Nuclear DNA Methylation and Chromatin Condensation Phenotypes Are Distinct Between Normally Proliferating/Aging, Rapidly Growing/Immortal, and Senescent Cells

Jin Ho Oh,Jian Tajbakhsh,Arkadiusz Gertych

doi:10.18632/oncotarget.942

Abstract

This study reports on probing the utility of in situ chromatin texture features such as nuclear DNA methylation and chromatin condensation patterns - visualized by fluorescent staining and evaluated by dedicated three-dimensional (3D) quantitative and high-throughput cell-by-cell image analysis - in assessing the proliferative capacity, i.e. growth behavior of cells: to provide a more dynamic picture of a cell population with potential implications in basic science, cancer diagnostics/prognostics and therapeutic drug development. Two types of primary cells and four different cancer cell lines were propagated and subjected to cell-counting, flow cytometry, confocal imaging, and 3D image analysis at various points in culture. Additionally a subset of primary and cancer cells was accelerated into senescence by oxidative stress. DNA methylation and chromatin condensation levels decreased with declining doubling times when primary cells aged in culture with the lowest levels reached at the stage of proliferative senescence. In comparison, immortal cancer cells with constant but higher doubling times mostly displayed lower and constant levels of the two in situ-derived features. However, stress-induced senescent primary and cancer cells showed similar levels of these features compared with primary cells that had reached natural growth arrest. With regards to global DNA methylation and chromatin condensation levels, aggressively growing cancer cells seem to take an intermediate level between normally proliferating and senescent cells. Thus, normal cells apparently reach cancer-cell equivalent stages of the two parameters at some point in aging, which might challenge phenotypic distinction between these two types of cells. Companion high-resolution molecular profiling could provide information on possible underlying differences that would explain benign versus malign cell growth behaviors.

Highlights

Cells that share the same genotype, may not necessarily present the same phenotype, including structural and functional properties that can be assayed with a plethora of existing technologies
Doubling times increased in primary cells as cells reached senescence, while doubling times stayed nearly constant for all cancer cell lines, as anticipated
The results confirm that proliferative capacity of primary cells rapidly decrease as they age in culture, while this capacity stays nearly constant for immortal cancer cell lines

Summary

Introduction

Cells that share the same genotype, may not necessarily present the same phenotype, including structural and functional properties that can be assayed with a plethora of existing technologies. Cancer cells usually display hypermethylation of a relatively small number of single gene promoters mostly in gene-rich genomic regions termed CpGislands, leading to silencing of certain tumor suppressors involved in cell-cycle regulation, DNA mismatch repair, cellular differentiation, and apoptosis This phenomenon is often coexistent with hypomethylation at the global DNA (gDNA) level, a large portion of which occurs in repetitive elements; potentially inducing activation of latent retrotransposons with the consequence of genomewide mutations and genome instability [13] alongside with substantial spatial reorganization of chromatin in cell nuclei, which can be visualized by light microscopy [14,15,16,17,18]. This differential topology can be be triggered by exposing cells to strong demethylating agents [19,20]

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