In order to identify novel candidates associated with prostate cancer metastasis, we compared the proteomic profile of the poorly metastatic human prostate cancer cell line LNCaP, with its highly metastatic variant LNCaP-LN3, by two-dimensional gel electrophoresis. A major protein spot (pI of 5.9 and molecular weight of 37 kDa) was seen in LNCaP cells, but not in LNCaP-LN3 cells and was identified as lactate dehydrogenase-B (LDHB), by tandem mass spectrometry. Furthermore, enzyme kinetic assays and zymography showed a higher LDH enzyme activity in LNCaP cells compared with LNCaP-LN3. Bisulphite-modified DNA sequencing showed promoter hypermethylation in LNCaP-LN3 cells but not in LNCaP, Du145, PC3, CWR22 or BPH45 cells. Treatment of LNCaP-LN3 cells with 5′-azacytidine caused re-expression of LDHB transcripts. In tissues, LDHB promoter hypermethylation occurred at a higher frequency in prostate cancer, 14/ 31 (45%), compared to adjacent nonmalignant or benign tissue, 2/19 (11%) (P<0.025). Immunohistochemistry showed a higher frequency of LDHB expression in benign or non-malignant tissues, 59/ 73 (81%), compared to cancer cases, 3/53 (6%) (P<0.001). Absent LDHB expression was also seen in 7/7 (100%) cases of metastatic cancer in bone. Our data are the first to show loss of LDHB expression in prostate cancer, the mechanism of which appears to involve promoter hypermethylation.
Prostate cancer is among the most commonly diagnosed cancers in men and is the second most common cause of cancer-related deaths (Greenlee et al., 2000; Gronberg, 2003). While certain tumours are slow growing and usually do not impact on patient survival, other tumours unpredictably develop into incurable metastatic disease (Chodak et al., 1994; Hamdy, 2001; Bhandari et al., 2005).
The molecular mechanisms underlying prostate cancer metastasis are poorly understood, partly because of the lack of reliable in vivo models. In an effort to develop better models for the study of prostate cancer metastasis, Pettaway et al. (1996), established metastatic variants by successive orthotopic implantation of the LNCaP cell line into the prostates of nude mice (Horoszewicz et al., 1980). One of the variants, LNCaP-LN3, had significantly higher incidences of lymph node and liver metastasis.
Recent advances in ‘proteomics’, the large-scale characterization of the protein complement of cells, tissues, fluids, etc., offers a promising approach for the identification of novel proteins associated with disease development (Banks et al., 2000; Graves and Haystead, 2002). Although previous proteomic studies on prostate cancer have identified a number of alterations associated with the development of cancer (Ahram et al., 2002; Meehan et al., 2002; Schulz et al., 2003; Cheung et al., 2004), studies aimed specifically at identifying protein alterations associated with the metastatic progression are limited. A recent study has compared the protein profile of a highly metastatic human prostate cancer cell line M12 with its poorly tumorigenic variant M12(F6), and identified a reduced level of vimentin in the poorly tumorigenic variant (Liu et al., 2003).
The aim of the present study was to identify novel protein targets associated with the metastatic progression of human prostate cancer using comparative proteomics on isogenetic prostate cancer cell lines with a low (LNCaP) and high (LNCaP-LN3) metastatic potential. Our findings and their significance are discussed.
LDHB protein and mRNA is absent in LNCaP-LN3 but expressed in LNCaP cells
Image analysis comparisons of the 2D-polyacrylamide gel electrophoresis (PAGE) gels displaying the protein profiles of the LNCaP and LNCaP-LN3 cells showed remarkable similarities with regards to ‘on/off’ differences. However, a major protein seen as a spot with a pI of 5.9 and a molecular mass of ∼37 kDa was reproducibly found to be absent in LNCaP-LN3 cells relative to LNCaP cells (Figure 1). MALDI-TOF-MS and ESI-MS/MS analyses of the protein spot identified it as being LDHB (gene identifier 4557032) (Figure 2). ESI-MS/MS data gave a probability score of 701, with 49% sequence coverage and 17 peptides matched. The expression of LDHB protein by LNCaP cells but not in LNCaP-LN3 cells was also validated by 2D Western blotting (Figure 3). Furthermore, direct sequencing of the RT–PCR product confirmed the presence of LDHB transcripts in the LNCaP cell line, but barely detectable levels in LNCaP-LN3 cells (Figure 4).
LDHB promoter is hypermethylated in LNCaP-LN3 but not LNCaP cells
Hypermethylation of CpG sites within the promoter region of tumour suppressor genes is a widely accepted mechanism associated with gene silencing (Das and Singal, 2004; Li et al., 2005). In order to understand the molecular mechanisms underlying the loss of LDHB expression in the LNCaP-LN3 cell line, we sequenced the LDHB promoter region using previously published primers (Maekawa et al., 2003), or using primer pair LDH-B-Fwd2/ LDH-B-Rev2 (Figure 5). This region selected for amplification has previously been related to promoter activity (Takeno and Li, 1989; Maekawa et al., 2003). Sequencing of the LDHB promoter region showed an absence of promoter hypermethylation in the LDHB-expressing LNCaP cells, whereas promoter hypermethylation was present in the nonexpressing LNCaP-LN3 cells (Figure 6a). LDHB promoter hypermethylation was also absent in Du145, PC3 and CWR22 prostate cancer cell lines and in the BPH45 cell line derived from a patient with benign prostatic hyperplasia (BPH) (Klingler et al., 1999).
Restoration of LDHB mRNA expression by 5′-azacytidine treatment
We next tested the ability of the DNA methyltransferase inhibitor, 5′-azacytidine (Das and Singal, 2004) to restore LDHB mRNA expression in the LNCaP-LN3 cell line. LNCaP-LN3 cells were treated with the demethylating agent 5′-azacytidine for 5 days and the expression of LDHB mRNA was determined by RT–PCR analysis. Following RT–PCR, re-expression of LDHB mRNA transcripts was seen in the treated LNCaP-LN3 cells (Figure 4).
LNCaP-LN3 cells have a lower overall LDH enzyme activity
To determine whether the loss of LDHB subunit seen in the LNCaP-LN3 cells affected overall LDH enzyme activity, kinetic enzyme assays were performed using the cytosolic protein fractions from the LNCaP and LNCaP-LN3 cells. These data showed that the initial rate of reaction for LNCaP cells was 23.8 absorbance units/g of protein, and for LNCaP-LN3 cells was 13.3 absorbance units/g of protein (data not shown). Thus, in LNCaP cells, the initial rate of reaction was 1.78 (23.8/13.3) times higher than that of LNCaP-LN3.
LDH isoenzyme patterns in prostate cancer cell lines
In order to determine the isoenzyme patterns in the LNCaP and LNCaP-LN3 cell lines, we performed LDH zymography as described in the Materials and methods. As expected, LNCaP-LN3 cells predominantly showed expression of the LDH-5 isoenzyme (LDH-A4), with a weaker expression of the LDH-4 (LDH-A3B1) isoenzyme, but no detectable levels of LDH isoenzymes LDH-1 (LDH-B4), LDH-2 (LDH-A1B3) or LDH-3 (LDH-A2B2), (Figure 7). In contrast, LNCaP cells showed expression of LDH isoenzymes -2, -3, -4 and -5. The finding of the LDH-4 isoenzyme in LNCaP-LN3 cells was most likely due to the expression of very low levels of LDH-B by a subpopultion of LNCaP-LN3 cells.
LDHB promoter hypermethylation is frequent in human prostate cancer
Sequencing of bisulphite-modified DNA extracted from tissues showed promoter hypermethylation in 14/31 (45%) cases of prostatic adenocarcinoma (Figure 6b) and 1/2 cases of metastatic cancer in bone. In contrast, methylation was seen in only 2/14 (14%) cases of adjacent nonmalignant epithelium, but not in any of the five cases of BPH tissues analysed (Figure 6c). The difference in the frequency of methylation between cancer and nonmalignant or BPH tissues was statistically significant (P<0.025, χ2).
Immunohistochemical expression of LDHB protein in human prostate
To assess the expression of LDHB protein in tissues, we performed immunohistochemistry on BPH, adjacent nonmalignant, premalignant, malignant and metastatic tissues arranged onto tissue microarrays. In nonmalignant or BPH tissue, LDHB staining was localized predominantly in the cytoplasm of basal cells with weaker staining of the luminal cells (Figure 8). Expression was seen in 59/73 (81%) cases of BPH and nonmalignant tissues, whereas in cancer tissues 50/53 (94%) cases showed absent expression, while the remainder 3/53 (6%) cases of cancer showed strong expression (Figure 8). The expression of LDHB in nonmalignant or BPH tissue, but absent expression in cancer tissue was statistically significant (P<0.001). Absent expression was seen in 7/7 (100%) cases of metastatic cancer in bone and 3/5 (60%) cases of high-grade prostatic intraepithelial neoplasia lesions (HG-PIN).
In an effort to identify novel proteins associated with prostate cancer metastasis, we performed comparative 2D-PAGE analysis on a poorly metastatic cell line (LNCaP) and its highly metastatic variant (LNCaP-LN3). Remarkably, the two cell lines showed virtually identical protein expression profiles with respect to ‘on/off’ changes. Possible reasons for this were that our protein extraction protocol selectively enriched for the cytosolic protein fraction. Furthermore, only proteins with a isoelectric point (pI), within the range 4–7 were resolved in the first dimension, thus proteins with a pI outside this range were not represented on the 2D gels. Nevertheless, a single protein spot with a pI of 5.9 and a molecular mass of ∼37 kDa was seen to be present in the poorly metastatic LNCaP cells, but was absent in the highly metastatic LNCaP-LN3 cells. Subsequent peptide mass fingerprint analyses identified this to be LDH subunit B, and its identity was further confirmed by 2D Western blotting and sequencing of the RT–PCR product. Thus, our data suggest that the LDHB subunit represents a candidate protein whose loss of expression may be associated with the metastatic progression of human prostate cancer.
Support for the involvement of LDHB in metastasis comes from a recent proteomic study showing downregulation of the LDHB subunit in a highly metastatic hepatocellular carcinoma cell strain (MHCC97-H) compared to a strain with a lower metastatic potential (MHCC97-L) (Ding et al., 2004). In addition to the involvement of LDHB in tumour metastasis, other studies have shown that it may be involved in the development of prostate cancer. For instance, measurements of LDH isoenzymes in prostatic fluid from patients with and without prostate cancer have shown a higher LDH-5/LDH-1 ratio in the prostatic fluid from patients with prostatic malignancy compared to patients with BPH (Grayhack et al., 1980). Taken together, our data suggest that loss of the LDHB subunit may play a role both in the development and metastatic progression of human prostate cancer.
LDH isoenzymes catalyse the interconversion of lactate and pyruvate in the glycolytic pathway and are key mediators of glycolysis (Holbrook et al., 1975). In mammals, two genes code for two LDH subunits; LDHA and LDHB. These subunits associate and give rise to five tetrameric forms of LDH isoenzymes: LDH-A4 (LDH-5), LDH-A3B1 (LDH-4), LDH-A2B2 (LDH-3), LDH-A1B3 (LDH-2) and LDHB4 (LDH-1) (Markert et al., 1975). Our finding of absent LDHB expression in a high frequency of prostate cancers and in the LNCaP-LN3 cell line could explain a number of key observations relating to the altered cellular metabolism and tumour behaviour seen in prostate and other cancers. For instance, previous studies have shown that malignant transformation is associated with an increase in glycolytic flux and increased cellular lactate excretion (Walenta and Mueller-Klieser, 2004; Walenta et al., 2004). Thus, loss of LDHB in prostate cancer cells is expected to eliminate LDH isoenzymes 1–4, leaving only the LDH-5 (LDH-A4) isoenzyme, the isoenzyme with the highest efficiency to catalyse the conversion of pyruvate to lactate, particularly under hypoxic conditions (Shim et al., 1997; Maekawa et al., 2003; Walenta and Mueller-Klieser, 2004; Walenta et al., 2004; Koukourakis et al., 2005). Other studies have shown that high tumour lactate levels can enhance the in vitro migration and invasion of cancer cells (Martinez-Zaguilan et al., 1996; Shi et al., 2001; Stern et al., 2002). Furthermore, the production of lactate in solid malignant tumours has been correlated with a high incidence of distant metastasis (Brizel et al., 2001). Although using the in vitro kinetic enzyme assay and zymography we showed a lower overall LDH enzyme activity in the LNCaP-LN3 variant compared to LNCaP cells, these results may not represent the in vivo scenario, where tumour cells frequently encounter hypoxic conditions (Vaupel and Harrison, 2004). Furthermore, the role of other factors such as HIF-1, a well-characterized hypoxia inducible gene that is known to upregulate LDHA levels under hypoxic conditions, cannot be appreciated in vitro (Firth et al., 1995; Semenza et al., 1996). Further studies, utilizing in vivo models, are required to elucidate the exact mechanism by which loss of LDHB contributes to tumour progression.
Numerous studies have shown that hypermethylation of normally unmethylated CpG dinucleotides located in a gene promoter is associated with gene silencing at the transcriptional level (Das and Singal, 2004; Li et al., 2005). A previous study has reported LDHB promoter hypermethylation in 3/20 (7%) of gastric cancer tissues but not in the corresponding healthy mucosa (Maekawa et al., 2003). However, our finding of LDHB promoter hypermethylation in 45% of human prostate cancers, but rarely in nonmalignant or benign tissues suggests that LDHB methylation is a frequent event associated with the development of prostate cancer. Interestingly, by immunohistochemistry, 14/73 (9%) of adjacent nonmalignant or benign tissues showed absent LDHB expression. This finding could indicate epigenetic silencing of LDHB at a very early stage of tumorigenesis, well before the development of any histological changes. Although another mechanism underlying the loss of LDHB could involve chromosome loss of heterozygosity, this mechanism is unlikely since previous cytogenetic and comparative genomic hybridization (CGH) studies on the LNCaP-LN3 and other prostate cancer cell lines did not detect alterations affecting chromosome 12p12.2–p12.1, to where the LDHB gene maps (Rethore et al., 1976; Chu et al. 2001).
In summary, our data have demonstrated the feasibility of using 2D-PAGE followed by mass spectrometry in an in vivo model of human metastatic prostate cancer to identify candidate protein(s) associated with tumorigenesis. The mechanism underlying the loss of LDHB protein expression appears to involve promoter hypermethylation. Thus, the role of LDHB promoter hypermethylation and the consequences of loss of protein expression warrants further investigation in prostate and other cancers.
Materials and methods
Archived paraffin-embedded tissues obtained by radical prostatectomy, cystoprostatectomy or prostatic resection from patients attending the Academic Urology unit, University of Sheffield were used. Cases of metastatic prostate cancer in bone were collected by 8 mm trephine under general anaesthesia. For sequencing of the LDHB gene promoter region, 31 cases of prostatic adenocarcinoma, 14 cases of matched nonmalignant tissues, two cases of metastatic cancer in bone and five cases of BPH were used. Tumour Gleason grades were: 2 (n=3); 3 (n=8); 4 (n=15) or 5 (n=5) (Table 1). For immunohistochemistry, tissue microarrays comprising 53 cases of prostatic adenocarcinoma of various Gleason grades (2–5); 40 cases of matched nonmalignant tissue, seven cases of metastatic cancer in bone, five cases of HG-PIN lesions and 33 cases of BPH were used. Twenty-eight of the 31 cases of adenocarcinoma used for sequencing were also used for immunohistochemistry (Table 1). Prior local ethics committee approval and informed patient consent was obtained.
Cell lines and culture
The human prostate cancer cell lines LNCaP, Du145 and PC3 were obtained from the American Type Culture Collection (Manassas, VA, USA). The LNCaP-LN3 metastatic variant of the LNCaP cell line was kindly provided by Dr Curtis Pettaway (University of Texas MD Anderson Cancer Centre) (Pettaway et al., 1996). The CWR22 prostatic cancer xenograft was passaged under UK Home Office regulations as described previously (Rehman et al., 2005). The BPH45 cell line derived from a patient with BPH has been described previously (Klingler et al., 1999).
Tissue microdissection and DNA extraction
Tissues were precisely microdissected by removing 0.6 mm cylindrical cores to a depth of 2–3 mm from relevant malignant, nonmalignant and benign areas of the paraffin blocks as previously described (Rehman et al., 2005), and the DNA was extracted according to previously published methods (Rehman et al., 1996).
LNCaP and LNCaP-LN3 cell lines were cultured to ∼90% confluence in T175 flasks. Cytosolic protein fractions were prepared using the NER-pierce cell lysis kit according to the manufacturer's instructions (Pierce, Northumberland, UK), with the addition of COMPLETE EDTA-freeTM protease inhibitor mixture (Roche Diagnostics, East Sussex, UK). The protein concentration was determined using the Coomassie G-250 protein assay (Pierce, Northumberland, UK), according to supplied instructions.
Analytical and preparative 2D-PAGE gels were performed as previously detailed (Allen et al., 2003; Rehman et al., 2004a). Analytical gels were scanned using a Powerlook III scanner (UMax, Ascot, UK) and analysed using the Phoretix two-dimensional software (Nonlinear Dynamics, Newcastle, UK). For the purpose of this study, only those protein expression differences seen as ‘on–off’ differences were scored.
MALDI-TOF-MS and ESI-MS/MS analyses
MALDI-TOF-MS and ESI-MS/MS analyses were performed using the commercially available Protein and Nucleic Acid Chemistry (PNAC) facility at the University of Cambridge, UK (http://www.bioc.cam.ac.uk/pnac/pnac.html), according to previously published methods (Himpel et al., 2001). Database searches were performed using the Mascot Software (Matrix Science, London, UK) to search the Swiss Prot/TrEMBL (www.ca.expasy.org) and NCBI (ncbi.nlm.nih.gov) databases.
Two-dimensional Western blotting
Proteins were extracted from prostate cancer cell lines using the Mammalian cell lysis kit (Sigma-Aldrich, Dorset, UK) according to the manufacturer's instructions. Protein extracts (100 μg each) were resolved in two-dimensions as described above and the gel was trimmed to include the region of interest. The proteins were then blotted onto immobilon PVDF transfer membranes for 1 h at 65 V. Blots were then blocked using 5% nonfat milk for 1 h, then incubated with a 1:2000 dilution of the polyclonal goat anti-human LDH antibody (Code ab2101, Abcam, Cambridge, UK), overnight at 4°C. After incubation with horseradish peroxidase-conjugated anti-goat secondary antibody at a 1:30 000 dilution (Dako, Cambridge, UK), the membranes were developed by DuraWest substrate detection kit (Pierce) and exposed to an X-ray film. Anti-actin antibody (Sigma-Aldrich, Dorset, UK) was used as a protein loading control.
RNA was extracted using TRI reagent (Sigma-Aldrich, Dorset, UK) according to the manufacturer's instructions. After precipitation, the pellet was washed three times with 75% ethanol. All RNA was quantified spectrophotometrically.
cDNA synthesis and LDHB reverse transcription PCR
RNA (2 μg) was reverse transcribed into cDNA using a reverse transcription kit (Invitrogen, Paisley, UK), with 250 ng of random primers according to the manufacturer's instructions. For LDHB RT–PCR, primers were designed manually using the LDHB transcript sequence from the Ensembl genome browser (ENST00000350669). The sequence of the forward primer was (5′ –3′): IndexTermTTGTGGTTTCCAACCCAGTGGACA (LDHB-F-RT1, mapping across exons 4–5) and the reverse primer was: IndexTermAAAATCCATCCATGGCAGCTGCTG (LDHB-R-RT1, mapping within exon 5). RT–PCR was performed using 20 pmol of each primer in 20 μl volumes as described previously (Rehman et al., 2005).
Sequencing of bisulphite-modified DNA and primer design
For sequencing of the LDHB promoter, two primer pairs were used. The sequences of the first primer pair were those previously published which gave a 282 bp PCR product with 14 CpG sites (Maekawa et al., 2003) (Figure 5). The second primer pair was designed using the methprimer web tool (http://www.urogene.org/methprimer), by importing the LDHB promoter sequence (GenBank accession no. X13794). The primer sequences were (5′–3′): IndexTermTTTGGTTTATAGGTAAGTTTGATGG (LDH-B-Fwd2) and IndexTermACTACTACCCTCTACCTTCTACTCCTC (LDH-B-Rev2) (Figure 5). The relative position of the LDH-B-Fwd2 primer was –3015 to –3039 and that of the LDH-B-Rev2 primer was –2843 to −2869, designating the ATG translation start site as +1. The 197 bp PCR product contained 14 CpG sites. The promoter region selected for sequence analysis has been shown to be related to promoter activity (Maekawa et al., 2003; Takeno and Li, 1989). All primers were purchased from MWG (www.mwg-biotech.com).
Sodium bisulphate-modified DNA was sequenced as previously described (Rehman et al., 2005) and the chromatograms were visualized using the Chromas software (http://www.technelysium.com.au/chromas.html).
The χ2 analyses were performed using the web χ2 calculator (http://www.georgetown.edu/faculty/ballc/webtools/web_chi.html).
Lactate dehydrogenase enzyme activity assay
LDH enzyme activity was assayed in the LNCaP and LNCaP-LN3 cell lines, by a modification of the method of Bergmeyer (1974). Briefly, the assay determines the activity of LDH by measuring the rate at which the enzyme catalyses the interconversion of pyruvate to lactate. The rate of reaction was determined by the decrease in absorbance at 340 nm, resulting from the oxidation of NADH to NAD+.
Fresh cytosolic proteins were isolated from cell lines using the Mammalian cell extraction kit (Pierce) according to supplied instructions. Following protein quantification (Bio-rad), the lysates were immediately used for the enzyme assay. A 0.26 mM β-NADH solution was mixed with 100 μl of 68 mM sodium pyruvate solution in a cuvette (1 cm path). The mixture was placed into a Smart-Spec 3000 photospectrometer (BioRad), and the absorbance at 340 nm was measured until a constant reading was obtained. To ensure reproducibility, this procedure was repeated three times for each cell line and using extracts from three separate extractions. Purified L-lactate dehydrogenase enzyme solution (Sigma-Aldrich) was used as a positive control (data not shown).
LDH isoenzyme analysis
LDH isoenzyme patterns were determined by previously published methods (Salplachta and Necas, 2000). Briefly, following protein quantification, 100 μg of protein per sample was loaded onto a 6% nondenaturing polyacrylamide gel and electrophoresed for 60 min at 200 V and at 4°C. Following electrophoresis, the gels were placed in 10 ml of staining solution containing 0.1 mol/l sodium lactate, 1.5 mmol/l NAD, 0.1 mol/l Tris-HCl (pH 8.6), 10 mmol/l NaCl, 5 mmol/ MgCl2, 0.3 mg Phenazinmethosulphate (PMS) and 2.5 mg nitrobluetetrazolium (NBT). The assay relies on the conversion of lactate to pyruvate, with the production of NADH and H+. The NADH then reduces PMS, which in turn reduces NBT to give an insoluble product, diformazan. To ensure maximum sensitivity for detection of the various isoenzymes, the reaction was allowed to proceed to completion. Protein extracted from mouse heart served as a positive control and was obtained from an animal killed as part of another study.
Immunohistochemistry and scoring of staining
Immunohistochemistry was performed essentially as described previously (Rehman et al., 2004b). Sections were incubated for 1 h with mouse monoclonal anti-human LDH-B-specific antibody (Cat. No L7016, Sigma, Dorset, UK) at a 1:200 dilution in 1/4 X Casein in 0.5 M TBS, 2 mM CaCl2, 0.012 % Tween 20, pH 7.5. Secondary antibody was biotinylated rabbit anti-mouse, used at a 1:400 dilution (Vector laboratories, Peterborough, UK). The anti-LDH-B antibody used has previously been shown to be LDH-B specific and nonreactive against LDH-A (Tsoi et al., 2001).
LNCaP-LN3 cells were cultured in T175 flasks with RPMI-1640 medium to 20% confluence. A final concentration of 1 μ M freshly prepared 5′-azacytidine (Sigma-Aldrich) was added to the culture medium. After 5 days of drug treatment, RNA was isolated using TRI reagent (Sigma) as described above.
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Financial support was provided by a National Cancer Research Institute (NCRI) grant (MRC 58152) to FC Hamdy. We thank Dr Len Packman (University of Cambridge) for peptide mass fingerprint analyses and database searches and Anne Fowles for technical assistance.
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Leiblich, A., Cross, S., Catto, J. et al. Lactate dehydrogenase-B is silenced by promoter hypermethylation in human prostate cancer. Oncogene 25, 2953–2960 (2006). https://doi.org/10.1038/sj.onc.1209262
- prostate cancer
- benign prostatic hyperplasia
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