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, 22 (41), 6319-31

Suppression of the Protein Tyrosine Phosphatase Receptor Type O Gene (PTPRO) by Methylation in Hepatocellular Carcinomas

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Suppression of the Protein Tyrosine Phosphatase Receptor Type O Gene (PTPRO) by Methylation in Hepatocellular Carcinomas

Tasneem Motiwala et al. Oncogene.

Abstract

A diet lacking folic acid and choline and low in methionine (folate/methyl deficient diet, FMD diet) fed to rats is known to produce preneoplastic nodules (PNNs) after 36 weeks and hepatocellular carcinomas (tumors) after 54 weeks. FMD diet-induced tumors exhibit global hypomethylation and regional hypermethylation. Restriction landmark genome scanning analysis with methylation-sensitive enzyme NotI (RLGS-M) of genomic DNA isolated from control livers, PNNs and tumor tissues was performed to identify the genes that are differentially methylated or amplified during multistage hepatocarcinogenesis. Out of the 1250 genes analysed, 2 to 5 genes were methylated in the PNNs, whereas 5 to 45 genes were partially or completely methylated in the tumors. This analysis also showed amplification of 3 to 12 genes in the primary tumors. As a first step towards identifying the genes methylated in the PNNs and primary hepatomas, we generated a rat NotI-EcoRV genomic library in the pBluescriptKS vector. Here, we describe identification of one methylated and downregulated gene as the rat protein tyrosine phosphatase receptor type O (PTPRO) and one amplified gene as rat C-MYC. Methylation of PTPRO at the NotI site located immediate upstream of the trancription start site in the PNNs and tumors, and amplification of C-MYC gene in the tumors were confirmed by Southern blot analyses. Bisulfite genomic sequencing of the CpG island encompassing exon 1 of the PTPRO gene revealed dense methylation in the PNNs and tumors, whereas it was methylation free in the livers of animals on normal diet. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed significant decrease in the expression of PTPRO in the tumors and in a transplanted rat hepatoma. The expression of PTPRO mRNA in the transplanted hepatoma after demethylation with 5-azacytidine, a potent inhibitor of DNA methyltransferases, further confirmed the role of methylation in PTPRO gene expression. These results demonstrate alteration in methylation profile and expression of specific genes during tumor progression in the livers of rats in response to folate/methyl deficiency, and further implicate the potential role of PTPRO as a novel growth regulatory gene at least in the hepatocellular carcinomas.

Figures

Figure 1
Figure 1
RLGS technique. (a) A schematic representation of the RLGS technique. DNA (unmethylated or methylated DNA at one or both alleles) are digested with NotI (cuts only unmethylated DNA) followed by endlabeling with (32P-γ)ATP, EcoRV digestion and separation in agarose tube gel (first dimension). DNA is further digested in gel with HinfI followed by electrophoresis in polyacrylamide slab gel (second deminsion). The dried gel is subjected to autoradiography. (b) Rat master RLGS profile using the NotI–EcoRV–HinfI enzyme combination. Directions for the first dimension and second dimension separations are indicated. The grids dividing the profile into rows (1–7) and columns (A–I) are also shown
Figure 2
Figure 2
Rat RLGS profiles from normal liver, PNN and tumor. Sections from RLGS profiles derived from control, PNN (36 weeks on FMD diet) and tumor (54 weeks on FMD diet). Chromosomal DNA isolated from the livers of rats on normals diet, livers bearing PNNs and tumors of animals on the deficient diet were subjected to RLGS-M analysis following the protocol described in Figure 1a. (a) Arrows indicate the position of the lost spot (3E41) in the PNN and tumor RLGS-M profile. (b) Arrows indicate RLGS spot (3C24) that was enhanced in the tumor profile
Figure 3
Figure 3
Cloning of RLGS spots. (a) Flow chart depicting the steps involved in the generation of a rat methylation (NotI–EcoRV) library, cloning and identification of spots of interest on the RLGS profile. (b) RLGS mixing gels with pooled DNA clones from rat NotI–EcoRV library. The RLGS fragment (3E41) was found in mixing gels from plate 12, row C and column 23. A corresponding section from normal liver is shown for comparison. (c) RLGS mixing gels with pooled DNA clones from a rat NotI–EcoRV library. Enhanced RLGS fragment (3C24) was found in mixing gels from plate 2, row F and column 20. A corresponding section from normal liver is shown for comparison
Figure 4
Figure 4
Southern blot analysis of clones corresponding to RLGS fragments 3E41 and 3C24. (a) Southern blot analysis of enhanced RLGS fragment 3C24. EcoRI-digested genomic DNA from normal liver, two preneoplastic lesions and one tumor were hybridized to 32P-labeled random-primed RLGS fragments 3C24, C-MYC and 2C31. (b) Genomic DNA from normal rat liver was digested with EcoRV alone or both NotI and EcoRV and hybridized with inserts from clones 3E41 (upper panel) and 2D18 (lower panel) (lanes 1 and 2). Similarly, genomic DNAs from PNNs and tumors were digested with EcoRV and NotI and hybridized with the same probes (upper and lower panels, lanes 3–6)
Figure 5
Figure 5
The different PTPRO variants. (a) Transcript variants of human PTPRO. Schematic representation of the different known isoforms of human PTPRO. The numbers on top represent exons. (b) Four different isozymes of PTPRO are encoded by six different mRNA variants
Figure 6
Figure 6
RT–PCR analysis of rat PTPRO. (a) Total RNA isolated from control livers, PNNs and tumors as well as from brain was converted to cDNA using random hexamers as primers using RT–PCR kit (Perkin-Elmer). The cDNA was then used for PCR with primers common to all PTPRO isoforms (panel 1, rPTP-3′), specific for full-length PTPRO (panel 2, rPTP-5′), truncated rat PTPRO (panel 3, rPTPt) or rat COX-1 (panel 4, rCOX-1). (b) Quantitative analysis of the rat PTPRO expression in tumors. The 32P-signal in each PCR product obtained with the rPTP-5′ primers was determined from the PhosphorImager analysis (Molecular Dynamics) and quantitated by a volume analysis program (Molecular Dynamics)
Figure 7
Figure 7
Bisulfite sequencing of exon 1 of rat PTPRO gene. (a) Nucleotide sequence of 3E41 clone corresponds to the rat PTPRO gene. The NotI site located in the immediate promoter and lost upon methylation in PNN and tumors is indicated. The arrow indicates the transcription start site. The underlined sequence corresponds to the position of the second set of nested PCR primers (rPTP-BS-F2 and rPTP-BS-R2) used to amplify CpG island of PTPRO gene from the bisulfite-converted genomic DNA. (b) Sequence analysis of PTPRO exon 1 methylation. Genomic DNAs from control livers, PNNs and tumors were treated with sodium bisulfite, and the CpG island of rat PTPRO was amplified using nested primers. The PCR products were cloned in TA vector and 10 individual clones were randomly selected for DNA sequence determination. Each row of boxes represents a clone of the particular sample. As indicated in the figure, the sequence included 18 CpGs between +168 and +360 with respect to the transcription start site. The numbers on top represent the positions of CpGs with respect to the transcription start site. The filled and open boxes represent methylated and unmethylated CpGs at a specific position in the particular clone. The numbers above each box denote positions of cytosines with respect to the transcription initiation (+1) site
Figure 8
Figure 8
Induction of PTPRO in Morris hepatoma upon 5-AzaC treatment. (a) RLGS sections from ACI rat liver and Morris hepatoma (transplated into ACI rats) depicting loss of spot corresponding to PTPRO. DNAs isolated from the transplanted hepatomas from the ACI rats and 5-AzaC treated tumor-bearing rats and the host livers were subjected to RLGS-M analysis as described in Figure 1a. (b) Total RNA was isolated from the brain, liver of ACI rats, Morris hepatoma 3924A and 5-AzaC-treated hepatoma and subjected to RT–PCR analysis with primers common to all PTPRO isoforms (rPTP-3′) or rat COX-1. (c) Sequencing of the CpG island of rat PTPRO amplified from bisulfite-treated DNA of the host liver and hepatoma with rPTP-BS-F2 (see Materials and methods). Arrows indicate positions of methylated CpGs in the hepatoma. (d) Bisulfite sequence analysis of PTPRO in the liver of ACI rats, Morris hepatoma 3924A and 5-AzaC-treated hepatoma. CpG island of rat PTPRO was amplified and cloned from liver, hepatoma and 5-AzaC-treated hepatoma as described (see Materials and methods). Four clones selected at random were subjected to automated sequencing. Each row of boxes represents an individual clone from the sample. Filled and open boxes represent methylated and unmethylated CpGs, respectively, within a particular clone. The positions of each cytosine methylated/unmethylated with respect to +1 site are represented above each box

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