Immortalization of T-cells is accompanied by gradual changes in CpG methylation resulting in a profile resembling a subset of T-cell leukemias [expression]

We have previously described gene expression changes during spontaneous immortalization of T cells, thereby identifying cellular processes important for cell growth crisis escape and long term proliferation. Here we analyze the same model to investigate the role of genome-wide methylation in the immortalization process at different time points pre- and post- crisis using high-resolution arrays. We show that over time in culture there is an overall accumulation of methylation alterations, with preferential increased methylation close to transcription start sites, island and shore regions. Most methylation alterations did not affect gene expression but among the affected genes, increased methylation close to transcription start site was associated with decreased gene expression. Interestingly, the pattern of CpG site methylation observed in post-crisis T-cell cultures was similar to clinical T-cell acute lymphoblastic leukemia (T-ALL) samples classified as CpG island methylator phenotype positive (CIMP+). These sites were highly overrepresented by polycomb target genes and involved in developmental, cell adhesion and cell signaling processes. The presence of nonrandom methylation events in in vitro immortalized T-cell cultures and diagnostic T-ALL samples indicates altered methylation of CpG sites with a possible role in malignant hematopoiesis. RNA was extracted with TRIZOL according to manufacturer instructions. Total RNA was amplified bu the Illumina TotalPrep RNA Amplification kit. Gene expression array analysis was performed on the cell cultures.