LINE-1 Hypomethylation in Cancer Is Highly Variable and Inversely Correlated with Microsatellite Instability

BACKGROUND: Alterations in DNA methylation in cancer include global hypomethylation and gene-specific hypermethylation. It is not clear whether these two epigenetic errors are mechanistically linked or occur independently. This study was performed to determine the relationship between DNA hypomethylation, hypermethylation and microsatellite instability in cancer. METHODOLOGY/PRINCIPAL FINDINGS: We examined 61 cancer cell lines and 60 colorectal carcinomas and their adjacent tissues using LINE-1 bisulfite-PCR as a surrogate for global demethylation. Colorectal carcinomas with sporadic microsatellite instability (MSI), most of which are due to a CpG island methylation phenotype (CIMP) and associated MLH1 promoter methylation, showed in average no difference in LINE-1 methylation between normal adjacent and cancer tissues. Interestingly, some tumor samples in this group showed increase in LINE-1 methylation. In contrast, MSI-showed a significant decrease in LINE-1 methylation between normal adjacent and cancer tissues (P<0.001). Microarray analysis of repetitive element methylation confirmed this observation and showed a high degree of variability in hypomethylation between samples. Additionally, unsupervised hierarchical clustering identified a group of highly hypomethylated tumors, composed mostly of tumors without microsatellite instability. We extended LINE-1 analysis to cancer cell lines from different tissues and found that 50/61 were hypomethylated compared to peripheral blood lymphocytes and normal colon mucosa. Interestingly, these cancer cell lines also exhibited a large variation in demethylation, which was tissue-specific and thus unlikely to be resultant from a stochastic process. CONCLUSION/SIGNIFICANCE: Global hypomethylation is partially reversed in cancers with microsatellite instability and also shows high variability in cancer, which may reflect alternative progression pathways in cancer

Agrobacterium-mediated transformation of tarwi (Lupinus mutabilis Sweet), a potential platform for the production of plant-made proteins

A robust, reproducible method of Agrobacterium-mediated transformation was developed for Lupinus mutabilis Sweet (tarwi), a large-seeded Andean legume. Initially, a regeneration and transformation protocol was developed using a plasmid which contained a bifunctional fusion gene conferring both \u3b2-glucuronidase (gus) and neomycin phosphotransferase activities, under the control of a constitutive 35S35SAMV promoter. The tissue explants consisted of longitudinal slices from embryonic axes of imbibed, mature seed. Using a series of tissue culture media for cocultivation, shoot initiation, shoot elongation, and rooting, kanamycin-resistant transgenic plants were recovered from approximately 1% of the explants. This transformation protocol was further used with a construct that contained the human adenosine deaminase (hADA) gene under the control of a legumin seed-specific promoter, also with a kanamycin resistance cassette for chemical selection. Changes made during the course of this study, which included adjustments to the antibiotic concentration during the shoot elongation and rooting phases plus the incorporation of techniques to improve ventilation in the tissue culture system, resulted in major improvements in shoot quality and, most significantly, rooting. The outcome was an increased frequency of transgenic plant recovery (7.4%), with a low (9.6%) rate of plants that escaped selection. The inheritance of the hADA gene was documented and showed the expected Mendelian segregation pattern. The produced hADA protein was a fully functional enzyme and localized only in the seed, as expected. Thus, this legume species is an excellent candidate for a nonfood plant host platform for the production of plant-made proteins.Peer reviewed: YesNRC publication: Ye

Seed specific expression and analysis of recombinant human adenosine deaminase (hADA) in three host plant species

The plant seed is a leading platform amongst plant-based storage systems for the production of recombinant proteins. In this study, we compared the activity of human adenosine deaminase (hADA) expressed in transgenic seeds of three different plant species: pea (Pisum sativum L.), Nicotiana benthamiana L. and tarwi (Lupinus mutabilis Sweet). All three species were transformed with the same expression vector containing the hADA gene driven by the seed-specific promoter LegA2 with an apoplast targeting pinII signal peptide. During the study, several independent transgenic lines were generated and screened from each plant species and only lines with a single copy of the gene of interest were used for hADA expression analysis. A stable transgenic canola line expressing the ADA protein, under the control of 35S constitutive promoter was used as both as a positive control and for comparative study with the seed specific promoter. Significant differences were detected in the expression of hADA. The highest activity of the hADA enzyme (Units/g seed) was reported in tarwi (4.26 U/g) followed by pea (3.23 U/g) and Nicotiana benthamiana (1.69 U/g). The expression of mouse ADA in canola was very low in both seed and leaf tissue compared to other host plants, confirming higher activity of seed specific promoter. Altogether, these results suggest that tarwi could be an excellent candidate for the production of valuable recombinant proteins.Peer reviewed: YesNRC publication: Ye

Figure 2

<p>Differential LINE-1 methylation among CIMP/MSI groups in primary colorectal carcinoma samples (CRCs). A) Colorectal tumor DNA and their normal appearing adjacent mucosa from sixty patients were evaluated for LINE-1 methylation. These tumors were previously evaluated for CpG island methylator phenotype (CIMP), using a panel of single-copy genes methylation analysis, and microsatellite instability (MSI) status, resulting in the identification of three CIMP/MSI groups. In normal appearing mucosa (top) little variation in LINE-1 methylation is observed between samples and CIMP/MSI groups (average methylation = 64.3%), while in tumor (bottom) several samples undergo high LINE-1 demethylation (25/60 tumor samples have methylation density bellow 55%), most notable in CIMP+/MSI-and CIMP-/MSI-groups. B) Relative LINE-1 demethylation in CRCs. Relative demethylation was calculated as the percent change of LINE-1 methylation in tumor compared to its normal appearing mucosa. Both CIMP+/MSI-and CIMP-/MSI-samples presented in average 16% demethylation for LINE-1, while no significant changes were observed for the CIMP+/MSI+samples. For the CIMP+group, 4–9% increase of methylation density for LINE-1 was observed for a small fraction of samples, most of them identified as CIMP+/MSI+samples.</p

Figure 1

<p>Quantitation of DNA methylation using bisulfite LINE-1 PCR and pyrosequencing. A) Diagram of the CpG island promoter (GenBank accession no. X58075, nucleotide position 108–520 bp) associated with the full length LINE-1. Each vertical line represents a single CpG site. The 3′UTR, 5′UTR and two ORFs of LINE-1 are shown at the top. Arrows indicate the location of primers used for bisulfite PCR (R-biot and F) and pyrosequencing (S). B) Representative LINE-1 pyrograms for normal peripheral blood lymphocytes (PBL) and breast cancer cell lines (MB-468 and SKBR3). The pyrogram quantitates C for methylated and T for unmethylated DNA. The shaded region represents the CpG site quantitated in LINE-1 elements, and the percent methylation is shown above the peak.</p

Figure 3

<p>Methylated CpG Island Amplication (MCA)/CpG island microarray for repetitive DNA sequences. A) Relative abundance of hypermethylated and hypomethylated repeats for each CIMP/MSI group. A higher number of hypermethylated compared to hypomethylated repeats was observed for the CIMP+/MSI+group, and a gradual change in representation of hypermethylated and hypomethylated repeats was seen for the CIMP+/MSI-and CIMP-/MSI-groups, resulting in an overrepresentation of hypomethylated repeats in microsatellite stable groups. B) Validation of microarray results for LINE repeats. Note that CIMP/MSI groups with higher demethylation, as determined by bisulfite-pyrosequencing of LINE-1, presented also a higher number of hypomethylated LINE repeats by microarray analysis, as represented by a lower hyper/hypomethylation ratio. C) Unsupervised hierarchical clustering was applied to methylation data from a set of 770 repetitive DNA sequences across sixteen colorectal tumors paired with their normal appearing mucosa DNA. The colorectal tumors dendrogram is shown, and the sample ID for each case is included in the right. The terminal branches are color coded to represent the CIMP/MSI status of the tumor sample (red, CIMP+/MSI+; blue, CIMP+/MSI-; green, CIMP-/MSI-). Overall, samples of the same CIMP/MSI group clustered together, reinforcing the different methylation fate for repetitive DNA sequences methylation in each group. LINE, long interspersed nuclear elements; SINE, short interspersed nuclear elements, LTR, long terminal repeats; DNA repeats; Satellite repeats.</p

Methylation density of LINE-1and clinical and demographic characteristics of 60 colorectal carcinomas and their normal appearing adjacent mucosa^*

*<p>Means of cancer methylation is significantly (P<0.001) lower than mean of adjacent normal for all categories except for MSI+cancers (p = 0.24)</p>a<p>Significant P values (<0.05) are underlined</p>b<p>Side information was not available for all cases</p><p>CI = confidence interval</p

Figure 4

<p>LINE-1 methylation variability in cancer cell lines. DNA samples of normal peripheral blood lymphocyte, normal colon mucosa and sixty-one cell lines from eight different tissues types were investigated for LINE-1 methylation using bisulfite PCR followed by pyrosequencing. The normal tissues presented high levels of LINE-1 methylation (above 70% in average), and a large variation in methylation levels was observed for cancer cell lines, with a minimum methylation density of 6.5% being observed for the leukemia cell line K562. Taken as a group, leukemia cell lines were moderately demethylated (average 56.1%), followed by ovary, colon, prostate and lung cancer cell lines (variation from 49.7% to 35.1%). Central nervous system (CNS), breast and the one liver cancer cell lines tested were deeply demethylated (bellow 30% in average). Dotted line represents average methylation in normal controls.</p