Frequent hypermethylation of the 5' CpG island of the mitotic stress checkpoint gene Chfr in colorectal and non-small cell lung cancer

Paul G. Corn1,*, Matthew K. Summers2,*, Franz Fogt1, Arvind K. Virmani3, Adi F. Gazdar3, Thanos D. Halazonetis4 and Wafik S. El-Deiry5,6

1 University of Pennsylvania Medical Center, Philadelphia, PA 19104, USA,
2 Graduate Program in Cell and Molecular Biology, University of Pennsylvania, Philadelphia, PA 19104, USA,
3 Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA,
4 The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA and
5 Laboratory of Molecular Oncology and Cell Cycle Regulation, Howard Hughes Medical Institute, Departments of Medicine, Genetics, Pharmacology, Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chfr, a mitotic stress checkpoint gene, regulates a prophase delay in cells exposed to agents that disrupt microtubules, such as nocodazole and taxol. In the present study, we report that Chfr is frequently methylated in cell lines derived from tumors of the colon (80%), brain (100%) and bone (100%). In addition, Chfr was methylated in 37% of primary colon adenocarcinomas and in 10% of primary non-small cell lung carcinomas. In normal colon tissue, but not lung, there was evidence for age-related methylation of Chfr, suggesting that in some cases the tumor may have arisen from a methylated clonal precursor. Methylation was associated with loss of Chfr mRNA and protein expression in cancer cell lines. In cells with methylated Chfr, treatment with the demethylating agent 5-aza-2'-deoxycytidine resulted in re-expression of Chfr, and partial restoration of the prophase checkpoint. These results suggest that epigenetic inactivation of Chfr may be responsible for many of the checkpoint defects observed in human cancers.

Abbreviations: 5Aza-dC, 5-aza-2'deoxycytidine; MSP, methylation-specific PCR; Rb, retinoblastoma gene; RT–PCR, reverse transcription-PCR


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chfr, a mitotic stress checkpoint gene, was recently cloned and localized to chromosome 12q24.33 (1). In mammalian cells exposed to drugs that disrupt microtubule structure, such as nocodazole or taxol, the Chfr protein mediates a delay of entry into metaphase that is characterized microscopically by delayed chromosome condensation. In addition, Chfr promotes cell survival in response to mitotic stress. The Chfr protein possesses an N-terminal forkhead-associated (FHA) domain, a central ring finger (RF) domain, and a C-terminal cysteine-rich (CR) region. Based on functional analysis of deletion mutants of Chfr, both the FHA and CR regions are required for its checkpoint function. More recent studies have shown that the Chfr protein has ubiquitin ligase activity that is dependent on the RF domain (2). While definitive targets for the ubiquitin ligase activity of Chfr remain to be identified in human cells, in Xenopus cell-free extracts Chfr can ubiquitinate Polo-like kinase 1 (Plk1), a protein kinase with multiple roles in mitosis (3).

In the initial study of Chfr, northern blot analysis of a series of eight colon, osteosarcoma and neuroblastoma cancer cell lines revealed that Chfr expression was absent in three (1). The molecular mechanism responsible for this aberrant Chfr expression, however, was not investigated. In addition, in the U2OS osteosarcoma cell line, a missense mutation was detected in the cysteine-rich region of Chfr that resulted in a loss of function phenotype. While many human cancers appear to be sensitive to mitotic stress, mutations of known mitotic spindle checkpoint genes are rare, including hsMAD2, hBUB1 and hBUB3 (49). Thus, the observation that Chfr was frequently inactivated in tumor cell lines prompted us to further investigate whether alterations of Chfr occur in primary tumors.

In this study, we examined whether hypermethylation of the 5' CpG island of Chfr occurs in human cancers. Aberrant methylation of 5' CpG islands located in promoter regions recruits a repressive transcription complex that results in silencing of the gene (10). In previous studies, this epigenetic process has been shown to inactivate a number of tumor suppressor genes including VHL (Von Hippel-Lindau), p16 and E-cadherin (1114). Here we show that loss of Chfr expression is associated with methylation of the 5' CpG island in cancer cell lines and can be restored after treatment with the demethylating agent, 5-aza-2'-deoxycytidine (5Aza-dC). This finding led us to screen for methylation changes in a series of primary colon, lung, breast, and renal carcinomas.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Source of tissue samples
Primary tumor specimens analyzed in this study were obtained from three principal sources. First, frozen tissue specimens consisting of four colon, two breast, one lung, and one kidney carcinoma were obtained from the Cooperative Human Tissue Network (Hospital of the University of Pennsylvania). Paired normal specimens were available for two of the four colon cancers, and for each of the breast, lung, and kidney tumors. Demographic data were not available for these specimens. Secondly, 27 paraffin-embedded tumor specimens were obtained from a group of patients with adenocarcinoma of the colon that were identified by a search through the pathology archives of the Presbyterian Medical Center at the University of Pennsylvania. This group had a mean age of 70 (range 44–87), and consisted of 60% males and 40% females. Patients with tumor-node-metastasis stages I–IV were represented in the group. There were two patients with stage I disease (7%), 11 patients with stage II disease (41%), 11 patients with stage III disease (41%) and three patients (11%) with stage IV disease. Hematoxylin and eosin stained sections were reviewed by a pathologist (F.F.) to confirm the presence of adenocarcinoma in each case. Normal specimens for 14 of the 27 cancers were obtained from adjacent tissue at least 5 cm away from the distal negative margin of the tumor. In addition, nine normal control specimens were obtained from age-matched patients with a history of diverticulosis/diverticulitis but without any history of a malignant disorder. For these control cases, colonic mucosa without any involvement by inflammatory disease was chosen for further analysis. Thirdly, 19 primary non-small cell lung cancers and paired normal tissues used in this study were described previously (15).

Cell lines and culture conditions
Cell lines analyzed in this study included colon (DLD1, HCT116, SW480, Lovo, HT29), osteosarcoma (U2OS, SAOS), esophageal (Bic1, Seg1), central nervous system (IMR5, TE671), leukemia (U937, KG1a), breast (MCF7, MDA468) and prostate (DU145) cancer cell lines. All colon, leukemia, breast and prostate cell lines were available from the American Type Culture Collection (Manassas, VA). DNA samples from Bic1 and Seg1 were kindly provided by Tsung-Teh Wu (The University of Texas, M.D. Anderson Cancer Center, Houston, TX). DNA samples from IMR5 and TE671 were kindly provided by Kenneth J.Cohen (Johns Hopkins University School of Medicine, Baltimore, MD). Cell lines were maintained in appropriate media and treated with 5-Aza-dC (Sigma, St Louis, MO) at a concentration of 5 µM for 3 days to achieve demethylation.

DNA extraction
Genomic DNA was isolated from paraffin-embedded tissue slides as described previously (16). Genomic DNA was isolated from frozen tissue specimens and cell lines by standard phenol and chloroform extraction (17).

Methylation analysis
Analysis of the genomic (GenBank accession number 13650132) and mRNA (GenBank accession number AF170724) sequences of Chfr revealed that the region spanning exon 1 (containing transcription start), intron 1, and exon 2 (containing translation start) contains a CpG island. Analysis of methylation patterns within the CpG island (sequence -281 to +51 bp relative to translation start) was determined after the chemical modification of genomic DNA with sodium bisulfite and methylation-specific PCR (MSP) as described previously (18). Two sets of MSP primers, primer sets 1 and 2, were used to examine portions of the CpG island that were nearest to the transcription and translation start site, respectively. For primer set 1, primer sequences for the methylated reaction were 5'-GTAATGTTTTTTGATAGCGGC-3' (sense) and 5'-AAT- CCCCCTTCGCCG-3' (antisense), and for the unmethylated reaction were 5'-GGTTGTAATGTTTTTTGATAGTGGT-3' (sense) and 5'-CAAATCCCCC- TTCACCA-3' (antisense). The product sizes of the methylated and unmethylated PCR products for pimer set 1 were 106 and 112 bp, respectively. For primer set 2, primer sequences for the methylated reaction were 5'-GTCGGGTCGGGGTTC-3' (sense) and 5'-CCCAAAACTACGACGACG-3' (antisense), and for the unmethylated reaction were 5'-TGGTT- GGGTTGGGGTTT-3' (sense), and 5'-ATCCCCAAAACTACAACAACA-3' (antisense). The product sizes for the methylated and unmethylated PCR products for primer set 2 were 150 and 155 bp, respectively.

Detection of Chfr expression
For analysis of Chfr mRNA, cytoplasmic RNA was isolated using TriZOL Reagent (InVitrogen, Carlsbad, CA) and reverse transcribed using SuperScript II (InVitrogen). The RT–PCR primers for Chfr were 5'-TGGAACAGTGATTAACAAGC-3' (sense, exon 4) and 5'-AGGTATCTTTGGTCCCATGG-3' (antisense, exon 6). Glyceraldehyde-3-phosphate dehydrogenase mRNA expression was analyzed as described previously (19). For analysis of Chfr protein, whole cell lysates were prepared from cell pellets using EBC buffer (50 mM Tris pH 8.0, 120 mM NaCl, 0.5% NP-40). One milligram of lysate was immunoprecipitated with polyclonal antisera against Chfr. Immunoprecipitates were resolved on 7.5% SDS–PAGE and detected with a monoclonal Chfr antibody. Both the rabbit polyclonal and mouse monclonal antibodies were raised against a partial length human Chfr protein encompassing amino acids 1–433.

Checkpoint analysis
A total of 4x105 HCT116 or DLD1, or 6x105 SAOS2 cells were seeded in 100 mm dishes. Cells were treated with 5 µM 5Aza-dC or vehicle every 24 h for a total of 72 h. At 59 h of treatment, 1 µM nocodazole or vehicle was added to the cells. After 13 h of nocodazole treatment, cells were collected and fixed in ice-cold 70% ethanol for at least 10 min. Samples were extracted on ice with 0.2% Triton X-100 for 5 min, diluted with 5 ml PBS and pelleted. The cells were blocked with 10% heat-inactivated FBS/PBS for 30 min at room temperature, re-suspended in 200 ml of 1% BSA/PBS containing polyclonal anti-phospho-S10 Histone H3 antibody (Upstate Biotechnology, Waltham, MA) at 1:200 and incubated overnight at 4°C. Samples were pelleted, washed in PBS, pelleted again and incubated in 200 ml 1% BSA/PBS containing Alexafluor 488 conjugated goat anti-rabbit antibody (Molecular Probes, Eugene, OR) at 7 µg/sample for 30 min at room temperature. The cells were washed as above and re-suspended in 1 ml of 0.1% Tween-20/2% FBS/PBS with 5 ng RNase and 5 µg propidium iodide for 1 h at 37°C. Samples were analyzed by flow cytometry using a Becton Dickinson FACScan with CellQuest software.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In a previous study, northern blot analysis of a series of eight colon, osteosarcoma and neuroblastoma cancer cell lines revealed that Chfr expression was absent in three (DLD1, HCT116, IMR5) and reduced in two others (HT29, U2OS) (1). To determine if aberrant 5' CpG island methylation might be responsible for transcriptional silencing of Chfr, we initially analyzed a series of cancer cell lines using MSP. Sequence analysis revealed that the Chfr CpG island region spans exon 1 (containing the transcription start site), intron 1 and exon 2 (containing the translation start site) of the gene (Figure 1AGo). For the initial MSP analysis, two sets of MSP primers, primer sets 1 and 2, were used to examine regions of the CpG island that were nearest the transcription and translation start sites, respectively (see Materials and methods). We found evidence of CpG island methylation in all of the cell lines shown previously to have absent or reduced Chfr expression (Figure 1BGo, upper panel). Using primer set 1, Chfr was fully methylated near the transcription start site in DLD1 (colon), HCT116 (colon), HT29 (colon), and partially methylated in U2OS (osteosarcoma) and IMR5 (neuroblastoma). Additional cancer cell lines from other tumor types were also analyzed. In total, four (80%) of five colon, two (100%) of two osteosarcoma, two (100%) of two central nervous system, and one (50%) of two leukemic cell lines showed evidence of CpG island methylation near the transcription start site. In contrast, solid tumor cell lines derived from esophageal (n = 2), breast (n = 2) and prostate (n = 1) were completely unmethylated by MSP (data not shown).



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Fig. 1. Methylation of the 5' CpG island of Chfr in cell lines. (A) Schematic of the Chfr CpG island spanning exon 1, intron 1 and exon 2. Bold vertical lines indicate individual CpG sites. TS, indicates the translation start site in exon 2. S1 and AS1 denote primer locations for MSP primer set 1, and S2 and AS2 denote primer locations for MSP primer set 2. Primer sets 1 and 2 detect methylation changes near the transcription and translation start sites, respectively. (B) MSP analyis of the Chfr CpG island. Visible product in lanes marked U indicates the presence of unmethylated alleles; visible PCR product in lanes marked M indicates the presence of methylated alleles. (Upper panel) MSP analysis of cancer cell lines using primer set 1. Chfr was fully methylated in DLD1 (colon), HCT116 (colon), HT29 (colon), and partially methylated in SAOS2 (osteosarcoma), U2OS (osteosarcoma), IMR5 (neuroblastoma) and U937 (leukemia). (Lower panel) MSP analysis of cancer cell lines using primer set 2. The same cell lines were methylated. Note that IMR5 and U937 were partially methylated near the transcription start site and completely methylated at the translation start site. Similarly for U2OS, the evidence for methylation was more pronounced near translation start. SW480 (colon) and DU145 (prostate) are unmethylated.

 
Using primer set 2, a similar pattern of Chfr methylation near the translation start site was also apparent (Figure 1BGo, lower panel). Interestingly, IMR5 and U937 were partially methylated near the transcription start site and completely methylated at the translation start site. Similarly for U2OS, methylation was more pronounced near the translation start site, although unmethylated alleles remained clearly evident. The heterogeneity of Chfr methylation in cancer cell lines as detected by MSP is consistent with regional variations in methylation within the CpG island, and has been described previously for other methylated genes, such as p73, E-cadherin and Timp-3 (tissue inhibitor of metalloproteinase-3) (2022).

In contrast to the above findings, Chfr was completely unmethylated in SW480 and predominantly unmethylated in SAOS2, two cancer cell lines that have been previously shown to express mRNA by northern analysis. Thus, the MSP data indicated a strong correlation between 5' CpG island methylation and Chfr expression, and further suggested that epigenetic silencing is a common mechanism for silencing Chfr in human tumor cell lines.

To directly test whether methylation was responsible for transcriptional silencing of Chfr, we treated cell lines with the demethylating agent 5Aza-dC. HCT116 and DLD1, two colon cancer cell lines that were fully methylated at Chfr, did not express Chfr transcript by RT–PCR. However, following treatment with 5Aza-dC, Chfr mRNA was readily detected in both cell lines (Figure 2AGo). Immunoprecipitation analysis of HCT116 cells confirmed that following 5Aza-dC treatment, Chfr transcription was associated with Chfr protein expression (Figure 2BGo). In contrast, Chfr protein was present at baseline in U2OS, a cell line that was predominantly unmethylated at the transcription start site, and expression levels were not significantly affected by 5Aza-dC treatment. The ability of 5Aza-dC to restore expression of Chfr confirmed the importance of methylation in the epigenetic silencing of this gene.



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Fig. 2. Expression analysis of Chfr. (A) Analysis of Chfr mRNA expression. The presence or absence of reverse transcriptase is indicated by + and -, respectively. Expression of glyceraldehyde-3-phosphate dehydrogenase was performed on all samples to ensure the integrity of the RNA preparations. Chfr is not expressed at baseline in HCT116 or DLD1, two cell lines that are completely methylated. After treatment with the demethylating agent 5Aza-dC (5AZA), Chfr expression is readily detected in both cell lines. (B) Analysis of Chfr protein expression. Cell lysates were immunoprecipitated with a polyclonal Chfr antibody, resolved by SDS–PAGE and immunoblotted with a monclonal Chfr antibody. Chfr protein is present at baseline in U2OS, a predominantly unmethylated cell line, and levels are unaffected by 5Aza-dC treatment. In contrast, Chfr protein is detected in the methylated cell line HCT116 only after treatment with 5Aza-dC.

 
To determine the functional consequences of restored Chfr expression by 5Aza-dC, the mitotic checkpoint was assayed in response to nocodazole, a mitotic stress agent. Normally, the Chfr checkpoint arrests cells prior to metaphase, and arrested cells do not exhibit phosphorylation of histone H3 at serine 10, a hallmark of mitosis (M.K.S. and T.D.H., unpublished observations and refs 1 and 23). When control SAOS2 cells expressing Chfr were treated with nocodazole, the histone phosphorylation profile of the G2/M cells was predominantly negative, consistent with an intact Chfr checkpoint (Figure 3Go). The histone phosphorylation profile of SAOS2 cells was not significantly affected by 5Aza-dC treatment. In contrast, when control HCT116 or DLD-1 cells that do not express Chfr were treated with nocodazole, a majority of the cells were highly positive for histone H3 phosphorylation. This evidence for progression to prometaphase in the presence of mitotic stress is consistent with a defective Chfr checkpoint. Interestingly, when HCT116 or DLD-1 cells were treated with 5Aza-dC to reactivate Chfr expression prior to nocodazole exposure, there was a marked decrease in histone H3 phosphorylated cells, indicating that fewer cells were progressing to prometaphase. The data are consistent with reactivation of Chfr expression causing reconstitution of the prophase checkpoint. However, because of the global changes in gene expression induced by treatment with 5Aza-dC, we cannot exclude the possibility that another gene(s) could also affect progression to prometaphase.



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Fig. 3. Analysis of the mitotic checkpoint. Cells were exposed to nocodazole, a mitotic stress agent, without (Control) or with (5Aza-dC) prior 5Aza-dC treatment. Cells were initially analyzed by flow cytometry for DNA content and G2/M gated cells were further analyzed for phosphorylation of histone H3. Distribution curves for the histone H3 phosphorylation analysis are shown. For each curve, the left peak indicates cells that are negative for H3 phosphorylation, and the right peak indicates cells that are positive for H3 phosphorylation. Cells that are positive for histone phosphorylation indicate progression to metaphase.

 
We next examined primary cancers for evidence of Chfr CpG island methylation near transcription start using primer set 1. By MSP, Chfr methylation was detected in 37% (11 of 30) of colon adenocarcinomas and in 10% (two of 20) of primary non-small cell lung carcinomas (Figure 4Go). In contrast, there was no evidence for Chfr methylation in one renal and two breast tumors (data not shown). Unlike with cancer cell lines, methylated primary tumors always displayed evidence of unmethylated PCR product. These unmethylated Chfr alleles most probably reflect the presence of normal cells present in the cancer specimen or heterogeneity of the methylation event within the tumor cell population itself.



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Fig. 4. Methylation of the 5' CpG island of Chfr in primary human tumors. (Upper panel) MSP analysis of primary colon cancers near transcription start. Chfr methylation is present in cases 1–4. For case 4, there is evidence for methylation in paired normal colon. (Lower panel) MSP analysis of primary non-small cell lung cancers near transcription start. Chfr methylation is evident in cases 1 and 4.

 
In the group of non-small cell lung carcinomas, methylation of the Chfr CpG island was tumor-specific, as paired corresponding normal tissue from each case was unmethylated. Among the 11 methylated colon cancer cases, paired normal tissue was available for seven. The Chfr methylation change was tumor-specific for 71% (five of seven), as the corresponding normal tissue was also unmethylated. In contrast, in the remaining two cases in which the tumor was methylated, paired normal tissue was also methylated, although the intensity of the methylated PCR product in the normal tissue was always less intense than in the tumor (Figure 4Go, upper panel, case 4). Chfr methylation of normal colon tissue without corresponding methylation of the tumor was never observed.

It has been proposed that methylated CpG islands in colon cancer can be divided into two general groups: those that become methylated in a tumor-restricted manner, and those that initially become methylated to a slight degree during normal cellular aging and subsequently become more extensively methylated in the cancer tissue (24,25). In this model, the methylation event is not by itself transforming, but does establish a pre-malignant state that is permissive for progression to cancer if additional genetic and epigenetic alterations take place. While the mean age of the patients in our study was 70 (range 44–87), the two in our study with Chfr methylation in both normal and tumor colon tissues were 83 and 86 years old, respectively, and represented the second and third oldest members of the study group. To further explore if Chfr methylation occurred in normal colon tissue, we also examined biopsies of normal colon from nine individuals with a history of diverticulitis/diverticulosis but without a history of malignancy. Chfr methylation occurred in 22% (two of nine) of these normal colon samples (data not shown). Interestingly, while the mean age of the patients in this group was 59 years (age range 45–74 years), the two patients with Chfr methylation were both 73 years old. Thus, our data suggest that Chfr CpG island methylation is associated with colon cancer tumorigenesis, and that a subset of tumors evolve from a methylated precursor that develops with normal aging.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During cell division, checkpoints have evolved to ensure that progeny cells receive the correct genetic information from one cell generation to the next (26,27). When checkpoint pathways are activated in response to adverse conditions, such as DNA damage or microtubule disruption, cell-cycle progression is delayed until the cellular injury has been repaired. In this way, checkpoints safeguard the genome. Thus, loss of checkpoint function would be expected to contribute to genetic instability, one of the hallmarks of human cancer cells (28). In support of this hypothesis, a number of genes involved in the DNA damage checkpoint are inactivated in human cancers, including p53, ATM, Chk2 and BRCA1 (29).

In contrast, while mitotic checkpoint defects are thought to be common in cancer, mutations of known mitotic spindle checkpoint genes are relatively rare, including hsMAD2, hBUB1 and hBUB3 (59). This discrepancy suggests that additional gene(s) or mechanisms may be responsible for the majority of mitotic checkpoint defects in human cancer. In the present study, we demonstrate that epigenetic inactivation of Chfr is a common event in human cancer cell lines, and in primary colon and lung cancers. During the preparation of this manuscript, another group reported methylation of Chfr in 19% (seven of 37) of primary lung cancers (30). Our results further indicate that Chfr methylation occurs in multiple different tumor types, and that epigenetic loss of the Chfr protein results in functional abrogation of the prophase checkpoint. Together, these studies suggest that the epigenetic inactivation of Chfr represents one of the more common molecular defects of a mitotic checkpoint gene to date.

Previous studies have demonstrated that methylation- associated silencing affects many of the critical molecular pathways involved in cellular immortalization and transformation, including perturbations of the cell cycle (p16, Rb), cellular adherence (E-cadherin, Timp-3), metabolic detoxification enzymes (glutathione S-transferase P1, GSTP1) and the DNA damage response pathway (hMLH1, BRCA1, MGMT, p14ARF) (31). Our data extend the biologic consequences of epigenetic inactivation to include abrogation of a mitotic checkpoint pathway. Although additional studies will be necessary to determine the overall frequency of Chfr inactivation in human cancers, our analysis of cancer cell lines and primary tumors also suggests that epigenetic inactivation of Chfr occurs more frequently in specific types of cancers (compare, for example, 42% for colon versus 10% for non-small cell lung cancers versus 0% for breast cancer). The data are consistent with previous observations that while defects in critical pathways are a common feature of many different human cancers, the pattern of genes involved and mechanisms responsible (genetic versus epigenetic) vary considerably between tumor types (31). For example, the pRb pathway is inactivated by p16 methylation more frequently in non-small cell than small cell lung cancers, while Rb mutations account for the majority of pRb defects in small cell tumors (32,33). Thus, in breast tumors, which are commonly aneuploid, we would postulate that mitotic checkpoint defects are probably a result of mechanisms other than aberrant methylation of Chfr.

Interestingly, we found evidence of age-related methylation of Chfr in normal colon tissue that was not seen in normal lung tissue. As an epigenetic phenomenon, age-related methylation has been described most commonly in colon epithelia (24,25). This may reflect unique molecular pathways involved in colon carcinogenesis that are not involved in cancers derived from other tissue types. Notably, sporadic colon cancer is a disease associated with aging, as the age-specific incidence rises steadily from the second to ninth decade of life, and 90% of cancers occur in people 50 years or older. In contrast, the development of lung cancer is directly associated with exposure to carcinogens in tobacco smoke (34). In lung carcinogenesis, the selection pressure for methylation changes may not be dependent on the aging process per se, but rather on the cumulative exposure to tobacco smoke (35). Consistent with this idea, aberrant promoter methylation of p16 has been detected in non-malignant bronchial epithelium of current and former smokers without cancer that was not present in individuals who never smoked (36). The inclusion of Chfr in future methylation studies should help elucidate when Chfr methylation occurs during lung cancer carcinogenesis. In addition, determining the methylation status of Chfr in lung cancers may help predict response rates to antimitotic chemotherapies such as the taxanes, which are commonly used in the treatment of non-small cell lung carcinoma (37). Since defects in the mitotic checkpoint may contribute to the sensitivity to these agents, tumors with methylated Chfr would be predicted to respond more favorably than unmethylated tumors.


    Notes
 
* These authors contributed equally to this work. Back

6 To whom correspondence should be addressed Email: wafik{at}mail.med.upenn.edu Back


    Acknowledgments
 
W.S.E-D. is an Assistant Investigator of the Howard Hughes Medical Institute. Supported in part by The Howard Hughes Medical Institute and NIH grants CA76391-05 (P.G.C.), CA089630 (T.D.H) and CA09677 (M.K.S.).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received July 17, 2002; revised September 30, 2002; accepted September 30, 2002.