Reduced mRNA and Protein Expression of the Genomic Caretaker RAD9A in Primary Fibroblasts of Individuals with Childhood and Independent Second Cancer

Background: The etiology of secondary cancer in childhood cancer survivors is largely unclear. Exposure of normal somatic cells to radiation and/or chemotherapy can damage DNA and if not all DNA lesions are properly fixed, the mis-repair may lead to pathological consequences. It is plausible to assume that genetic differences, i.e. in the pathways responsible for cell cycle control and DNA repair, play a critical role in the development of secondary cancer. Methodology/Findings: To identify factors that may influence the susceptibility for second cancer formation, we recruited 20 individuals who survived a childhood malignancy and then developed a second cancer as well as 20 carefully matched control individuals with childhood malignancy but without a second cancer. By antibody microarrays, we screened primary fibroblasts of matched patients for differences in the amount of representative DNA repair-associated proteins. We found constitutively decreased levels of RAD9A and several other DNA repair proteins in two-cancer patients, compared to onecancer patients. The RAD9A protein level increased in response to DNA damage, however to a lesser extent in the twocancer patients. Quantification of mRNA expression by real-time RT PCR revealed lower RAD9A mRNA levels in both untreated and 1 Gy c-irradiated cells of two-cancer patients. Conclusions/Significance: Collectively, our results support the idea that modulation of RAD9A and other cell cycle arrest and DNA repair proteins contribute to the risk of developing a second malignancy in childhood cancer patients

Reduced DNA damage response of two-cancer patients.

<p>Differential induction of RAD9A (left side) and DDIT (right side) at 1 h and 4 h after 1 Gy γ-irradiation in fibroblasts of two-cancer patients (gray boxes) and one-cancer patients (dotted boxes). Protein expression was measured by antibody microarrays (normalized by log10 transformation and z scores). Box plots show the distribution of z ratios of treated vs. untreated cells of the same patients. The median is represented by horizontal lines. The bottom of the box indicates the 25^th percentile, the top the 75^th percentile. The T bars extend from the boxes to at most 1.5 times the height of the box. Outliers are shown as open circles. The DNA-damage induced increase in the 2C group is lower than that in the 1C group for RAD9A at 4 h after irradiation (−1.44x, p = 0.012) and for DDIT3 at 1 h after irradiation (−1.13x, p = 0.019).</p

Reduced expression of DNA repair-associated proteins in two-cancer patients.

<p>The relative expression levels in fibroblasts of 2C versus 1C patients are −1.36x (p = 0.017) for BRCA1, −1.27x (p = 0.011) for DDIT3, −1.16x (p = 0.021) for MSH6, −1.18x (p = 0.003) for TP53, −1.38x (p = 0.040) for RAD9A, and −1.37 (p = 0.009) for RAD51. Protein expression was measured by antibody microarrays (normalized by log10 transformation and z scores). Box plots show the distribution of z ratios in matched 2C vs. 1C patients. The median is represented by horizontal lines. The bottom of the box indicates the 25^th percentile, the top the 75^th percentile. The T bars extend from the boxes to at most 1.5 times the height of the box. Outliers are shown as open circles.</p

Western blot showing reduced RAD9A protein levels in a two-cancer patient.

<p>The gel on the left side shows Coomassie blue staining of nuclear and cytoplasmic protein extracts (30 µg each) from untreated fibroblasts of two-cancer patient 2C-7 and the matched one-cancer patient 1C-7. The corresponding gel on the right side is stained with anti-RAD9A antibody, which recognizes a 45 kDA nuclear protein. The calculated 2C/1C RAD9A protein ratio is 0.6.</p

Reduced mRNA expression of <i>RAD9A</i> in two-cancer patients.

<p><i>RAD9A</i> mRNA levels in untreated and irradiated (1 h, 4 h and 24 h after 1 Gy) fibroblasts of two-cancer patients, compared to matched one-cancer patients. mRNA was quantified by real-time RT PCR (normalized with the ΔΔCT method and two endogenous control genes). Box plots show the distribution of expression ratios in matched 2C vs. 1C patients. The median is represented by horizontal lines. The bottom of the box indicates the 25^th percentile, the top the 75^th percentile. The T bars extend from the boxes to at most 1.5 times the height of the box. Outliers are shown as open circles, extreme outliers as triangles. Two-cancer patients show reduced <i>RAD9A</i> mRNA levels without induction of DNA damage (−2.40x, p = 0.004) as well as at 1 h (−2.54x, p = 0.003), 4 h (−2.62x, p = 0.003), and 24 h (−2.54x, p = 0.003) after irradiation.</p

Constitutive expression differences of DNA repair-associated proteins in fibroblasts of 2C vs. 1C patients, measured by antibody microarrays.

<p>Constitutive expression differences of DNA repair-associated proteins in fibroblasts of 2C vs. 1C patients, measured by antibody microarrays.</p

Representative antibody microarray.

<p>Different amounts (approximately 1.5, 1.0 and 0.5 pg) of anti-RAD9A antibody (2 ng/µl) are spotted in triplicates onto nitrocellulose-coated slides and incubated with fluorescent-labeled nuclear protein extract of untreated fibroblasts from two-cancer patient 2C-7 and the matched one-cancer patient 1C-7, respectively. Anti-ACTB serves as positive and spotting buffer as negative control. The measured 2C/1C RAD9A protein ratio is 0.5.</p