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[Cancer Research 63, 1914-1919, April 15, 2003]
© 2003 American Association for Cancer Research

Molecular Biology and Genetics

A Peroxisome Proliferator-activated Receptor-{gamma} Agonist and the p53 Rescue Drug CP-31398 Inhibit the Spontaneous Immortalization of Breast Epithelial Cells1

Brittney-Shea Herbert2, Virginia P. Pearce, Linda S. Hynan, Denise M. LaRue, Woodring E. Wright, Levy Kopelovich and Jerry W. Shay

Departments of Cell Biology [B-S. H., V. P. P., D. M. L., W. E. W., J. W. S.] and Psychiatry and Academic Computing Services [L. S. H.], The University of Texas Southwestern Medical Center, Dallas, Texas 75390, and CADRG, DCP, National Cancer Institute, Bethesda, Maryland 20892 [L. K.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell immortalization is a critical and rate-limiting step in cancer progression. Agents that inhibit cell immortalization may have utility for novel molecular chemopreventive strategies. Preimmortal breast epithelial cells derived from a patient with the Li-Fraumeni Syndrome (LFS) can spontaneously immortalize in vitro at a measurable and reproducible frequency. In the present study, these cells were treated in vitro with low (nM) concentrations of potential and otherwise clinically validated chemopreventive agents, including several nonsteroidal anti-inflammatory drugs, rosiglitazone maleate, and the p53 rescue drug CP-31398. Rosiglitazone maleate (P < 0.05) and CP-31398 (P < 0.05) significantly inhibited the frequency of spontaneous immortalization of LFS breast epithelial cells compared with untreated controls. Nonsteroidal anti-inflammatory drugs, including specific cyclooxengenase-2 inhibitors, only moderately inhibited the spontaneous immortalization of preimmortal LFS breast epithelial cells. The significant effects of the p53 rescue drug CP-31398 correlated with the increase in cellular death induced by telomere shortening-induced DNA damage signals, including increases in p53 and p21 protein levels. Because immortalization is one step in cancer progression, these studies show the potential usefulness of a cell-based model system to screen the effects of known and potentially novel chemopreventive agents, using cell immortalization as an end point.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spontaneous immortalization of human cells in vitro is an extremely rare event, requiring mutations in several genes and cellular pathways normally involved in cellular senescence (1, 2, 3) . Normal cells have a limited life span and undergo replicative senescence, in which cells cease to proliferate (4, 5, 6) . Senescence occurs when cells contain at least some critically short telomeres. The maintenance of functional telomeres (consisting of TTAGGG repeats in humans) is essential for protecting the chromosome ends from being recognized as damaged DNA (7) . Because of the end replication problem, where lagging strand synthesis cannot copy all of the way to the very end of the lagging strand, almost all normal human cells gradually lose telomeric DNA as they age (8, 9, 10) . This growth arrest is believed to be triggered by a DNA damage repair signaling program (11) . Cells that lose critical cell cycle checkpoint functions escape this initial growth arrest and divide until they enter crisis, when telomere lengths become extremely short, and chromosome end fusions and apoptosis occur (12 , 13) . Immortal cells escape crisis, a period of balanced cell growth and death usually followed by a decrease in the total number of surviving cells, when telomerase or another mechanism to maintain telomere stability is activated (14 , 15) . Once telomerase is activated, it preferentially elongates the critically short telomeres, stabilizes telomere lengths, and permits continued cell division. This hypothesis is supported by the observation that ectopically introduced telomerase activity can extend telomeres and indefinitely prolong cellular life span (16 , 17) . Over 90% of breast carcinomas have been shown to contain telomerase activity, whereas adjacent normal tissues do not express telomerase activity (18, 19, 20) .

Although normal human breast epithelial cells in vitro rarely spontaneously immortalize,LFS-derived3 (p53 +/-, telomerase silent) breast epithelial cells have been shown to spontaneously immortalize at a relatively high and reproducible frequency of ~5 x 10-7 (21 , 22) . Telomerase inhibitors have been shown to inhibit the spontaneous immortalization of LFS-derived breast epithelial cells by preventing the activation of telomerase (22) . The fact that the LFS-derived breast epithelial cells can reproducibly and spontaneously immortalize allows investigations into the effects of different agents on the immortalization frequency in vitro. In the present study, we surveyed a panel of potentially novel chemopreventive agents, including several NSAIDs, a peroxisome PPAR{gamma} agonist, and the p53 rescue drug CP-31398, for their effects on the spontaneous immortalization of LFS-derived breast epithelial cells. Here, we show that treatment of LFS-derived breast epithelial cells just before crisis with low (nM range), nontoxic dosages of some, but not all, chemopreventive agents reduces the frequency of spontaneous immortalization.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
The chemopreventive agents nimesulide (a specific COX-2 inhibitor) and rosiglitazone maleate (a PPAR{gamma} agonist) were received from the DCP Repository (McKesson BioServices, Rockville, MD). NS-398 (a specific COX-2 inhibitor) was purchased from Cayman Chemical, and celecoxib (a specific COX-2 inhibitor) was purchased from LKT Laboratories. Pfizer (Dr. Farzan Rastinejad) kindly provided the p53 rescue drug CP-31398. Sulindac sulfide (a nonselective COX-1 and COX-2 inhibitor) was a gift from Merck Research Laboratories. The drugs were dissolved in DMSO as stock solutions with the exception of rosiglitazone maleate, which was dissolved in ethanol. The final concentrations of solvents were no >0.1%.

Cell Culture.
The HMECs used for these experiments were derived from a 31-year-old LFS patient’s noncancerous breast tissue [containing a germ-line mutation at codon 133 in one of the two alleles of the p53 gene (Met to Thr [M133T]) that affects wild-type p53 protein conformation] and grown under serum-free conditions as described previously (21 , 22) . These cells undergo crisis around PD level 50–60 (21 , 22) . As is the case for other mammary epithelial cells grown under these conditions, the cells had undergone a "self-selection stage" during which the culture was overgrown by cells that lacked p16 expression, probably attributable to methylation (23 , 24) .

Determination of the Frequency of Immortalization.
The frequency of immortalization was estimated using a fluctuation analysis as described previously (21 , 22 , 25 , 26) , in which cells were maintained in 10 different series for the last 10 doublings of their life span. Immortalization was expressed as the number of immortal cell lines obtained per number of culture series. Frequency was expressed as the probability of obtaining an immortal cell line based on the total number of cells plated at each passage (not per cell division) and is calculated by taking the total number of independent immortalization events (series yielding immortal cell lines) and dividing by the total number of cells plated (the number of series times the number seeded per dish).

Statistical Analysis.
The data were collected as three different experiments for the cells treated with chemopreventive agents (Table 1)Citation . A two-tailed Fisher’s exact test was performed to examine the association between treatment and experiment to determine whether the data across the three experiments for each treatment could be combined in additional analyses. Because there was no statistically significant association between experiment and treatment for the chemopreventive agents (P > 0.99), the data for each treatment across three experiments were combined. Comparisons of immortalization events for the nine chemopreventive treatments to either of the two controls (untreated or solvent) were performed using two-sided {chi}2 tests of independence. When significant, these were followed with Tukey-type post hoc multiple comparison tests for proportions (27) to examine which treatments were statistically and significantly different. The comparisons of interest were each of the chemopreventive treatments versus either the untreated or solvent controls. Statistical analyses were not performed on the average frequencies of immortalization (Table 1)Citation . The {alpha} level for all statistical tests was set to 0.05, and the Fisher exact and {chi}2 analyses were performed using the SAS program (Version 8.2; SAS Institute, Cary, NC). The Tukey-type post hoc multiple comparisons tests for proportions were programmed in Microsoft Excel, and significance was determined by consulting "Critical Values of the q Distribution" by Harter (reprinted in Ref. 27 ), where exact Ps are not available.


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  		Table 1 Effects on the spontaneous immortalization of LFS-derived breast epithelial cells by chemopreventive agents

 
Western Analysis.
Whole cell lysates were prepared from logarithmically growing cells by lysis using 2% SDS in 50 mM Tris-HCl. Total protein concentration was determined using the bicinchoninic acid assay (Pierce, Rockford, IL) according to manufacturer’s instructions. Samples (50–100 µg) were electrophoresed on a 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membrane (Millipore Corp., Marlborough, MA). The blots were washed in PBS-T, then blocked in PBS-T plus 5% powdered milk for 1 h. Monoclonal antibodies to actin (Santa Cruz Biotechnology, Santa Cruz, CA), COX-1 (Santa Cruz Biotechnology), COX-2 (Santa Cruz Biotechnology), PPAR{gamma} (a gift from David Manglesdorf, University of Texas-Southwestern Medical Center at Dallas), and p53 (Ab-6; Oncogene Science) were added to PBS-T plus milk. After washing, appropriate secondary antibodies coupled to horseradish peroxidase (Amersham, Buckinghamshire, United Kingdom) were added. After washing, specific protein bands were detected using the chemiluminescent substrate, enhanced chemiluminescence (Amersham), and exposed to X-ray films.

Transient Transfection and Luciferase Reporter Assay to Measure PPAR{gamma} Activation.
The reporter plasmids, pPPRE3-TK-LUC (luciferase gene under the control of the herpes simplex virus thymidine kinase promoter), and three PPAR{gamma} response elements used in this assay have been described (28) . A 250-ng measurement of pCMX-ß-GAL (ß galactosidase) was used as an internal control. For each experiment, LFS-HMECs maintained in culture media without antibiotics were plated 1.5 x 105/well in Falcon six-well dishes. LFS-HMECs were transfected with the indicated DNAs by using FuGENE 6 (Roche Molecular Biochemicals), according to manufacturer’s guidelines. Cells were washed, fed with culture media, treated with or without rosiglitazone maleate, and harvested 24 h later. Cell extracts were assayed for luciferase activity, using the Promega luciferase assay system, and values were normalized with ß galactosidase. Triplicate samples were measured in each experiment. Data are presented as fold increase over control ± SD of two experiments.

Terminal Restriction Fragment Assay.
Measurements of telomere lengths were performed as described previously (29) . Briefly, HMEC samples were lysed, and the proteins were digested in 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 100 mM EDTA (pH 8.0), 1% Triton X-100, and 2 mg/ml proteinase K for 2 h at 55°C followed by inactivation of proteinase K for 30 min at 70°C and dialysis in 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA (pH 8.0) at 4°C overnight. Genomic DNA was digested to completion with multiple restriction enzyme mix (~1 unit/µg AluI, CfoI, HaeIII, HinfI, MspI, and RsaI; Boehringer Mannheim). The digested DNA was separated on a 0.7% agarose gel in 1x TAE buffer [0.04 M Tris-acetate and 0.002 M EDTA (pH 7.6)]. The gel was denatured for 20 min in 0.5 M NaOH and 1.5 M NaCl, rinsed with distilled water for 10 min, dried on Whatman 3MM paper under vacuum for 1 h at 55°C, and neutralized for 15 min in 1.5 M NaCl and 0.5 M Tris-HCl (pH 8.0). The gel was probed with a radiolabeled telomeric (CCCTAA)4 probe for 16 h at 42°C in 5x SSC buffer, 5 x Denhardt’s solution, 10 mM Na2HPO4, and 1 mM Na2H2P2O7. The gel was then washed twice at room temperature for 15 min each in 0.1x SSC and exposed to a phosphor screen (PhosphorImager; Molecular Dynamics, Sunnyvale, CA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Final Concentrations of the Agents Used Do Not Induce Toxicity.
Initial studies tested a range (1 nM to 100 µM) of chemopreventive agent concentrations. Fig. 1Citation shows the dose response of LFS-derived breast epithelial cells treated 72 h with 1 nM to 100 µM of specific COX-2 inhibitors (celecoxib, nimesulide, and NS-398), a nonselective COX-1 and COX-2 inhibitor (sulindac sulfide), and the PPAR{gamma} agonist rosiglitazone maleate, compared with untreated control cells. Final concentrations (10–100 nM) of the chemopreventive agents used in these studies did not affect normal growth rates or induce toxic effects on the long-term culture of the preimmortal cells. Final concentration of CP-31398 (2 µg/ml) used in the experiments also did not affect normal growth rates (data not shown).



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  		Fig. 1. Dose response of growth inhibition by chemopreventive agents. Concentrations of chemopreventive agents used for the studies (indicated by asterisks) were chosen to be below the levels that affected cell proliferation and were not toxic to the cells. Data indicate inhibition percentage of LFS-HMEC growth treated with the NSAIDs celecoxib, nimesulide, NS-398, sulindac sulfide, and PPAR{gamma} agonist rosiglitazone maleate for 72 h as compared with control cells.

 
Effect on the Spontaneous Immortalization of LFS-derived Breast Epithelial Cells by Chemopreventive Agents.
Treatment with some, but not all, chemopreventive agents reduced the frequency of spontaneous immortalization of the LFS-derived breast epithelial cells. Cells were continuously treated 5–10 PDs before crisis until the fluctuation analyses ended. Table 1Citation shows the effects of the chemopreventive agents on the frequency of spontaneous immortalization of LFS-derived breast epithelial cells. The NSAID sulindac sulfide showed no significant effect, but there was a modest reduction in the frequency of immortalization seen with the other three COX-2-specific inhibitors (celecoxib, nimesulide, and NS-398). A frequency of 3–4 x 10-7 compared with a frequency of 5–5.33 x 10-7 for untreated or solvent control (Table 1)Citation was, according to our analyses, not significant. Increasing the concentrations of the NSAIDs 10-fold to 100 nM did not significantly improve the inhibition of spontaneous immortalization (a frequency of 2.33–3 x 10-7 compared with a frequency of 5–5.33 x 10-7 for untreated or solvent control; Table 1Citation ). This may reflect the fact that by Western analyses, LFS-HMECs show an increase in COX-1 and a decrease in COX-2 protein levels after immortalization (Fig. 2)Citation .



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  		Fig. 2. Western blot analysis of COX-1 and COX-2 protein levels in LFS-HMECs and immortalized LFS-HMECs. Loading control was determined by Western blot analysis of actin. Human and mouse fibroblast lysates were included to show the species specificity of the antibody.

 
Expression and Activation of PPAR{gamma} by Rosiglitazone Maleate in LFS-HMECs.
The PPAR{gamma} agonist rosiglitazone maleate markedly reduced the frequency of spontaneous immortalization (1.33 x 10-7 compared with a frequency of 5.33 x 10-7 for untreated control). Western analysis with an antibody that detects both subtypes of PPAR{gamma} shows that PPAR{gamma} is present at roughly equal level in both the preimmortal and immortal LFS-HMECs, and treatment with 10 nM rosiglitazone maleate did not affect PPAR{gamma} expression levels after 24 h (Fig. 3A)Citation . Transient transfections with a pPPRE3-TK-LUC reporter plasmid showed only minimal activation of the endogenous PPAR{gamma} after 24-h treatment with rosiglitazone maleate in the LFS-HMECs (Fig. 3B)Citation . This result indicates that rosiglitazone maleate may mediate its effects through a PPAR{gamma}-independent pathway in LFS-derived HMECs.



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  		Fig. 3. Expression of PPAR{gamma} and effects by rosiglitazone maleate in LFS-HMECs. A, Western blot analysis of protein lysates from LFS-HMECs and immortalized LFS-HMECs without (-) or with (+) treatment of 10 nM rosiglitazone maleate for 24 h. The PPAR{gamma} antibody recognizes both PPAR{gamma}1 and PPAR{gamma}2 isoforms. Data are representative of three experiments. In B, LFS-HMECs were transfected with 5 ng of pPPRE3-TK-LUC reporter gene. The effects of rosiglitazone maleate on the luciferase activity activated by PPAR{gamma} were measured, and data are presented as the fold increase over control (±SD) of two experiments done in triplicate. Cells received no treatment (Control) or treatment with 10 nM and 1 µM rosiglitazone maleate. Luciferase counts were normalized to ß-galactosidase.

 
Induction of Telomere Shortening-induced DNA Damage Signals after Treatment with the p53 Rescue Drug CP-31398.
CP-31398 also significantly reduced the frequency of spontaneous immortalization compared with solvent control (Table 1)Citation . Treatment with CP-31398 resulted in more cellular death late in the crisis period, compared with untreated or solvent controls. No toxicity from the treatment with CP-31398 was observed before or early in the crisis period. As shown in Fig. 4Citation , increase of p53 and p21 protein levels (Fig. 4A)Citation , in addition to telomere shortening to a critical size (Fig. 4B)Citation , correlated with the increase in cellular death (data not shown) and inhibition of spontaneous immortalization in the LFS-HMECs during the fluctuation analyses.



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  		Fig. 4. Telomere shortening-induced DNA damage signals in LFS-HMECs after treatment with p53 rescue drug CP31398 correlate with inhibition of the spontaneous immortalization. A, Western blot analysis of p53 and p21 in treated LFS-HMECs during the fluctuation analyses, as well as analyses of p53 in treated immortalized LFS-HMECs and MCF-7 breast carcinoma cells. Data are representative of three experiments. B, telomere shortening to a critical length over time as evidenced by telomere restriction fragment length analyses. LFS-HMEC DNA from PD levels 30–50 were digested with six restriction fragment enzymes that do not cut within the telomeric region and probed with a telomere-specific radiolabeled probe.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is widely accepted that chemopreventive agents may prevent or hinder the occurrence of cancer (30) . Unfortunately, results from clinical trials take many years to generate. It is therefore attractive to design and test agents that act on specific molecular targets and develop preclinical models with a measurable end point to examine the effects of potential chemopreventive agents and their mechanisms of action (30) . In the present study, we used an in vitro cell culture model system of HMECs that were derived from a patient with LFS to study the effects of several novel chemopreventive agents on the frequency of spontaneous immortalization, a measurable end point for the critical step toward cancer progression (22) . We studied the effects of four NSAIDs, a peroxisome PPAR{gamma} agonist, and the P53 rescue drug CP-31398 on the frequency of spontaneous immortalization of LFS-derived breast epithelial cells.

NSAIDs block the biosynthesis of prostaglandins by inhibiting the COX activity of the enzyme prostaglandin G/H-synthase (for review, see Ref. 31 ). COX-1 and COX-2, two COX isoforms, are expressed in a variety of normal tissues, and COX-2 has been implicated as having a role in breast cancer as well as other cancers by stimulating cell growth through prostaglandins, suppressing apoptosis by increasing Bcl-2 and enhancing angiogenesis and cell invasiveness (reviewed in Ref. 32 ). The ability of NSAIDs to reduce transformation can be confined to specific oncogenic pathways, such as those involving RAS (33) , which can activate COX-2 (32) . Although most chemopreventive studies have been performed with prostate and colon cancer, the expressions of COX-1 and COX-2 were found to be high in human breast cancer (34) . Hwang et al. found that COX-2 expression was particularly high in breast epithelial tumors, whereas COX-1 was primarily localized to the adjacent stromal cells, which also contained some COX-2. The fact that the NSAIDs, including specific COX-2 inhibitors (celecoxib, nimesulide, and NS-398), had only a modest effect on the frequency of spontaneous immortalization in the HMECs used in this study may be attributable to the low doses used in this study (nM range) compared with those used in anticancer studies (µM range), suggesting that the anticancer effects in culture cells are only seen at doses that are toxic for the mammary epithelial cells used in this study. More importantly, the system used in this study measures the frequency of immortalization in vitro and, thus, only one step in carcinogenesis. It would not address the in vivo chemopreventive activity of agents that affected other stages in becoming tumorigenic. The relationship between epithelial cells and their underlying stromal cells is not represented in this particular LFS-HMEC in vitro system. Because NSAIDs, particularity COX-2 inhibitors (such as celecoxib, nimesulide, and NS-398 used in the present study), appear to mediate their effects on stromal cells (reviewed in Ref. 35 ), the chemopreventive predictability of NSAIDs would be best suited by in vivo or other studies. Celecoxib, NS-398, and sulindac sulfone were all found to prevent mammary carcinogenesis in mice and rats (36, 37, 38) . Our investigations were limited to the specific effects of potential chemopreventive agents on the immortalization frequency of LFS-derived breast epithelial cells.

Rosiglitazone maleate (rosiglitazone), a member of the TZD class of drugs, modulates the activity of the peroxisome PPAR{gamma}, a nuclear hormone receptor (39 , 40) . PPAR{gamma} regulates diverse physiological and pathophysiological gene expression, more specifically, cell proliferation and differentiation (39 , 41) . PPAR{gamma} binds to a target gene as a heterodimer with a retinoid X receptor and can act as a ligand-dependent transcription factor (39) . PPAR{gamma}, normally expressed in adipose tissue, is highly expressed in tumors, and studies show reduced tumor cell growth and/or tumor cell arrest with certain PPAR{gamma} ligands, such as rosiglitazone (39 , 42) . Furthermore, studies of ligand-mediated activation of PPAR{gamma} in cultured breast cancer cells also show reduction in growth rate and increased differentiation and suggest chemotherapeutic applications for rosiglitazone in breast cancer treatment (42, 43, 44, 45) . Because cell differentiation and reduced cell growth may influence the immortalization process, we sought to examine rosiglitazone-mediated effects on the spontaneous immortalization frequency in breast epithelial cells from an individual predisposed to cancer (HMECs derived from an LFS patient). Our in vitro results, using the LFS-derived HMECs to screen for immortalization frequency, suggest the chemopreventive potential of nontoxic doses of rosiglitazone in individuals predisposed to breast cancer. However, like other members of the TZD class of drugs (ciglitizone and troglitazone), our results indicated that rosiglitazone may be working via a PPAR{gamma}-independent pathway. These TZD class PPAR{gamma} agonists were shown to sensitize a variety of cell types to the death receptor-mediated apoptosis, further suggesting an alternative target for these ligands (46) .

The high frequency of p53 mutations in cancer, especially breast cancer, makes the rescue of the tumor suppressor function an attractive mechanism for chemoprevention (47) . The Pfizer compound CP-31398 has been reported to stabilize the core domain of p53 in vitro and be an effective anticancer drug by rescuing destabilized mutants of p53 (48) . Because the breast epithelial cells derived from a patient with LFS contain an inherited mutation in p53 and lose the second functional allele when they spontaneously immortalized, we investigated if CP-31398 could rescue the mutant p53 and prevent the spontaneous immortalization of these cells. Treatment of the LFS-derived breast epithelial cells with CP-31398 significantly reduced the frequency of spontaneous immortalization. We also observed an increase in cellular death compared with untreated cells undergoing crisis, which occurred near the end of the fluctuation analyses. Other investigators have reported a nonspecific toxic effect when using CP-31398 (49) . They concluded that the nonspecific effects of CP-31398 might override the p53-specific effects in cells. Our use of preimmortal HMECs in these studies suggests a different interpretation. The activation of p53 by "too short" telomeres is a key part of the DNA damage response that occurs during replicative aging. Stabilizing a mutant p53 in normal healthy cells may be nontoxic, because the p53 would be expected to lack the post-translational modifications that occur during the DNA damage response. However, as the LFS-HMECs approach their proliferation limits, the telomere shortening-induced DNA damage signals activate the stabilized p53 and produce the very late "toxic" effects we observed. This would be analogous to what has been observed in mouse studies, in which an extra copy of an activated mutant p53 produced toxicity to the tumor cells, whereas an extra copy of a wild-type p53 did not (50) . Rather than being nonspecific, this would reflect a direct action mechanism of the drug. The telomere shortening in the LFS-HMECs as they reached crisis and the increase of p53/p21 protein levels seen in our studies suggest that stabilization of p53 by CP-31398 can increase telomere shortening-induced DNA damage signals, as evidenced by an increase in cellular death.

The data presented in this study provide insights into the effects of different chemopreventive agents on the spontaneous immortalization of LFS breast epithelial cells. Treatment with rosiglitazone maleate and the p53 rescue drug CP-31398 notably reduced the frequency of spontaneous immortalization of LFS-derived breast epithelial cells. The use of NSAIDs, including specific COX-2 inhibitors (celecoxib, nimesulide, and NS-398), had only a marginal effect on the spontaneous immortalization of preimmortal LFS breast epithelial cells at the concentrations tested. In summary, these studies show the usefulness of a human cellular-based model system to dissect some of the cellular pathways of known and potentially novel chemopreventive agents, using cell immortalization as an end point.


   ACKNOWLEDGMENTS
 
We thank Dr. Farzan Rastinejad (Pfizer Central Research) for the gift of CP-31398, Merck Research Laboratories for providing us with the sulindac sulfide, Dr. David Manglesdorf (University of Texas-Southwestern Medical Center at Dallas) for the PPAR{gamma} antibody, and Jason Sherrell for technical assistance.


   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 Supported by Contracts N01-CN-0501763-63 and N01-CN-15015-40 from the National Cancer Institute and a postdoctoral fellowship from the Department of Defense Breast Cancer Research Program [B-S. H.]. Research funding for statistical consulting was provided in part by the Simmons Cancer Center. Back

2 To whom requests for reprints should be addressed, at Department of Cell Biology, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-9039. Phone: (214) 648-2662; Fax: (214) 648-8694; E-mail: brittney-shea.herbert{at}utsouthwestern.edu Back

3 The abbreviations used are: LFS, Li-Fraumeni syndrome; HMEC, human mammary epithelial cell; PD, population doubling; NSAID, nonsteroidal anti-inflammatory drug; TZD, thiazolidinedione; PBS-T, PBS plus 0.05% Tween; COX, cyclooxengenase; PPAR, peroxisome proliferator-activated receptor. Back

Received 9/ 6/02. Accepted 3/ 5/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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J. S. Butler and S. N. Loh
Folding and misfolding mechanisms of the p53 DNA binding domain at physiological temperature
Protein Sci., November 1, 2006; 15(11): 2457 - 2465.
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