, such as NAC, dimercaprol, and 2-oxo-4-thiazolidine-carboxylic acid, induced apoptosis in transformed MEFs but not in normal MEFs. p53 was induced after treatment with antioxidants and, moreover, was required for initiation of apoptosis (6) . These activities appear to be distinct from the induction of apoptosis by antioxidants in conjunction with metal ions (12) .

The involvement of p53 in the initiation of apoptosis is complex. There is evidence that it plays a role in the extrinsic pathway by up-regulating both FAS ligand (13) and its receptor (14) . There is also convincing support for the participation of p53 in the intrinsic, or mitochondrial, pathway based on its ability to transactivate BAX (15) and p53 AIP1, which causes loss of the mitochondrial transmembrane potential (16) . Finally, a recent study has shown that after hypoxia, p53 is itself translocated to the mitochondria, where it induces apoptosis (17) .

The effect of two thiol-containing antioxidants, NAC and PEN, on normal and transformed human cells is the focus of this report. Each is classified as a general antioxidant by virtue of its free sulfhydryl group. Although their chemical structures are similar, they are used for different purposes clinically. NAC is prescribed for chronic bronchitis because of its antimucolytic activity. PEN has been used to treat Wilson’s disease (a copper storage disease), because it chelates copper in addition to zinc. We show here that both of these antioxidants are able to induce apoptosis in transformed cells, whereas normal cells are resistant. PEN, found to be the more effective of the two agents, was investigated further using 12 additional cell lines derived from tumors.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
Human foreskin fibroblasts (LG1) and the MSU 1.0 cell line derived from them (18 , 19) were maintained in DMEM/high glucose with 10% fetal bovine serum (Life Technologies, Inc., Rockville, MD). Hydrocortisone (1 µg/ml) was added to this medium for the MSU 1.0 derived cell lines, MSU 1.0.1, PH2M, PH3M, PH2MT, and PH3MT. Human foreskin fibroblasts obtained from the Department of Dermatology at Yale School of Medicine were also grown in DMEM/high glucose containing 10% FCS. Tumor cell lines were obtained from ATCC (Rockville, MD). Cell lines with wild-type p53 included: MCF-7 (mammary gland adenocarcinoma), A549 (lung carcinoma), HT1080 (fibrosarcoma), LNCap (prostate carcinoma), and HCT-116 (colon carcinoma). HCT-15 cells (colon adenocarcinoma) are heterozygous for p53, with one allele containing alanine instead of proline at codon 153. p53-null cell lines were H1299 (non-small cell lung carcinoma), Saos-2 (osteosarcoma), A253 (epidermoid carcinoma), and Calu-1 (lung carcinoma). Cells were cultured in media suggested by ATCC, except for MCF-7 cells, which were grown in DMEM/F12, and HT1080 cells, which were grown in DMEM. RKO cells (colon carcinoma) express wild-type p53, whereas RC10.2 cells, an RKO clone resulting from transfection of these cells with HPV16 E6, are effectively p53 null at the protein level. These were grown in McCoy’s 5a medium without and with G418, respectively, and were obtained from K. Cho, University of Michigan, Ann Arbor, MI. IMR90 lung fibroblasts and the MycER cells derived from them (obtained from R. DePinho, Harvard Medical School, Boston, MA) were grown in DMEM/high glucose containing 10% fetal bovine serum. Primary keratinocytes obtained from the Department of Dermatology at Yale School of Medicine were grown in KGM-2 (BioWhittaker, San Diego, CA). All cell media contained 100 units/ml penicillin and 100 µg/ml streptomycin (Life Technologies, Inc.). Cells, media, and p53 status are shown in Table 1Citation .


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  		Table 1 Cells and medium

 
Treatment of Cells with NAC or PEN.
Cells were seeded at 2 x 106/100 mm dish the day before treatment. NAC and PEN (Sigma Chemical Co., St. Louis, MO) were dissolved in cell culture media and neutralized if necessary. For the caspase 3 assay, cells were treated for 24 h with the antioxidant, then harvested and solubilized as described (10) and frozen at -80°C. For SDS-PAGE, cells were pelleted, washed, and solubilized in 50 mM TrisCl (pH 8.0), 150 mM NaCl, 1% NP40, 10 µg/ml each of aprotinin and leupeptin, 20 µM phenylmethylsulfonyl fluoride, 2 µg/ml each of pepstatin, antipain, and chymostatin, and 10 µg/ml cytochalasin B. Protein assays were carried out using the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL).

Immunoblots.
Equal amounts of protein (25 or 50 µg) were run on 10% Ready Gels (Bio-Rad, Hercules, CA) and transferred to nitrocellulose. After blocking, blots were screened with antibody to p53 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Horseradish peroxidase-conjugated secondary antibody was from Pierce. Detection was carried out using SuperSignal West Pico Chemiluminescent Substrate (Pierce) and HyperfilmECL (Amersham Life Science, Piscataway, NJ).

Caspase 3 Assay.
Caspase activity was assayed using acetyl-DEVD-paranitroanilide as substrate (Bachem, King of Prussia, PA), which was dissolved in DMSO at a concentration of 10 mM. Acetyl-DEVD-paranitroanilide (2 µl) was added to 98 µl of cell extract containing 50 µg of protein in 20 mM Pipes (pH 7.2), 100 mM NaCl, 1 mM EDTA, 10 mM DTT, 0.1% Chaps, and 10% sucrose. The absorbance at 405 nm was read after a 4-h incubation at 37°C.

{alpha}-PFT Treatment.
{alpha}-PFT (Calbiochem, San Diego, CA) was dissolved in DMSO and added at the same time as PEN. Cells were incubated for 24 h in the presence of both agents and processed for the caspase 3 assay as described above.

Cloning/Transfections.
HPV16 E6 cDNA from the pCMVneo 16 E6 vector (generously provided by K. Cho) was cloned into pcDNA4/TO (Invitrogen, Carlsbad, CA) to create pcDNA4/TO/HPV16 E6. Site-directed mutagenesis was carried out to generate a more efficient ribosome binding site and reverse a mutation that occurred during storage. To express HPV16 E6 using an inducible system, MSU 1.0 cells were first transfected with pcDNA6/TR (Invitrogen), which encodes the tetracycline repressor, and then with pcDNA4/TO/HPV16 E6. MSU 1.0 cells were seeded at a density of 3 x 106 cells/100 mm dish; for each dish, 24 µg of DNA and 85 µl of Lipofectamine 2000 (Life Technologies, Inc.) were used. Transfectants were selected with 100 µg/ml Zeocin and 1 µg/ml blastocidin. Six clones, 2C8, 3C8, 5C8, 1C9, 3C9, and 8C9, were selected to verify the absence of p53 induction after PEN treatment. These clones were then treated with PEN for 20 h and assayed for caspase 3 activation as described above.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Acquisition of Sensitivity to Thiol-Antioxidants at an Early Stage of Transformation of Human Fibroblasts.
Our laboratory had demonstrated previously that transformed MEFs are sensitive to thiol-containing antioxidants, whereas normal fibroblasts were resistant (6) . We were interested in finding out whether this was also true for human fibroblasts and, if so, at what stage of transformation this apoptotic response occurred. Human fibroblasts ranging from normal primary derived to tumorigenic were tested for sensitivity toward two small thiol antioxidants, NAC and PEN.

A survival assay was used first; cells were treated with 50 mM NAC or 15 mM PEN for 24 h, harvested, and counted with trypan blue. Normal fibroblasts (LG1) were resistant to both thiol compounds, whereas LG1 cells immortalized with v-MYC (MSU 1.0) were 4- and 20-fold more sensitive at the same doses of NAC or PEN, respectively (Fig. 1)Citation . In addition, LG1 and MSU 1.0 cells were treated for 24 h with PEN, the more potent of the two agents, and then cultured for an additional 48 h in normal media before staining with crystal violet. Fig. 2Citation shows that few of the v-MYC immortalized cells (MSU 1.0) remained 72 h after treatment. In contrast, normal cells treated with the same concentration of PEN were indistinguishable from untreated cells.



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  		Fig. 1. Cell death after antioxidant treatments. Cells were treated for 24 h with NAC or PEN, then trypsinized and counted with trypan blue to determine cell survival. Untreated cells were given fresh media only. Open bars, untreated; stippled bars, 50 mM NAC; solid bars, 15 mM PEN. SE is shown for two experiments.

 


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  		Fig. 2. Immortalized human fibroblasts are sensitive to thiol antioxidants. LG1 primary derived cells (A and B) and immortalized MSU 1.0 (C and D) cells were untreated (A and C) or treated for 24 h with 15 mM PEN and allowed to grow for an additional 48 h in normal medium (B and D). Untreated cells were stained with crystal violet when confluent, at 48 h for LG1 and 36 h for MSU 1.0. Treated cells were stained at 72 h after the initial treatment.

 
To determine whether cell death was caused by apoptosis, we then measured the activation of caspase 3, one of the key effector caspases. Cleavage of the substrate DEVD-pNA by caspase 3 was followed by the increase in absorbance of free pNA at 405 nm. After treatment with NAC or PEN for 24 h, cells were harvested, and extracts were prepared for measurement of caspase 3 activity. Caspase 3 activation in v-MYC-transformed cells (MSU 1.0), was 12- and 480-fold higher than for primary derived cells (LG1) on treatment with NAC and PEN, respectively (Fig. 3)Citation . MSU 1.1 cells, which were derived from the MSU 1.0 cells and are less growth factor dependent, exhibited intermediate levels of caspase activity. Two clones derived from the MSU 1.1 cells after transfection with H-RAS (PH2M and PH3M) and their tumor-derived counterparts (PH2MT and PH3MT) had become resistant to treatment with NAC and PEN.



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  		Fig. 3. Human fibroblasts acquire sensitivity to antioxidants at the stage of v-MYC immortalization. Cells were treated for 24 h with the indicated concentrations of NAC (A) or PEN (B), harvested, and lysed. Caspase 3 activity was plotted as pmol pNA cleaved/µg/h after subtracting the absorbance for untreated cells. LG1, normal primary derived human fibroblasts ({blacksquare}); MSU 1.0, v-MYC immortalized, nontumorigenic ({bullet}); MSU 1.1, growth factor-independent clone of MSU 1.0 ({blacktriangleup}); PH3M and PH2M, HRAS transformed, tumorigenic ({square}, {circ}); and PH2MT and PH3MT ({triangleup}, x) tumor-derived lines.

 
Because both NAC and PEN are antioxidants, but only PEN has chelating activity (20) , it was possible that its ability to chelate zinc, an inhibitor of caspases (10) , might be responsible for its greater potency. Treating primary fibroblasts and v-MYC-transformed cells with the cell permeable zinc chelator, N,N,N'N'-Tetrakis-(2-pyridylmethyl)-Ethylenediamine, caused apoptosis at micromolar concentrations; however, there was negligible preference for the transformed cells (data not shown).

Overexpression of MYC Does Not Account for the Sensitivity of Transformed Human Fibroblasts to PEN.
Overexpression of MYC has been reported to sensitize fibroblasts to apoptosis (21) , so we asked whether it was possible that MYC was responsible for the sensitivity to PEN exhibited by the MSU 1.0 cells, which are transformed with v-MYC. Human lung fibroblasts (IMR90), with and without an inducible MycER, were treated with 15 mM PEN for 24 h, harvested, lysed, and assayed for caspase-3 activity. Hydroxytamoxifen (4-OHT) was added to the culture medium 24 h before the addition of PEN to induce c-MYC. Caspase 3 activity was measured for cells with and without the expression vector and with and without induction.

As shown in Table 2Citation , PEN treatment of IMR90 cells did not activate caspase 3 under any conditions. In contrast, in MycER cells (IMR90 cells containing an inducible c-MYC gene), caspase 3 activity increased 3–4-fold when either 4-OHT or PEN was added. However, PEN-induced caspase activation in these cells only doubled on expression of c-MYC. A 2-fold increase indicates that this oncogene is not sufficient to account for the sensitivity (480-fold difference in caspase 3 activity) of the MSU 1.0 cells to PEN.


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  		Table 2 Effect of PEN on IMR90 and MycER fibroblastsa

 
Human Cell Lines Derived from Tumors Exhibit Sensitivity to PEN.
To establish whether sensitivity to PEN was a general characteristic of transformed human cells, including fully transformed cells, 12 additional tumor cell lines were obtained from ATCC. These included both p53-wild-type and p53-null cell lines. Two assays were used to monitor apoptosis: caspase 3 activation and Annexin V binding. The third assay used was trypan blue uptake, which measures cell viability (Fig. 4)Citation .



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  		Fig. 4. Cells from human tumor cell lines are preferentially sensitive to PEN, and sensitivity is correlated with the presence of wild-type p53. In A, cells were treated with 15 mM PEN for 24 h before harvesting, washed with PBS, solubilized, and assayed for protein. Equal amounts of protein (50 µg) were used to measure caspase 3 activity. In B, cells were treated for 16 h with 15 mM PEN, harvested, washed, and incubated with Annexin V and propidium iodide. The percentage of apoptotic cells was determined by flow cytometry after correcting for necrotic cells. In C, cells were treated with 15 mM PEN for 24 h, harvested, and counted with trypan blue. MCF-7 cells do not express caspase 3 and are labeled C3-.

 
After treatment with PEN, caspase 3 activation occurred in 10 of 11 caspase-proficient cell lines, whereas negligible caspase 3 was expressed by the primary derived cells (Fig. 4A)Citation . The MCF-7 cells served as a useful negative control because they do not express caspase 3, as a consequence of a 47-bp deletion in exon 3 (22) . MCF-7 cells, which contain wild-type p53, undergo apoptosis when treated with Taxol (23) , but PEN was not able to induce apoptosis. p53 status also appeared to influence the sensitivity of tumor cells to PEN; cell lines containing wild-type p53 were on average twice as sensitive to treatment as p53-null cell lines (Fig. 4A)Citation . The sensitivity of tumor cell lines to PEN indicates that the resistance of the MYC plus H-RAS-transformed clones PH2M and PH3M, along with their tumor-derived counterparts, PH2MT and PH3MT (Fig. 3)Citation , was a RAS-specific phenomenon and not an inevitable consequence of progressive tumorigenicity.

Annexin V binding revealed a similar pattern of selectivity toward transformed cells (Fig. 4B)Citation . Most of the tumor cell lines underwent apoptosis, again with the exception of the Calu-1 cells. In contrast, in the two normal fibroblast cell types (LG1 cells, which are the parent of the MSU 1.0 cells, and normal fibroblasts obtained from a different donor), <7% of the cells underwent apoptosis. Again, MCF-7 cells served as a negative control. Interestingly, the Annexin assay indicated a larger difference than the caspase 3 assay did between tumor cell lines with and without p53. Three cell types did not fit this pattern. HCT116 cells, derived from a colon carcinoma, have a frameshift in the BAX gene (24) and no functional BAX gene product; the extent of apoptosis was low in these cells, although they contained wild-type p53. HCT-15 cells contain one mutant p53 allele, with alanine replacing proline at position 153; these cells also have a mutation in the proofreading domain of DNA polymerase {delta}, leading to an increased mutation rate detected at several loci (25) . The extent of apoptosis was intermediate in these cells. Conversely, H1299 cells, which are p53 null, underwent apoptosis to the same extent as several cell types in the p53-wild-type group.

The third assay, trypan blue uptake, is not an apoptosis assay but instead reflects cell death; cells with disrupted membranes take up dye. Again, all tumor cell lines exhibited some cell death in response to treatment with PEN, whereas normal cells, including two additional cell types, IMR90 lung fibroblasts and keratinocytes, were resistant (Fig. 4C)Citation . In this assay also, there was a clear difference between transformed cells containing wild-type p53 and those without p53, with an average difference of 2.5-fold.

On the basis of these three assays, transformed cells and tumor cell lines are sensitive to treatment with the antioxidant PEN, whereas normal cells are not. In addition, cell lines containing wild-type p53 are more sensitive than p53-null cell lines.

p53 Is Induced by Thiol Antioxidants.
Previous studies carried out in our laboratory on MEFs showed that although apoptosis was induced only in transformed cells, p53 was induced by NAC in both normal and transformed cells (6) . Human fibroblasts behaved similarly when treated with NAC (Fig. 5)Citation . In normal (LG1), MYC-transformed (MSU 1.0), and one of the tumor-derived clones (PH2MT), p53 was induced within 2 h of treatment. Treatment with PEN caused a more pronounced induction at one-fifth the concentration of NAC (Fig. 5)Citation . Strikingly, and unlike NAC, PEN discriminated between the normal and transformed fibroblasts, with only a moderate induction of p53 in normal fibroblasts compared with the robust induction in the MYC-transformed cells (Fig. 6)Citation .



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  		Fig. 5. Induction of p53 in human fibroblasts after treatment with NAC or PEN. LG1, MSU 1.0, and PH2MT cells were treated with thiol compounds for the indicated times, harvested, and lysed; 50 µg of protein were run on 10% SDS-polyacrylamide gels. After transfer to nitrocellulose, blots were screened with antibody to p53. A, LG1 cells, 50 mM NAC. B, MSU 1.0 cells, 50 mM NAC. C, PH2MT cells, 50 mM NAC. D, MSU 1.0 cells, 10 mM PEN.

 


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  		Fig. 6. Treatment with PEN discriminates between normal and transformed cells in p53 induction. Cells were treated for 0, 3, 5, or 7 h with 15 mM PEN, harvested, washed in PBS, and frozen. Cells were lysed after all time points were collected and assayed for protein, and 25 µg were run on 10% gels. After transfer to nitrocellulose, blots were screened with p53 antibody.

 
p53 Plays a Role in PEN-induced Apoptosis.
Although the presence of wild-type p53 correlated with the sensitivity of the tumor cell lines to PEN-induced apoptosis and was induced to significantly higher levels in transformed compared with normal fibroblasts, the results do not demonstrate a requirement for p53. Therefore, to test p53 involvement, two approaches were used: (a) the addition of a p53 inhibitor to the culture medium along with PEN; and (b) expression of a viral protein that binds and degrades p53.

PFT-{alpha} is a p53 inhibitor that has been shown previously to inhibit apoptosis by 25–60% in transformed mouse cells after treatment with various cytotoxic agents (26) . MSU 1.0 cells were treated with PFT-{alpha}, which was added to the medium at the same time as PEN. After treatment for 24 h, caspase 3 activation was measured (Fig. 7)Citation . With increasing concentrations of PFT-{alpha}, caspase 3 activation was diminished until a plateau at 70 µM PFT-{alpha} was reached. At this concentration, PFT-{alpha} began to precipitate out of solution, preventing measurement of the inhibition at higher concentrations. These results indicate that >=45% of the caspase 3 activation induced by PEN treatment is p53 dependent.



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  		Fig. 7. Effect of the p53 inhibitor, PFT-{alpha}, on PEN-induced caspase 3 activity in v-MYC-transformed cells. Cells were treated with PFT-{alpha} at the time of PEN addition. Cells were harvested 24 h later, washed with PBS, and assayed for protein, and equal amounts of protein (50 µg) were used to measure caspase 3 activity.

 
An alternative to using a chemical inhibitor of p53 is to transfect cells with HPV16 E6, which binds to and degrades p53 (27) . Stable transfectants of the MYC-transformed cell line MSU 1.0 were generated to inducibly express HPV16 E6. To express HPV16 E6 using an inducible system, MSU 1.0 cells were first transfected with pcDNA6/TR, which encodes the tetracycline repressor, and then with pcDNA4/TO/HPV16 E6. Six clones expressing HPV16 E6 were screened for p53 expression after induction with PEN. Clones were treated with PEN in parallel with the parent MSU 1.0 cells. Two clones, 2C8 and 3C8, which showed no induction of p53 after treatment with PEN, were tested in a caspase 3 assay. The ability of PEN to activate caspase 3 was decreased 84.5 and 84.3%, respectively (Table 3)Citation , as would be expected if p53 is required for PEN-induced apoptosis.


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  		Table 3 Caspase 3 activity in MSU 1.0 cells expressing HPV16 E6a

 
Inhibition of caspase 3 activation by either PFT-{alpha} or degradation of p53 by HPV16 E6 confirms the requirement for p53 in PEN-induced apoptosis in transformed human fibroblasts and tumor cell lines.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the studies reported here, two thiol antioxidants, NAC and PEN, are shown to induce p53 and cause apoptosis in transformed fibroblasts and tumor cell lines but not in normal fibroblasts or keratinocytes. PEN is the more effective of these two agents and, interestingly, induces p53 to much higher levels in the MYC-transformed fibroblasts compared with the primary derived fibroblasts. It appears that BAX expression is necessary, because H116 cells, which do not express BAX, do not undergo apoptosis in response to PEN treatment.

Successful treatment of tumors by radiation and/or chemotherapy is often correlated with the presence of wild-type p53 (28) . Studies in many laboratories have shown that p53 is induced by treatments that cause some type of cell stress, including strand breaks or other DNA damage (29, 30, 31) , nutrient deprivation (32) , and hypoxia (33) . In addition to increases in the amount of p53 protein, its sequence-specific binding has been shown to be activated by the accumulation of short DNA single strands (34) and by various DNA-damaging agents (35) . Although the specific role p53 plays in apoptosis has not been clearly defined, overexpression of the wild-type protein has been shown to induce apoptosis in p53-null Saos-2 and H1299 tumor cell lines using an inducible wild-type p53 vector (36) ; the extent of apoptosis was correlated with the amount of p53 induced. However, this is not the case for all cell types, because expression of a temperature-sensitive p53 in GHFT1 cells was not sufficient to induce apoptosis; a DNA damage signal was also required (37) .

Most evidence linking p53 with apoptotic processes is based on its ability to up-regulate genes in the apoptotic pathway, such the FAS receptor and its ligand (13 , 14) , BAX (15) , and p53 AIP1 (16) . However, a full apoptotic response is believed to require both transactivation-dependent and -independent processes. The interaction of other proteins with p53 is important for regulation of its half-life (38 , 39) , as well as its activity in various cellular processes. The COOH terminus of p53 acts as a negative regulator; binding of an antibody or peptide to this domain activates the transcriptional activity of p53 (40) . Therefore, it is likely that interactions with cellular proteins not yet identified activate p53 in vivo.

The expression of oncogenes predisposes cells to undergo apoptosis (41) , but how antioxidants cooperate with oncogenes and p53 to cause apoptosis is unknown. In addition to up-regulating p53, each antioxidant could theoretically create a more reduced environment for the cysteine groups in p53 and potentiate its activity (42 , 43) . Although both NAC and PEN are antioxidants, PEN can also chelate zinc and copper. Because zinc inhibits caspases (10) , chelation could theoretically activate caspases and cause apoptosis. But in this case, PEN would not be predicted to induce apoptosis only in transformed cells. In fact, treatment of primary fibroblasts and MYC-transformed cells with a cell permeable zinc chelator, N,N,N'N'-Tetrakis-(2-pyridylmethyl)-Ethylenediamine, caused apoptosis at µM concentrations with a negligible preference for the transformed cells. These observations suggest it is not the chelating activity which is responsible for the preferential apoptosis. Alternatively, PEN has a relatively long half-life in the cell (20) , so it may be a more effective antioxidant than NAC because of its persistence.

Several other groups have shown that antioxidants can induce apoptosis in human cells (3, 4, 5 , 44) . In two of these studies, the mechanism is most likely different from the one used by thiol antioxidants. Caffeic acid-induced apoptosis is associated with glutathione depletion and is inhibited by NAC (44) . Treatment of human epidermoid carcinoma cells with epigallocatechin-3-gallate, the most active constituent in green tea, causes cell cycle arrest (3) , whereas this is not the case after thiol treatment of v-MYC-transformed cells.3 Moreover, Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid), a chain-breaking antioxidant that does not contain sulfur, did not induce cell death in transformed murine fibroblasts (6) or activate caspase 3 in human fibroblasts (data not shown). Therefore, it seems likely that redox-regulatory properties of thiol-containing antioxidants act through p53 to invoke apoptosis selectively in human cells that are transformed.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Therapeutic Radiology, Yale School of Medicine, 333 Cedar Street/HRT 309, New Haven, CT 06520-8040. Phone: (203) 785-2988; Fax: (203) 785-6309; E-mail: douglas.brash{at}yale.edu. Back

2 The abbreviations used are: MEF, mouse embryo fibroblasts; PEN, penicillamine; MycER, MYC-estrogen receptor fusion; ATCC, American Type Culture Collection; NAC, N-acetylcysteine; 4-OHT, 4-hydroxytamoxifen; PFT-{alpha}, pifithrin-{alpha}. Back

3 P. A. H. and D. E. B., unpublished observations. Back

Received 9/24/01. Accepted 12/28/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. DiMascio P., Murphy M. E., Sies H. Antioxidant defense systems: the role of carotenoids, tocopherols, and thiols. Am. J. Clin. Nutr., 53: 194S-200S, 1991.[Abstract/Free Full Text]
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