Epigenetics in radiotherapy: Where are we heading?

Radiotherapy is an important component of anti-cancer treatment. However, not all cancer patients respond to radiotherapy, and with current knowledge clinicians are unable to predict which patients are at high risk of recurrence after radiotherapy. There is therefore an urgent need for biomarkers to guide clinical decision-making. Although the importance of epigenetic alterations is widely accepted, their application as biomarkers in radiotherapy has not been studied extensively. In addition, it has been suggested that radiotherapy itself introduces epigenetic alterations. As epigenetic alterations can potentially be reversed by drug treatment, they are interesting candidate targets for anticancer therapy or radiotherapy sensitizers. The application of demethylating drugs or histone deacetylase inhibitors to sensitize patients for radiotherapy has been studied in vitro, in vivo as well as in clinical trials with promising results. This review describes the current knowledge on epigenetics in radiotherapy

The emerging role of GATA transcription factors in development and disease

The GATA family of transcription factors consists of six proteins (GATA1-6) which are involved in a variety of physiological and pathological processes. GATA1/2/3 are required for differentiation of mesoderm and ectoderm-derived tissues, including the haematopoietic and central nervous system. GATA4/5/6 are implicated in development and differentiation of endoderm- and mesoderm-derived tissues such as induction of differentiation of embryonic stem cells, cardiovascular embryogenesis and guidance of epithelial cell differentiation in the adult

Analysis of Promoter CpG Island Hypermethylation in Cancer: Location, Location, Location!

The genetic and epigenetic alterations that underlie cancer pathogenesis are rapidly being identified. This provides novel insights in tumor biology as well as in potential cancer biomarkers. The somatic mutations in cancer genes that have been implemented in clinical practice are well defined and very specific. For epigenetic alterations, and more specifically aberrant methylation of promoter CpG islands, evidence is emerging that these markers could be used for the early detection of cancer as well as prediction of prognosis and response to therapy. However, the exact location of biologically and clinically relevant hypermethylation has not been identified for the majority of methylation markers. The most widely used approaches to analyze DNA methylation are based on primer- and probe-based assays that provide information for a limited number of CpG dinucleotides and thus for only part of the information available in a given CpG island. Validation of the current data and implementation of hypermethylation markers in clinical practice require a more comprehensive and critical evaluation of DNA methylation and limitations of the techniques currently used in methylation marker research. Here, we discuss the emerging evidence on the importance of the location of CpG dinucleotide hypermethylation in relation to gene expression and associations with clinicopathologic characteristics in cancer

Proliferative regulation by MK3 in cancer cell lines.

<p>(A) Proliferation curves (left) of U-2OS cells expressing a retroviral <i>MK3</i> vector (<i>MK3</i>^<i>WT</i><i>OE</i>; filled circles) or an empty vector (con; open circles); overexpression of GST-MK3 (<i>MK3</i>^<i>WT</i><i>OE</i>) in U-2OS cells detected with and GST or a MK3-antiserum (right panel). Cell counts at t = 2 through t = 8 were normalized to cell counts at t = 0 for each transduced cell culture individually (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118840#sec013" target="_blank">Methods</a> section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (B) Phase contrast images showing cell morphology in U-2OS/<i>MK3</i>^<i>WT</i><i>OE</i> cells and control cells. (C) Protein expression levels of the check-point regulator proteins TP53 and p21^CIP1/WAF1 (P21) in U-2OS/<i>MK3</i>^<i>WT</i><i>OE</i> cells; loading control b-Actin (bAct). (D) DNA profile analysis of U-2OS/<i>MK3OE</i> versus control cells (4–6 days post-transduction; representative experiment). <i>MK3</i>^<i>WT</i>overexpression elicits an intra S-phase arrest: table shows a substantially increased S-phase occupancy. (E) Immunohistochemical staining for phosphorylated H2A.X (γH2A.X) and phosphorylated KAP1pSer824 (pKAP1; arrows) to visualize DNA damage in U-2OS/<i>MK3</i>^<i>WT</i><i>OE</i> cultures; control (top panels) or <i>MK3</i>^<i>WT</i><i>OE</i> (bottom panels).</p

Functional links between MK3 and PRC in proliferative life span.

<p>TIG3 cells were sequentially transduced with either <i>Bmi1</i>.<i>ires</i>.<i>GFP</i> (<i>Bmi1OE</i>) or <i>GFP</i> (con) virus, and <i>MK3</i>/<i>puromycin</i> (<i>MK3OE</i>) or control <i>puromycin</i> virus (con) at 48 hrs intervals. Retroviral vectors expressing murine <i>Bmi1/</i>GFP reporter were transduced first (or empty vector control), followed by a MK3/<i>puromycin</i> resistance marker (or empty vector control). Transduction of <i>Bmi1OE</i> and control transduced cells was simultaneously carried out with the same <i>MK3</i>^<i>WT</i><i>OE</i> viral preparation (or control virus) to minimize inter-experimental variation. Cells were grown on selection medium and proliferation capacity was tested ± 2–3 weeks post-transduction. (A) Proliferation curves of normal human TIG3 fibroblasts transduced with a retroviral <i>MK3</i>^<i>WT</i><i>overexpression</i> vector (<i>MK3</i>^<i>WT</i><i>OE</i>; black symbols) or <i>shcon</i> vector (white symbols), in conjunction with either an empty vector control (con; circles) or a murine <i>Bmi1</i> expression vector (<i>Bmi1OE</i>; triangles). (B) Proliferation curves of normal human TIG3 fibroblasts transduced with a retroviral <i>MK3</i> knock-down vector (<i>shMK3</i>; black symbols) or <i>shcon</i> vector (white symbols), in conjunction with either an empty vector control (con; circles) or a murine <i>Bmi1</i> expression vector (<i>Bmi1OE</i>; squares). Cell counts at t = 2 through t = 8 (A, B) were normalized to cell counts at t = 0 for each transduced cell culture individually (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118840#sec013" target="_blank">Methods</a> section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (C) Comparative morphology of TIG3 cells expression <i>Bmi1</i> and/or <i>MK3</i> versus control cells as recorded by GFP fluorescent imaging ± 3 weeks after transduction (D) Immunoblot analysis of EZH2, CBX4, RNF2 and H3K27me3 in <i>MK3</i>^<i>WT</i><i>OE</i>, <i>Bmi1OE</i>, <i>Bmi1OE/MK3</i>^<i>WT</i><i>OE</i> and <i>control</i> TIG3 cell lysates. (E) Expression analysis of BMI1, MK3, and TP53 at the indicated time points in (corresponding to experiment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118840#pone.0118840.g004" target="_blank">Fig 4C</a>). Cells were grown on selection medium and analysed at 1 or 4 weeks after serial transduction; expression vectors and antibodies are indicated in the figure. Early and late samples were loaded on the same gel for BMI1 analysis; corresponding sections are shown separately.</p

<i>MK3</i>^<i>WT</i> overexpression induces a proliferative arrest in normal human fibroblasts.

<p>(A) Retroviral expression systems were applied to enhance (<i>MK3</i>^<i>WT</i><i>OE</i>) or remove (<i>shMK3</i>) MK3 expression. Western blot shows MK3 proteins: endogenous (MK3^endo) and overexpressed (GST:MK3). (B) Proliferation curves of TIG3 cells transduced with: a retroviral <i>MK3</i> expression vector (<i>MK3</i>^<i>WT</i><i>OE</i>; filled circles), an empty vector (con; open circles) or a murine <i>Bmi1</i> expression vector (open triangles); proliferation was determined at 1 week (top panel) or ±4 weeks (bottom panel) after retroviral transduction of TIG3 cells. Cell counts at t = 2 through t = 8 were normalized to cell counts at t = 0 for each transduced cell culture individually (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118840#sec013" target="_blank">Methods</a> section for details); statistical significance was determined by two-tailed Student’s t-test and is presented relative to the empty vector control (* p < 0.05). (C) Quantification of DNA profiles (BrdU pulse-labeling and S-phase quantification by FACS) in TIG3/<i>MK3</i>^<i>WT</i><i>OE</i> cells at approximately 1 week post-transduction. <i>RasV12</i>-transduced cells were used as a positive control. (D) <i>MK3</i>^<i>WT</i><i>OE</i> reduces <i>de novo</i> DNA synthesis in TIG3/<i>MK3</i>^<i>WT</i><i>OE</i> cells; cell counts: control (con) 699 BrdU-positive cells/10 fields; <i>MK3</i>^<i>WT</i><i>OE</i>: 442 BrdU-positive cells/9 fields.</p

Functional links between MK3 and PRC in proliferative life span.

<p>(A) Expression levels of the PRC proteins EZH2, CBX8 and PHC1 in senescing TIG3/<i>MK3</i>^<i>WT</i><i>OE</i> cells at ±3–4 weeks post-transduction; loading control b-Actin (bAct). (B) Expression levels of the PRC proteins EZH2, CBX8 and PHC1 in senescing TIG3/<i>shMK3</i> cells at ±2–3 weeks post-transduction; loading control b-Actin (bAct). (C) Chromatin immunoprecipitation (ChIP) analysis of MK3, H3K27me3, CBX8, PHC1 enrichment in <i>MK3</i>^<i>WT</i><i>OE</i> (<i>MK3</i>^<i>WT</i><i>OE</i>) expressing and control (<i>con</i>) TIG3 cells; PRC1-target loci (p<i>16 promoter</i>, <i>p16exon1</i>, <i>HOXA10</i>, <i>HOXA11)</i> and non-target loci (p15exon1, p14exon1) are indicated below the figure. Enrichments are presented as percentages of total input. Negative control HA antiserum. Experiments were performed three times; results of one representative experiment are shown (statistical significance: * p<0.05, ** p<0.01; t-test).</p