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[Cancer Research 65, 8747-8753, October 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

ATM and p21 Cooperate to Suppress Aneuploidy and Subsequent Tumor Development

Kate C. Shen1, Henry Heng2, Yaolin Wang4, Shu Lu1, Guo Liu1, Chu-Xia Deng5, S.C. Brooks3 and Y. Alan Wang1

1 Barbara Ann Karmanos Cancer Institute, 2 Center for Molecular Medicine, and 3 Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, Michigan; 4 Department of Tumor Biology, Schering-Plough Research Institute, Kenilworth, New Jersey; and 5 Genetics of Development and Disease Branch, National Institute of Digestive and Kidney Diseases, NIH, Bethesda, Maryland

Requests for reprints: Y. Alan Wang, Barbara Ann Karmanos Cancer Institute, 110 East Warren Avenue, Detroit, MI 48201. Phone: 313-833-0715; Fax: 313-831-7518; E-mail: wangya{at}karmanos.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The DNA damage checkpoint protein kinase mutated in ataxia telangiectasia (ATM) is involved in sensing and transducing DNA damage signals by phosphorylating and activating downstream target proteins that are implicated in the regulation of cell cycle progression and DNA repair. Atm–/– cells are defective in cellular proliferation mediated by the Arf/p53/p21 pathway. In this report, we show that increased expression of p21 (also known as Waf1 or CDKN1a) in Atm–/– cells serves as a cellular defense mechanism to suppress further chromosomal instability (CIN) and tumor development because Atm–/–p21–/– mice are predisposed to carcinomas and sarcomas with intratumoral heterogeneity. It was found that Atm-deficient cells are defective in metaphase-anaphase transition leading to abnormal karyokinesis. Moreover, Atm–/–p21–/– primary embryonic fibroblasts exhibit increased CIN compared with either Atm–/– or p21–/– cells. The increased CIN is manifested at the cellular level by increased chromatid breaks and elevated aneuploid genome in Atm–/–p21–/– cells. Finally, we showed that the role of p21 in a CIN background induced by loss of Atm is to suppress numerical CIN but not structural CIN. Our data suggest that the development of aneuploidy precedes tumor formation and implicates p21 as a major tumor suppressor in a genome instability background.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Genome instability and aneuploidy are the hallmarks of most of the human cancers. The predominant form of genome instability in human cancer is chromosome instability, which is characterized by gains or losses of whole chromosomes (aneuploidy) and chromosomal structural aberrations (amplification, deletions, and chromosomal translocations) (1, 2). There are various nonmutually exclusive mechanisms for the maintenance of chromosomal stability (3). Chromosomal structural aberrations are largely attributed to the telomere dysfunction in cancer cells that lead to breakage-fusion-bridge translocations, whereas aneuploidy is thought largely due to a defective mitotic spindle checkpoint (2, 4–6). Aneuploidy is frequently observed in clinical tumor samples and is correlated with poor tumor prognosis. However, it is still not clear whether aneuploidy is the consequence or the cause of tumor development (7–9).

Genome integrity is constantly monitored by the coordinated action of cell cycle checkpoints and DNA repair systems (10). Inactivation of genes involved in the maintenance of genome integrity leads to genome instability, early onset of aging, and predisposition to cancer (11, 12). Ataxia telangiectasia, a genome instability syndrome, is characterized by progressive cerebellar degeneration, immune deficiencies, and premature aging. In addition, ataxia telangiectasia patients are sensitive to ionizing radiation and have increased incidence of tumorigenesis in the lymphoid organs (13–15). The protein kinase mutated in ataxia telangiectasia (ATM) is essential in sensing DNA damage and in activating cell cycle checkpoints. ATM protein is a member of the phosphatidylinositol 3-kinase–related kinase family of proteins containing a highly conserved COOH-terminal phosphatidylinositol 3-kinase domain (13–15). In response to DNA damage, activated ATM phosphorylates p53 as well as other downstream target proteins involved in cell cycle checkpoint regulation. Ataxia telangiectasia cells are defective in multiple cell cycle checkpoint regulation, including G1-S, intra-S, and G2-M (13–15). However, it is still uncertain whether ATM is normally involved in proper mitotic progression and/or plays a role in spindle checkpoint regulation. In addition, the mechanism of ATM in suppression of chromosome instability remains to be determined.

P53 is a transcription factor that modulates downstream target genes, which in turn regulate cell cycle arrest and apoptosis in response to DNA damage (16). Because p21 is a main target regulated by p53, and p53-mediated up-regulation of p21 has been implicated in G1-S checkpoint regulation, it was surprising then that p21-deficient mice were not prone to tumorigenesis, suggesting that p21-mediated G1-S cell cycle arrest in response to DNA damage is dispensable for p53-dependent tumor suppression (17–20). The potential function of p21 in tumor suppression and aneuploidy development was implicated recently in a p53-R172P knock-in mouse model, which displayed reduced tumorigenesis compared with p53-null mice (21). P53-R172P is an equivalent of a rare mutant form of human p53 (R175P) found in tumors and is completely defective in induction of apoptosis yet retains the ability to induce cell cycle arrest (22, 23). Importantly, tumors arising from these p53-R172P knock-in mice displayed diploid phenotype in contrast to the aneuploidy found in tumors derived from p53-null mice (21). In this study, we show that p21 acts as a tumor suppressor specifically in a genome instability background and Atm and p21 cooperate to impede tumorigenesis by suppressing aneuploidy development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal studies. Both Atm and p21 knockout mice used in this study are in the mixed genetic background of 129SvEv and Black Swiss (19, 24). The double knockout mice were generated as described previously (25). Histopathologic analysis was done as described (25).

Metaphase analysis. Active proliferating cells of early passage mouse embryonic fibroblasts (MEF; p1-p2) were treated with 0.15 µg/mL colcemid for 1 hour. Cells were trypsinized and washed with PBS. We slowly added hypotonic buffer (0.075 mol/L KCl at 37°C) to the cell pellet while vortexing the cells gently. Cells were subsequently fixed in 3:1 methanol/glacial acetic acid and the fixed metaphase chromosome preparation was placed onto a clear slide and stained with Giemsa solution (Karyomax, Life Technologies, Carlsbad, CA).

Spectral karyotyping analysis. The spectral karyotyping analysis was done as described previously (26). Images were acquired using the SkyVision spectral imaging system and the SkyView 1.2 software (Applied Spectral Imaging, Carlsbad, CA) was employed for imaging analysis.

Time-lapse imaging analysis. The histone H2B fused with green fluorescence protein (H2B-GFP) expression vector (Clontech, San Diego, CA) was transfected into immortalized p21–/– (p20) or Atm–/–p21–/– MEFs (p20) using Fugene 6 reagent (Roche, Indianapolis, IN). Transfected cells were selected with 1 µg/mL blasticidin and the stable transfected clones were pooled. A layer of mineral oil was added on top of the cultured cells in 10% fetal bovine serum/DMEM during imaging. Under these conditions, it was possible to only monitor cell cycle progression for a maximal of 3 hours. Chromosome segregation was monitored on a heating plate (38.9°C) with an inverted microscope and a 32x objective lens (Zeiss Axiovert 35). Under UV excitation, we identified cells at metaphase and monitored mitotic progression every 5 minutes on average. Mitotic progression was documented with a Dage CCT300 digital camera and MCID imaging software (Imaging Research, Inc., St. Catherines, Ontario, Canada).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Atm–/– cells are defective in cellular proliferation mediated by the Arf/p53/p21 pathway (25, 27–30). To determine whether Atm and p21 cooperate to suppress tumor development, we created 39 Atm–/–p21–/– mice in a mixed genetic background (129SvEv and Black Swiss), which were observed for signs of malignancy. As reported previously (25), 14 of these mice developed thymic lymphomas with delayed kinetics compared with Atm–/– mice. Interestingly, multiple primary tumors of different cell lineages were observed in ~50% of the Atm–/–p21–/– mice at a mean age of 7 months (Table 1). The increased frequency of tumor development in Atm–/–p21–/– mice is not merely due to a delayed onset of thymic lymphoma development because Atm–/–p21–/– mice developed embryonic type tumor as early as 3 months without thymic lymphoma (Table 1). The tumor spectrum (including sarcomas, myeloid leukemia, hepatomas, and teratomas) observed in Atm–/–p21–/– mice was found to be similar to that observed in p53–/– mice (31, 32). None of these tumors have been observed in either Atm–/– (n = 23) or p21–/– (n = 15) mice (Fig. 1A; Table 1). Interestingly, we noted that there were more carcinomas in this cohort of mice at older ages (Table 1). Up to a quarter of the tumors were carcinomas (Fig. 1B), which was reminiscent of the tumor spectrum observed in Tert–/–p53–/– mice (33). Indeed, aberrant mitoses with anaphase bridge and unequal chromosome segregation were observed in these tumor samples (Fig. 1C). Collectively, these results show that Atm and p21 cooperate to suppress tumor development. Although ataxia telangiectasia patients and Atm-deficient mice with short telomeres developed premature aging (34), we did not observe increased aging phenotype in these Atm–/–p21–/– mice (data not shown).


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  		Table 1. Tumor distribution

 


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  		Figure 1. Atm and p21 cooperate to suppress tumor development. A, histopathologic analysis of representative Atm–/–p21–/– tumors. Magnification, x200. a, hepatocarcinoma; b, hemangioma; c, osteosarcoma; d, teratoma. B, pie distribution of the tumor spectrum from a total of 53 tumors. C, aberrant mitoses in Atm–/–p21–/– tumors. Three aberrant mitotic cells were presented. Arrows, anaphase bridge (left and middle). The frequency of anaphase bridge was 27% and 39% for hepatocarcinoma and sarcoma, respectively. Right, unequally segregated chromosomes.

 
To characterize the mechanism of cooperative tumor suppression by Atm and p21, it was first ascertained whether a consistent alteration in the genome structures might contribute to tumor development in Atm–/–p21–/– mice. Spectral karyotyping analysis was done using the primary tumor cell lines derived from Atm–/–p21–/– mice. We found that tumor-derived metaphases were extremely heterogeneous and none of the tumor cell lines (n = 3) exhibited recurrent chromosomal abnormalities. In addition, heterogeneous chromosomal profiles were found among individual tumors (Fig. 2). The chromosomal instability (CIN) observed in this setting suggests that the faithful segregation of chromosomes might be compromised in Atm-deficient cells.



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  		Figure 2. Intratumoral heterogeneity shown by spectral karyotyping analysis. Early passage cells from sarcoma (7211) were analyzed by spectral karyotyping. A, representative image of the spectral karyotyping analysis. B, complex chromosomal translocations. C, heterogeneity of intratumoral cells. Karyotypes of eight metaphases are presented. T, translocation; Tc, complex translocation.

 
To determine whether ATM is directly involved in regulating chromosome segregation, we generated immortalized p21–/– and Atm–/–p21–/– MEFs expressing H2B-GFP to facilitate the monitoring of chromosome segregation during mitosis (35). Time-lapse imaging analysis was done with these H2B-GFP–expressing cells to study the metaphase-anaphase transition that is regulated by spindle assembly checkpoint. Whereas most of the p21–/– cells (n = 11) exhibited normal chromosomal separation (Fig. 3B), increased chromosomal segregation defects were observed in Atm–/–p21–/– cells (n = 18; Fig. 3C). Although 1 of 11 p21–/– metaphases analyzed had a misaligned metaphase plate, the metaphase-anaphase transition progressed normally and was completed in 24 minutes (data not shown). In contrast, 9 of 18 Atm–/–p21–/– cells exhibited defects during metaphase-anaphase transition although karyokinesis was attempted multiple times (Fig. 3C). Chromatid separation was occasionally initiated in misaligned metaphase chromosomes leading to a defective cytokinesis. In these cells, loss of cytoplasm occurred in the two daughter cells as evidenced by a visible circle of newly formed cell membrane surrounding the still condensed chromatin (Fig. 3C, b). Although an anaphase bridge was observed in 1 of the 11 p21–/– cells, 6 of the remaining 9 Atm–/–p21–/– cells showed such features (data not shown). Accordingly, it was found that Atm–/–p21–/– cells have a prolonged mitotic progression (64.3 ± 12 minutes, n = 9), measured from metaphase to decondensed chromatin, compared with control p21–/– cells (30.7 ± 4 minutes, n = 11; P < 0.02; Fig. 3A). It is not clear whether p21 deficiency would influence the mitotic behavior in Atm-null cells; we therefore independently generated immortalized Atm–/– MEFs. As shown in Fig. 3D, defective mitotic segregation was also observed in Atm–/– cells. Our results show clearly that Atm is required for proper metaphase-anaphase transition during mitosis. This result is consistent with the observation that ATM was activated in response to mitotic stress in several cell lines examined (data not shown).



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  		Figure 3. Atm is required for proper mitotic progression. H2B-GFP fusion expression vector was introduced into p21–/–, Atm–/–, and Atm–/–p21–/– cells and selected for blasticidin resistance. Pooled clones were analyzed by time-lapse imaging. A, prolonged mitotic progression in Atm–/–p21–/– cells (n = 9) compared with p21–/– cells (n = 11). P < 0.02, two-tailed Student's t test assuming unequal variance. The duration from metaphase to chromatin decondensation was measured. The average duration was 30.7 ± 4 minutes for p21–/– cells and 64.3 ± 12 minutes for Atm–/–p21–/– cells. B, p21–/– cells undergo normal metaphase-anaphase transition and complete the mitosis. C, Atm–/–p21–/– cells were defective in metaphase-anaphase transition (a-c). a, in these serial images, cells attempted to undergo karyokinesis. b, mitotic spindle was misaligned. Metaphase-anaphase transition occurred on the side of the cells and apparently completed cytokinesis with the loss of large amount of cytoplasm. c, unsegregated chromosomes showed dynamic movement but could not undergo cytokinesis as illustrated by these serial images taken in <1-minute interval. D, time-lapse imaging analysis of Atm–/– cells in which mitotic chromosomes failed to segregate properly.

 
To further characterize the mechanism of cooperative tumor suppression by Atm and p21, we carried out a detailed karyotypic analysis of the MEFs derived from wild-type (WT), p21–/–, Atm–/–, and Atm–/–p21–/– cells. Chromosome numbers of all metaphases from the above cell types were scored. WT MEFs exhibited a background level of aneuploid metaphases (Fig. 4A), which was also described recently (36, 37). This observation may partially explain why murine fibroblasts were more easily immortalized in normal cell culture conditions compared with cells derived from humans. p21–/– MEFs had the same level of aneuploidy as WT MEFs, although the numbers of tetraploid and polyploid cells were slightly increased (Fig. 4A and B). In contrast, a significant increase in number of aneuploid cells was observed in Atm–/– MEFs (Fig. 4A and B). More importantly, the increased aneuploidity observed in Atm-deficient cells was further exacerbated by the loss of p21 (Fig. 4A and B). These results show that Atm and p21 cooperate to suppress the development of aneuploid genome and thereby maintain genome stability.



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  		Figure 4. Atm and p21 cooperate to maintain genome stability. A and B, cytogenetic analysis of MEFs. A, chromosome numbers were determined from metaphase spreads. Ploidy distributions were calculated as a percentage of metaphases analyzed. Total metaphases analyzed: WT MEFs, n = 73; p21–/– MEFs, n = 30; Atm–/– MEFs, n = 114; Atm–/–p21–/– MEFs, n = 134. B, increased aneuploidy in Atm–/–p21–/– MEFs. Data in A were recalculated to determine the distribution of diploid (2N), tetraploid (4N), polyploidy (PN), and aneuploidy (AN) in these MEFs. C, increased CIN in Atm–/–p21–/– MEFs. Metaphases were analyzed for the presence of chromatid breaks, multiradial chromosomes, centrimeric fusions, ring chromosomes, and double minutes. Columns, mean chromatid breaks per metaphase. Metaphase analyzed: WT MEFs, n = 47; p21–/– MEFs, n = 30; Atm–/– MEFs, n = 45; Atm–/–p21–/– MEFs, n = 60. D, increased chromosomal gain and/or loss in Atm–/–p21–/– MEFs were revealed by spectral karyotyping analysis. G/L, gain or loss of chromosomes; TL, nonreciprocal translocation. Note that about the same frequencies of nonreciprocal translocations were observed in Atm–/– and Atm–/–p21–/– MEFs, whereas increased frequencies of chromosomal deletions and/or amplifications were observed in Atm–/–p21–/– MEFs compared with Atm–/– MEFs. Total metaphase analyzed: Atm–/– MEFs, n = 26; Atm–/–p21–/– MEFs, n = 38.

 
An analysis of the metaphase preparations for chromosomal aberrations showed a strikingly high frequency of chromosomal aberrations in Atm–/–p21–/– versus Atm–/– MEFs (91.6% versus 66.6%). The mean chromatid breaks for the Atm–/–p21–/– MEFs was 2-fold greater than that in Atm–/– MEFs (Fig. 4C), whereas chromatid breaks were rarely observed in WT or p21–/– MEFs. Most interestingly, there was an increase of double minute chromosome fragments in Atm–/–p21–/– MEFs compared with Atm–/– MEFs (28.3% versus 4.4%). Therefore, loss of p21 augmented CIN in an Atm-deficient background. Chromatid exchanges characterized by triradials and quadriradials occurred in a similar frequency in both Atm–/– and Atm–/–p21–/– MEFs (4.4% versus 1.6%), suggesting that loss of p21 did not affect chromosomal translocations in the Atm–/– background.

To further extend the cytogenetic analysis, a detailed examination of chromosome structures was done with spectral karyotyping. Chromosomal aberrations were scored for gain or loss of chromosomes and chromosomal translocations. Consistent with the cytogenetic analysis, we found that aberrant metaphases occurred more frequently in Atm–/–p21–/– MEFs than in Atm–/– MEFs (Fig. 4D). The increased frequency of chromosomal aberrations observed in Atm–/–p21–/– metaphases was mostly due to the increased rate of chromosomal losses or gains in Atm–/–p21–/– MEFs (Fig. 4D). Of the abnormal metaphases examined, none displayed consistent chromosome lesions. Nonreciprocal chromosomal translocations (NRTs) were observed in 34% of the metaphases analyzed in Atm–/– MEFs. However, loss of p21 in an Atm–/– background did not facilitate further increases of NRT (Fig. 4D). These data indicate that loss of p21 selectively exacerbates chromosomal rearrangements (i.e., aneuploidy versus NRT) in an Atm deficiency–induced genome instability background.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because aneuploidy is a common feature in almost all human cancers, a better understanding of the molecular mechanisms of aneuploidy and CIN will have tremendous effect on clinical prognosis of human cancer and on drug development targeting the CIN pathways. Whether aneuploidy is the consequence or the cause of cancer development remain uncertain. The results of this study show that aneuploidy occurs early in the neoplastic process, which may start during embryogenesis, and is suppressed by Atm and p21. Furthermore, the observations in this report indicate that Atm and p21 also cooperate to suppress tumor development. The tumor spectrum observed in Atm–/–p21–/– mice can be divided into two groups: the early tumor spectrum was mostly lymphomas and sarcomas, which was similar to that observed in p53–/– mice (31, 32), and the late-onset carcinomas, which are not frequently observed in mouse models of cancer, except in a telomerase-deficient animal model and recently developed p53 mutant knock-in mouse models for Li-Fraumeni syndrome (33, 38, 39). The development of carcinomas in the Atm–/–p21–/– mice is of particular interest in its epistatic correlation with Tert–/–p53–/– mice because ATM activates p53 in response to DNA damage and is also involved in telomere maintenance (34, 40, 41). The role of p21 in tumor suppression seems to be context dependent. We have shown previously that p21 was required for survival of thymic lymphomas in Atm–/– mice and thymic lymphoma development was delayed in Atm–/–p21–/– mice (25). This observation was supported by experiments showing that radiation-induced lymphomas were significantly decreased in p21–/– mice than those in control group (42). However, p21 did not seem to play a role in lymphomagenesis induced by Moloney murine leukemia virus (43). In addition, previous studies showed that loss of p21 accelerated the development of pituitary tumors in Rb+/– mice and in p18–/– mice (44, 45), mammary tumors in Ras but not Myc transgenic mice (46–48), and intestinal tumors in Apc-haploinsufficient mice (49). Furthermore, loss of p21 modulated tumor spectrum in INK4a/ARF–null mice but had no effect in Wrn-null mice (50, 51). It was also shown that p21-null mice developed spontaneous tumors with an average onset of 16 months in about half of the mice analyzed (42). That report differs from our current analysis showing that no tumor was seen in p21-null mice for up to 18 months (data not shown). Nonetheless, our data are consistent with the earlier report showing that tumorigenesis was not observed in that particular strain of p21-null mice (19, 44, 45, 50). The difference in spontaneous tumorigenesis observed in these two strains of mice could be due to genetic background or housing environment. Indeed, it was reported that up to 60% of the p21-null mice died of autoimmune disease due to abnormal proliferation of memory T lymphocytes (42). Regardless, our data clearly show for the first time a synergistic interaction of Atm and p21 in tumor suppression in vivo.

One of the main features of telomere dysfunction is nonreciprocal chromosome translocation generated by fusion-bridge-breakage of nonhomologous chromosomes leading to structural CIN (2). Indeed, structural CIN was observed in Atm–/– MEFs but was not further increased in Atm–/–p21–/– MEFs, suggesting that p21 was not involved in suppression of p53-dependent early telomere crisis. The role of p21 in tumor suppression is underscored by the observation that p21 was specifically involved in suppression of numerical CIN in the Atm–/– background. We found that Atm was involved in proper metaphase-anaphase progression and was activated by mitotic stress independent of the DNA damage checkpoints (data not shown). The numerical CIN we have observed in Atm–/– cells is therefore likely due to the defect in metaphase-anaphase progression.

The observation of low levels of aneuploidy in normal MEFs in this study is of particular interest as the basal level of aneuploidy in normal rodent cells was also observed previously by others but has not been discussed extensively (52–54). Whether a low level of aneuploidy could be detected in human cells remains to be determined. Loss of Atm causes increased aneuploidy above the basal level in normal cells (this study) and leads to activation of p53-mediated G1-S checkpoint because loss of p53 in the Atm-deficient background rescued cell proliferation defect and suppressed p21 expression in Atm-null cells (27, 29, 30). The importance of p21 induction in a genome instability background is clearly shown in this study. The function of p21 in this setting is to specifically suppress numerical chromosome instability and therefore suppress further CIN development in a genome instability background. This finding is consistent with the observation that tumors derived from p53R172P knock-in mice, which retained the p21-mediated G1-S checkpoint but were defective in apoptosis induction, tend to be diploid compared with those derived from p53-null mice (21). The role of p53 in suppressing aneuploidy development in mouse cells was in stark contrast with the result obtained from p53 knockout human colon cancer cells or fibroblasts (55). The difference between mouse and human cells in basal level of aneuploidy could partly be explained by the sensitivity of mouse cells to oxidative stress in cell culture. Consistent with this notion, it was recently shown that mouse cells cultured in a serum-free medium had greatly reduced aneuploidy compared with those culture in serum-containing medium (37). Taken together, the data presented here show clearly that a major function of ATM in maintaining genome stability is carried out through the regulation of faithful segregation of chromosomes by suppressing both structural CIN and numerical CIN, whereas p21 safeguards against further genome instability by suppressing numerical CIN. Therefore, our results showed that aneuploidy levels above those in normal cells are actively and subsequently suppressed by Atm and p21, respectively. This study therefore reveals multiple levels of failsafe mechanisms to suppress aneuploidy and subsequent tumorigenesis. Consistent with our experimental observations and conclusion, it was recently shown that mutation of the spindle checkpoint gene Bub1b is directly implicated in aneuploidy and subsequent cancer development in human patients with Mosaic variegated aneuploidy (56). Further analysis of Atm and p21 function in this Atm–/–p21–/– mice and derived MEF cells will lead to better understanding of aneuploidy in human cancer cells. These Atm–/–p21–/– mice may also serves as a good in vivo model to evaluate novel agents for inhibiting aneuploidy during cancer development.


   Acknowledgments
 
Grant support: DAMD-17-02-1-0619, P30 ES06639, and RO1 CA89526 (S.C. Brooks and Y.A. Wang).

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.

We thank Dr. R. Cardiff for help with histopathologic analysis. This work was initiated in Dr. Leder's laboratory and we are grateful for his generous support.

Received 4/28/05. Revised 7/ 7/05. Accepted 7/19/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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