CHFR promoter hypermethylation in colon cancer correlates with the microsatellite instability phenotype
Received January 17, 2005; revised and accepted February 16, 2005
Johann C. Brandes,
Manon van Engeland 1,
Kim A.D. Wouters 1,
Matty P. Weijenberg 2 and
James G. Herman *
Cancer Biology Program, Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231, USA, 1 Department of Pathology and 2 Department of Epidemiology, University Maastricht, 6200 MD Maastricht, The Netherlands
* To whom correspondence should be addressed: The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, 1650 Orleans Street, Baltimore, MD 21231, USA. Tel: +1 410 955 8774; Fax: +1 410 614 9884; Email: hermaji{at}jhmi.edu
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Abstract
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A subset of sporadic colon cancers has been shown to have microsatellite instability caused by an epigenetic inactivation of the MLH1 gene by hypermethylation of the the CpG island in its promoter region. We report here that in colorectal cancer, inactivation of the MLH1 gene is frequently accompanied by hypermethylation of the CpG island in the promoter of the mitotic gene checkpoint with forkhead and ring finger domains (CHFR). This was first observed in the colon cancer cell lines HCT-116, DLD-1, RKO and HT29. Among the 61 primary colon cancer samples studied, hypermethylation of the MLH1 and the CHFR promoter was found in 31% of the tumors. In 68% of all primary cancers (13/19) with MLH1 promoter hypermethylation, hypermethylation of the CHFR promoter was observed as well (P-value < 0.0001, Fisher's two-sided exact). Hypermethylation of the HLTF, MGMT, RASSF1, APC, p14 and p16 promoter regions were also frequent events, being observed in 48% (28/58), 40% (26/64), 21% (14/64), 50% (31/62), 43% (26/60) and 56% (35/63), respectively. However, methylation of these genes was not associated with methylation of either MLH1 or CHFR. The observed methylation profile was unrelated to Duke's stage. The coordinated loss of both mismatch repair caused by methylation of MLH1 and loss of checkpoint control associated with methylation of CHFR suggests the potential to overcome cell cycle checkpoints, which may lead to an accumulation of mutations.
Abbreviations: CIN, chromosomal instability; MIN, microsatellite instability; MNNG, N-methyl-N'-nitro-N-nitroguanidine; MSP, methylation-specific PCR.
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Introduction
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Genetic instability is one of the hallmarks of human cancer. In colon cancer, tumors with chromosomal instability (CIN) can be distinguished from those with microsatellite instability (MIN) (1). While the former frequently show aneuploidy, the karyotype in the latter is usually preserved. These tumors as a result of defects in DNA mismatch repair, show instability in AT rich tandem repeats, so called microsatellites, which are interspersed into the genome. Mismatch repair genes are an evolutionary highly conserved family. The bacterial gene MutS detects the DNA damage, whereas the MutL gene creates nicks in the DNA, marking it for repair. In mammals, MutS homologues are MSH2, MSH 3 and MSH6 and MutL homologues are MLH1, PMS2 and MLH3 [reviewed in (2)]. MIN can either be a result of inherited mutations as in the HNPCC syndromes, mainly affecting the genes MLH1 and MSH2. It can, however, also be a result of spontaneous mutations or, more frequently, epigenetic silencing of these genes. Approximately 15% of spontaneous colon cancers show MIN. It has been reported that epigenetic silencing of the MLH1 gene by hypermethylation of CpG islands in its promoter region is frequently observed in sporadic colon cancers (3). This is in contrast to the absent hypermethylation of the MSH2 promoter in most cancers.
In addition to the correction of base-pair mismatches and the correction of deletion/insertion loops, new data suggest a role of MMR genes in the activation of cell cycle checkpoints and the induction of apoptosis. MMR deficient cells, for example, are unable to undergo G2/M arrest, when challenged with methylating agents such as N-methyl-N'-nitro-N-nitroguanidine (MNNG) (2). The purpose of this study was to examine a correlation of MLH1 deficiency with other G2/M checkpoint regulators. CHFR is a G2/M checkpoint gene that has recently been described by Scolnik and Halazonetis (4). It has received special attention because of its frequent inactivation in multiple different human cancers. This gene possesses a forkhead and a RING finger domain and functions as an ubiquitin ligase (5). CHFR delays entry into the mitotic M-phase, when cells are challenged with an inhibitor of microtubular assembly (4). It ubiquinates and thus inhibits the Plk1 kinase, which in turn is an important activator of the cdc2 kinase, the key regulator of G2/M phase entry (6).
CHFR expression has been reported to be silenced by CpG island hypermethylation in different tumor cell lines and primary tumors. The degree of CHFR promoter hypermethylation varies in different human cancers from 20% in NSCLC (7), to 30% in esophageal cancers (8) to
40% in colorectal cancers (9). CHFR promoter hypermethylation in colon cancer is dependent on the function of two DNA methyltransferases, DNMT1 and DNMT3b, since cells deficient in these enzymes, re-express CHFR (9). Treatment with the methyltransferase inhibitor 5 aza-cytidine also restores the CHFR expression (9). Somatic mutations have been described as an additional mechanism for the loss of CHFR function in three cases of lung cancer (10). However, the predominant mechanism for loss of function for CHFR in cancer appears to be epigenetic silencing associated with the promoter region hypermethylation.
While much progress has been made in determining how the CHFR gene expression is regulated and in identifying at least one downstream target of its ubiquitin ligase activity, the biologic significance of these findings is largely unknown. It appears likely that a disruption of a mitotic checkpoint would result in the disruption of an orderly chromosomal segregation and hence in CIN. However, a recent study failed to demonstrate a statistically significant correlation between CIN and the loss of CHFR function (11). On the other hand, a large number of colon cancer cell lines with the MSI+ phenotype showed a loss of CHFR expression in that study (11), perhaps suggesting a different association between loss of CHFR function and genomic stability. Based on these findings, we hypothesized that deficient MMR resulting in defective DNA repair would normally lead to a G2/M arrest. Disruption of the G2/M checkpoint by CHFR silencing would then allow such cells to proliferate even if unable to repair mismatches in DNA, creating a potentially mutation producing state. Here, we show a strong, statistically significant correlation between hypermethylation of CHFR and the MLH1 promoter regions in support of this hypothesis.
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Materials and methods
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Tumor samples and DNA extraction
After identification by a pathologist, genomic DNA from paraffin-embedded formalin-fixed tissues from tumors was microdissected and extracted using proteinase K (Qiagen) and the Puregene® DNA Isolation Kit (Gentra Systems) and stored at 4°C. This set of samples has been previously characterized for promoter methylation of RASSF1 (12,13). The clinical characteristics of this cohort are as described (12). Cell lines used are the colorectal cancer cell lines RKO, HCT-116, DLD-1, LoVo, SW480, HT29 and CACO2 (as previously described) (3).
Bisulfite modification
DNA bisulfite modification procedure was performed according to previously described methods (14). Up to 1 µg of genomic DNA was diluted to a total volume of 50 µl with water, and sodium bisulfite treatment was carried out for 16 h at 50°C. Bisulfite-treated DNA samples were then purified with a Wizard DNA Clean-Up System (Promega, Madison, WI), and desulfonated before ethanol precipitation.
Nested methylation-specific PCR (MSP)
The methylation status of the promoter regions for MLH1, CHFR, HLTF, APC, p16 and p14 was determined by the method of MSP further modified as a nested two-step approach in order to increase the sensitivity of detecting allelic hypermethylation at targeted sequences and to facilitate the examination of multiple gene loci. This approach has been used to examine multi-gene methylation correlates with non-nested approaches (13) and the presence of methylation correlates with loss of gene expression (15). Fifty to one hundred nanograms of bisulfite-treated DNA was used for step one of the nested MSP, which was carried out with primer sets (sense and anti-sense) for these genes with an annealing temperature of 56°C. Step one primers flanked the CpG-rich promoter regions of the respective targeted genes. PCR products of step one were diluted to 1:500 and subjected to the second step of MSP that incorporated one set of primers for each gene. Step two primers were designed to recognize bisulfite-induced modifications of unmethylated cytosines.
The primer sequences and PCR conditions for this nested-MSP approach have been published previously for MLH1 (16) and for p14, p16, MGMT-1, APC (13). This set of samples has been previously characterized for promoter methylation of RASSF1 (12,13). For CHFR, the primers are: external upstream primer, 5'-TTTTYGTTTTTTTTGTTTTAATATAATATGG-3', external downstream primer, 5'-CRCRCACCAAAAACRACAACRAAAAC-3', product size 167 bp, internal methylated upper primer, 5'-GTTATTTTCGTGATTCGTAGGCGAC-3', internal methylated downstream, 5'-CGAAACCGAAAATAACCCGCG-3', product size 100 bp, and internal unmethylated upper primer, 5'-GATTGTAGTTATTTTTGTGATTTGTAGGTGAT-3', internal methylated downstream, 5'-AACTAAAACAAAACCAAAAATAACCCACA-3', product size 115 bp. For HLTF, the primers are: external upstream primer, 5'-ATYGTAGGTATYGTAGTYGTATTTTTGGG-3', external downstream primer, 5'-CCACAAAACACAAAAAAAAAAACAACTCC-3', product size 155 bp, internal methylated upper primer, 5'-GTGGTTTTTTCGCGCGTTC-3', internal methylated downstream, 5'-CGCTACCATTCAAAAACGACG-3', product size 94 bp, and internal unmethylated upper primer, 5'-GGTTTTGTGGTTTTTTTGTGTGTTT-3', internal methylated downstream, 5'-CCCCACTACCATTCAAAAACAACA-3', product size 103 bp. Each step of the nested MSP utilized a 25 µl reaction volume, 0.5 µl of Jump Start Red Taq DNA polymerase (Sigma, St Louis, MO), and 1 µl of DNA template and included 30 cycles at the following annealing temperatures: MLH1, 60°C, CHFR, 59°C, HLTF, 59°C.
DNA isolated from normal peripheral lymphocytes from healthy individuals served as a negative methylation control. Human placental DNA was treated in vitro with SssI methyltransferase (NEB, Beverly, MA) to create a completely methylated DNA at all CpG-rich regions. In vitro methylated DNA (IVD) served as the positive methylation control. MSP products were analyzed on 6% polyacrylamide gel electrophoresis, and were determined to have methylation if a visible band was observed in the methylation reaction.
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Results
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CHFR promoter hypermethylation in colon cancer cell lines
In order to test our assay for the methylation status of CHFR, we studied the methylation of the CpG island in the CHFR promoter in several colon cancer cell lines that were used in previous reports. We found CHFR promoter hypermethylation in all colon cancer lines that display the MSI+ phenotype (HCT-116, DLD-1, RKO, LoVo). Three colon cancer cell lines with CIN phenotype were also studied. The lines HT29 and CACO2 had methylation of the CHFR promoter, whereas the line SW480 did not (Figure 1). These findings are consistent with the results of previous studies (11). CHFR transcription has been shown previously to be silenced in HCT 116 (4, 11), LoVo (11) and DLD-1 (4). CHFR expression is suppressed but not completely silenced in HT29 (4,11) and it is fully expressed in CACO2 and SW480 (11).

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Fig. 1. MSP of CHFR in colon carcinoma cell lines. (u) lanes represent amplification of unmethylated alleles, and (m) lanes methylated alleles. In vitro methylated DNA (IVD) and normal human peripheral lymphocytes (NL) serve as the positive and negative methylation controls, respectively, and a water control was included as well (not shown). Normal lymphocytes are unmethylated in this 5' CpG island of the CHFR gene, as is SW480. CACO2 and HT29 are partially methylated, while HCT-116, DLD-1, RKO and LoVo all are completely methylated.
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CHFR, MLH1, HLTF, MGMT, RASSF1, p16, p14 and APC promoter hypermethylation in primary colon cancer
Next, we studied the methylation status of the CHFR and the MLH1 promoter through MSP, since MSI in primary colorectal cancer is most commonly due to promoter region methylation (3). We detected the hypermethylation of the MLH1 promoter in 19/61 (31%) of the primary colorectal cancer samples studied (Figure 2). In two cases it was not possible to amplify the bisulfite-treated DNA with MLH1-specific primers, most probably due to poor DNA quality. The CHFR promoter was hypermethylated in 19 of the 62 tumors examined (31%). We observed a strong correlation between methylation of CHFR and MLH1. Thirteen tumors had hypermethylation of both promoters, while 35 tumors were not methylated at either promoter region. Six cases had hypermethylation of the MLH1 promoter observed that was not accompanied by hypermethylation of the CHFR promoter, and likewise, CHFR was only methylated in six tumors without methylation of MLH1. The correlation between methylation of the MLH1 and CHFR promoters was highly statistically significant (P < 0.0001, Fisher's exact test, two-sided).

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Fig. 2. Methylation of primary colorectal cancer. Representative examples of methylation-specific PCR for CHFR, MLH1 and HLTF. (u) lanes represent amplification of unmethylated alleles, and (m) lanes methylated alleles. In vitro methylated DNA (IVD) and normal human peripheral lymphocytes (NL) serve as the positive and negative methylation controls, respectively, and a water control was included as well (not shown). Methylation is seen for CHFR in tumors 32, 34 and 39; for MLH1 in tumors 34 and 39; and for HLTF in tumors 31, 32, 33, 36 and 37.
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The correlation of methylation of CHFR with MSI in cell lines suggested a biologic explanation for the association between MLH1 inactivation and CHFR methylation. However, it is possible that this coincident methylation of two genes was merely the result of a CIMP (CpG island methylator) phenotype, as proposed by Toyota et al. (17). To examine this possibility, we studied the methylation of additional genes that have been shown to be silenced by promoter hypermethylation in colorectal cancers. CDKN2A (p16) is a member of the original panel of CIMP genes and its methylation had been linked to methylation of MLH1, but not to MIN caused by genetic mutations in the MLH1 gene (Figure 3). HLTF is a member of the SWI/SNF family of chromatin remodeling proteins, and reported to be hypermethylated in 43% of primary colon cancers and to be associated with the CIMP phenotype (18). MGMT is involved in the repair of O-methyl-guanine residues, and was of additional interest here since it is another DNA repair enzyme, but one which targets a different type of DNA damage than MLH1. It is also frequently inactivated by promoter hypermethylation, occurring in
40% of all cases (19). Loss of MGMT function had been associated with a higher incidence of p53 mutations (20). RASSF1 is a putative tumor suppressor gene and has been shown to be silenced by promoter hypermethylation in
20% of spontaneous colorectal carcinomas (12). APC methylation is a frequent event in sporadic colorectal cancers, as is methylation of p14 but have not been extensively examined in connection with CIMP.

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Fig. 3. Summary of results of methylation analysis for 62 primary colorectal carcinomas. A statistically significant correlation was observed between MLH1 and CHFR promoter hypermethylation (P < 0.0001, Fisher's exact test, two-sided). There was no statistically significant correlation between CHFR or MLH1 promoter hypermethylation with any of the other genes or the clinical stage.
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In examining these genes, we found no statistically significant correlation between promoter hypermethylation of any of our control genes with hypermethylation of either the MLH1 or the CHFR promoter. The HLTF promoter was methylated in 28 of 58 tumors (48%). When compared with MLH1 promoter hypermethylation, there was no statistical significance (P-value: 0.7772, Fisher's two-sided exact). There was a trend towards correlation to CHFR promoter hypermethylation but this did not reach a statistical significance (P-value: 0.094, Fisher's two-sided exact). The p16 promoter was methylated in 35 of 63 tumors (55%). Hypermethylation of the MGMT promoter was observed in 26 of 64 tumors (40%), but was not associated with hypermethylation of either CHFR or MLH1. RASSF1 was methylated in 14 of 64 tumors (21%), p14 was methylated in 26 of 60 tumors (43%) and APC was methylated in 31/62 tumors (50%). Methylation of neither of these genes was associated with methylation of either CHFR or MLH1 (P-value: non-significant). Thus, the only association of methylation between genes examined was between CHFR and MLH1, which was highly associated (P < 0.0001).
Correlation of CHFR and MLH1 promoter hypermethylation with the clinical stage
Previous studies have suggested that both the hypermethylation of the CHFR and the MLH1 promoter are early events in carcinogenesis. In fact, both events have been observed in pre-malignant lesions at high frequency (9,21). In order to rule out the possibility that the observed correlation between CHFR and MLH1 promoter hypermethylation is related to late changes in carcinogenesis and thus merely being an expression of progressive epigenetic dysregulation, rather than being a driving force in carcinogenesis, we examined the correlation between the clinical stage and the hypermethylation of the CHFR and the MLH1 promoter. Our findings indicate that there is no statistically significant correlation between the clinical stage and the observed phenotype. We found hypermethylation of both the CHFR and the MLH1 promoter in five cancers in Dukes stage A, two cancers in stage B, three cancers in stage C and another three cancers in stage D. Consistent with previous studies, this is further evidence that CHFR and MLH1 promoter hypermethylation is an early event in carcinogenesis.
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Discussion
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Our findings reveal a strong relationship between the hypermethylation of CpG islands in the promoter region of the CHFR and the MLH1 gene. One possible explanation for this would be the presence of a CIMP phenotype (17) as suggested by Bertholon et al. (11). The fact that we did not find a significant correlation with hypermethylation of the promoters of other genes such as MGMT, HLTF, RASSF1a, p14, APC and in particular p16, a gene important in the original description of CIMP, argues against this hypothesis as the sole explanation for the coincident methylation of CHFR and MLH1.
Additional support for the observed correlation between MLH1 and CHFR promoter hypermethylation is provided by the analysis of colon carcinoma cell lines. CHFR methylation was observed in cell lines with MIN (MSI+) phenotype, which was conferred either by mutation or deletion of the MLH1 gene (HCT-116, DLD-1) and MSH2 (LoVo) or by methylation of the MLH1 promoter (RKO). While frequently observed in sporadic colon cancers in which the MSI+ phenotype is conferred by epigenetic silencing of the MLH1 promoter, the CIMP phenotype appears to be unrelated to colon cancers that arise on the basis of HNPCC that is due to germ line mutations in mismatch repair genes. Despite this different mechanism for defects in mismatch repair, we still find frequent methylation of the CHFR promoter in those cell lines. However, CHFR methylation was observed in two cell lines without MIN (HT29 and CACO2), and a correlation of CHFR methylation with either CIN or MIN was not made in colon cancer cell lines in a previous study (11).
We have also compared our previous data for methylation of other genes in colon cancer cell lines and any relationship to MSI. The methylation status for the promoters of p14, p16, RASSF1 and MGMT has been reported before for the cell lines used in this study (22). All cell lines are methylated for the p16 promoter. p14 methylation was only found in MSI+ phenotype cell lines. This association has been described previously (23) and has been explained by the relative low incidence of p53 mutations in MSI+ tumors. Of the MSI+ cell lines in our study, only DLD1 harbors a p53 mutation, whereas all MSI cell lines in this study have a p53 mutation. With the exception of partial methylation in the HCT-116 line, no methylation was observed for the APC gene in any of the cell lines studied (24). When compared with the CHFR methylation in our analysis, the methylation status of none of the candidate genes reached a statistical significance. HLTF promoter methylation is absent in RKO and HT29 (18). Since both lines have methylation of the CHFR promoter, this also argues against a statistically significant correlation between CHFR and HLTF methylation in the cell lines.
The particular correlation of CHFR and MLH1 suggests that the loss of CHFR expression may allow cells, which are deficient or have decreased levels of MLH1 expression to progress through the G2/M cell cycle check point without delay. This would, thus, provide a survival advantage for these cells and lead to increased mutations of target genes, since these alterations would not be repaired before replication. However, not all primary colon cancers with MLH1 promoter hypermethylation were also hypermethylated at the CHFR promoter. This could possibly be explained either by additional, not yet determined, disruptions in the G2/M checkpoint in those cells, or by the fact, that while loss of CHFR might provide a survival advantage for MMR deficient cells, this might not be an absolute requirement for cell survival. The mechanism by which this is achieved remains to be elucidated. The important functions of both CHFR and MLH1 suggest some possible reasons for this association.
In addition to the repair of DNA mismatches, MMR genes are also important in the regulation of the G2/M checkpoint in response to DNA damage. They are involved in the detection of DNA damage caused by alkylation, methylation and by treatment with cisplatin. The best studied system is the response to MNNG, which converts guanine into O-methyl-guanine (25). Normally, this results in G2/M arrest. However, cells that lack MLH1 or MSH2 or have decreased levels of expression are resistant to MNNG or cisplatin and fail to undergo G2/M arrest or apoptosis (26). MMR genes have been associated with many DNA damage signaling genes such as the BASC complex (27) and MSH2 has been co-precipitated with the ataxia teleangiectasia mutated (ATM) gene (28). However, the exact role that the individual genes play in these processes remains unknown.
CHFR has been identified as a G2/M checkpoint, which delays entry into mitosis when cells are challenged with a microtubular inhibitor such as noconazole (4). CHFR acts as an ubiquitin ligase and has been shown in a Xenopus extract to facilitate ubiquitination and degradation of Plk-1 (6), thus inhibiting entry into mitosis. While certainly intriguing, it has not been proven yet that this function is the same in a mammalian cell. It is believed that CHFR interacts through its RING finger domain with phosphoproteins, but the exact mechanism by which CHFR activity is regulated has not been determined yet. CHFR has not been implicated so far as being involved in the control of the G2/M checkpoint as a result of DNA damage. However, given its possible function as regulator of Plk-1, CHFR is a very attractive gene with a potential gatekeeper function for entry into mitosis in general and could very well serve as a downstream target for alternative cellular signaling processes. Further work in this respect is needed.
In order to interpret the findings of concurrent hypermethylation of the MLH1 and the CHFR promoter, one will have to consider the timing at which these processes occur. If those abnormalities present a driving force in carcinogenesis rather than just being an epiphenomenon of dysregulated epigenetic control in late carcinogenesis, they should occur early. Since colonic adenomas are premalignant lesions, this question could be directly addressed. In precancerous lesions, hypermethylation of CpG islands in both the MLH1 promoter (21) and the CHFR promoter (9) is frequently observed, at frequencies that are comparable to those that we observed in colon cancers. Since it is not expected that this hypermethylation is reversed during subsequent carcinogenesis, it is likely that the correlation, which we observed between CHFR and MLH1 promoter hypermethylation, is also present at this earlier stage. Here, we show that the hypermethylation of the CHFR and the MLH1 promoter are equally common in the different clinical stages of colon cancer, which further supports this hypothesis.
In summary, we show a correlation between hypermethylation of CpG islands in the promoters of both the CHFR and the MLH1 gene. Most primary colon cancers with MLH1 promoter hypermethylation and all cell lines with the MSI phenotype studied (HCT-116, DLD-1, RKO, LoVo) also have hypermethylation of the CHFR promoter, suggesting a survival advantage that is conferred to these cells by loss or downregulation of CHFR.
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Acknowledgments
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Conflict of Interest Statement: J.G.H. is a consultant for OncoMethylome Sciences. None of the other authors have any conflicts of interest.
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