Transcriptomic and proteomic study of cancer cell lines exposed to actinomycin D and nutlin-3a reveals numerous, novel candidates for p53-regulated genes.

Transcriptomic analyses have revealed hundreds of p53-regulated genes; however, these studies used a limited number of cell lines and p53-activating agents. Therefore, we searched for candidate p53-target genes by employing stress factors and cell lines never before used in a high-throughput search for p53-regulated genes. We performed RNA-Seq on A549 cells exposed to camptothecin, actinomycin D, nutlin-3a, as well as a combination of actinomycin D and nutlin-3a (A + N). The latter two substances synergise upon the activation of selected p53-target genes. A similar analysis was performed on other cell lines (U-2 OS, NCI-H460, A375) exposed to A + N. To identify proteins in cell lysates or those secreted into a medium of A549 cells in control conditions or treated with A + N, we employed mass spectrometry. The expression of selected genes strongly upregulated by A + N or camptothecin was examined by RT-PCR in p53-deficient cells and their controls. We found that p53 participates in the upregulation of: ACP5, APOL3, CDH3, CIBAR2, CRABP2, CTHRC1, CTSH, FAM13C, FBXO2, FRMD8, FRZB, GAST, ICOSLG, KANK3, KCNK6, KLRG2, MAFB, MR1, NDRG4, PTAFR, RETSAT, TMEM52, TNFRSF14, TRANK1, TYSND1, WFDC2, WFDC5, WNT4 genes. Twelve of these proteins were detected in the secretome and/or proteome of treated cells. Our data generated new hypotheses concerning the functioning of p53. Many genes activated by A + N or camptothecin are also activated by interferons, indicating a noticeable overlap between transcriptional programs of p53 and these antiviral cytokines. Moreover, several identified genes code for antagonists of WNT/ß-catenin signalling pathways, which suggests new connections between these two cancer-related signalling systems. One of these antagonists is DRAXIN. Previously, we found that its gene is activated by p53. In this study, using mass spectrometry and Western blotting, we detected expression of DRAXIN in a medium of A549 cells exposed to A + N. Thus, this protein functions not only in the development of the nervous system, but it may also have a new cancer-related function.

Common Variants in Osteopontin and CD44 Genes as Predictors of Treatment Outcome in Radiotherapy and Chemoradiotherapy for Non-Small Cell Lung Cancer.

Osteopontin (OPN)-CD44 signaling plays an important role in promoting tumor progression and metastasis. In cancer, OPN and CD44 overexpression is a marker of aggressive disease and poor prognosis, and correlates with therapy resistance. In this study, we aimed to evaluate the association of single nucleotide polymorphisms (SNPs) in the OPN and CD44 genes with clinical outcomes in 307 non-small cell lung cancer (NSCLC) patients treated with radiotherapy or chemoradiotherapy. The potential impact of the variants on plasma OPN levels was also investigated. Multivariate analysis showed that OPN rs11730582 CC carriers had a significantly increased risk of death (p = 0.029), while the CD44 rs187116 A allele correlated with a reduced risk of locoregional recurrence (p = 0.016) in the curative treatment subset. The rs11730582/rs187116 combination was associated with an elevated risk of metastasis in these patients (p = 0.016). Furthermore, the OPN rs1126772 G variant alone (p = 0.018) and in combination with rs11730582 CC (p = 7 × 10-5) was associated with poor overall survival (OS) in the squamous cell carcinoma subgroup. The rs11730582 CC, rs187116 GG, and rs1126772 G, as well as their respective combinations, were independent risk factors for unfavorable treatment outcomes. The impact of rs11730582-rs1126772 haplotypes on OS was also observed. These data suggest that OPN and CD44 germline variants may predict treatment effects in NSCLC.

DNA Double-Strand Break Response and Repair Gene Polymorphisms May Influence Therapy Results and Prognosis in Head and Neck Cancer Patients.

Radiotherapy and cisplatin-based chemotherapy belong to the main treatment modalities for head and neck squamous cell carcinoma (HNSCC) and induce cancer cell death by generating DNA damage, including the most severe double-strand breaks (DSBs). Alterations in DSB response and repair genes may affect individual DNA repair capacity and treatment sensitivity, contributing to the therapy resistance and poor prognosis often observed in HNSCC. In this study, we investigated the association of a panel of single-nucleotide polymorphisms (SNPs) in 20 DSB signaling and repair genes with therapy results and prognosis in 505 HNSCC patients treated non-surgically with DNA damage-inducing therapies. In the multivariate analysis, there were a total of 14 variants associated with overall, locoregional recurrence-free or metastasis-free survival. Moreover, we identified 10 of these SNPs as independent predictors of therapy failure and unfavorable prognosis in the whole group or in two treatment subgroups. These were MRE11 rs2155209, XRCC5 rs828907, RAD51 rs1801321, rs12593359, LIG4 rs1805388, CHEK1 rs558351, TP53 rs1042522, ATM rs1801516, XRCC6 rs2267437 and NBN rs2735383. Only CHEK1 rs558351 remained statistically significant after correcting for multiple testing. These results suggest that specific germline variants related to DSB response and repair may be potential genetic modifiers of therapy effects and disease progression in HNSCC treated with radiotherapy and cisplatin-based chemoradiation.

Germline Variants in Angiogenesis-Related Genes Contribute to Clinical Outcome in Head and Neck Squamous Cell Carcinoma.

Fibroblast growth factor (FGF)/FGF receptor (FGFR), and platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR) systems, as well as some matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), are involved in various steps of angiogenesis. Data indicate that common germline variations in angiogenesis-regulating genes may modulate therapy results and cancer progression. However, whether these variants affect clinical outcome in head and neck squamous cell carcinoma (HNSCC) is unclear. Hence, we assessed the relationship between FGF/FGFR, PDGF/PDGFR, MMP, and TIMP genetic variants and treatment outcomes in HNSCC patients receiving radiotherapy (RT) alone or combined with cisplatin-based chemotherapy. In multivariate analysis, FGF2 rs1048201 CC homozygotes showed a higher risk of death (p = 0.039), while PDGFRA rs2228230 T was strongly associated with an increased risk of locoregional relapse (HR 2.49, p = 0.001) in the combination treatment subgroup. In the RT alone subset, MMP2 rs243865 TT carriers had a higher risk of locoregional recurrence (HR 2.92, p = 0.019), whereas PDGFRB rs246395 CC homozygotes were at increased risk of metastasis (HR 3.06, p = 0.041). The MMP2 rs7201 C and TIMP2 rs7501477 T were associated with a risk of locoregional failure in the entire cohort (p = 0.032 and 0.045, respectively). Furthermore, rs1048201, rs2228230, rs246395, rs243865, rs7201, and rs7201/rs7501477 were independent indicators of an unfavorable outcome. This study demonstrates that the FGF2, PDGFRA, PDGFRB, MMP2, and TIMP2 variants may contribute to treatment failure and poor prognosis in HNSCC.

Transcriptome Analysis of Cells Exposed to Actinomycin D and Nutlin-3a Reveals New Candidate p53-Target Genes and Indicates That CHIR-98014 Is an Important Inhibitor of p53 Activity.

Co-treatment with actinomycin D and nutlin-3a (A + N) strongly activates p53. Previously we reported that CHIR-98014 (GSK-3 kinase inhibitor), acting in cells exposed to A + N, prevents activation of TREM2-an innate immunity and p53-regulated gene associated with Alzheimer's disease. In order to find novel candidate p53-target genes and genes regulated by CHIR-98014, we performed RNA-Seq of control A549 cells and the cells exposed to A + N, A + N with CHIR-98014 or to CHIR-98014. We validated the data for selected genes using RT-PCR and/or Western blotting. Using CRISPR/Cas9 technology we generated p53-deficient cells. These tools enabled us to identify dozens of candidate p53-regulated genes. We confirmed that p53 participates in upregulation of BLNK, APOE and IRF1. BLNK assists in activation of immune cells, APOE codes for apolipoprotein associated with Alzheimer's disease and IRF1 is activated by interferon gamma and regulates expression of antiviral genes. CHIR-98014 prevented or inhibited the upregulation of a fraction of genes stimulated by A + N. Downregulation of GSK-3 did not mimic the activity of CHIR-98014. Our data generate the hypothesis, that an unidentified kinase inhibited by CHIR-98014, participates in modification of p53 and enables it to activate a subset of its target genes, e.g., the ones associated with innate immunity.

Polymorphisms in EGFR Gene Predict Clinical Outcome in Unresectable Non-Small Cell Lung Cancer Treated with Radiotherapy and Platinum-Based Chemoradiotherapy.

For non-small cell lung cancer (NSCLC), radiotherapy (RT) and platinum-based chemotherapy (CHT) are among the main treatment options. On the other hand, radioresistance and cytotoxic drug resistance are common causes of failure. The epidermal growth factor receptor (EGFR) plays an important role in radioresponse and therapy resistance. We hypothesized that single nucleotide polymorphisms (SNPs) in the EGFR gene might affect individual sensitivity to these treatments, and thus, therapy outcome and prognosis. The association between functional EGFR SNPs and overall (OS), locoregional recurrence-free (LFRS), and metastasis-free (MFS) survival was examined in 436 patients with unresectable NSCLC receiving RT and platinum-based CHTRT. In a multivariate analysis, the rs712830 CC homozygotes showed reduced OS in the whole group (p = 0.039) and in the curative treatment subset (p = 0.047). The rs712829 TT genotype was strongly associated with decreased LRFS (p = 0.006), and the T-C haplotype was a risk factor for locoregional recurrence in our patients (p = 0.003). The rs2227983 GG alone and in combination with rs712829 T was an indicator of unfavorable LRFS (p = 0.028 and 0.002, respectively). Moreover, significant independent effects of these SNPs on OS, LRFS, and MFS were observed. Our results demonstrate that inherited EGFR gene variants may predict clinical outcomes in NSCLC treated with DNA damage-inducing therapy.

Association of Genetic Variants in ANGPT/TEK and VEGF/VEGFR with Progression and Survival in Head and Neck Squamous Cell Carcinoma Treated with Radiotherapy or Radiochemotherapy.

Angiogenesis is essential for growth, progression, and metastasis of solid tumors. Vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) and angiopoietin (ANGPT)/ tyrosine kinase endothelial (TEK) signaling plays an important role in regulating angiogenesis. Very little is known about the effects of single-nucleotide polymorphisms (SNPs) in angiogenesis-related genes on treatment outcome in head and neck squamous cell carcinoma (HNSCC). Therefore, we evaluated the association between SNPs in ANGPT1, ANGPT2, TEK, VEGF, VEGFR1, and VEGFR2 genes and five clinical endpoints in 422 HNSCC patients receiving radiotherapy alone or combined with chemotherapy. Multivariate analysis showed an association of ANGPT2 rs3739391, rs3020221 and TEK rs639225 with overall survival, and VEGF rs2010963 with overall and metastasis-free survival. VEGFR2 rs1870377 and VEGF rs699947 affected local recurrence-free survival in all patients. In the combination treatment subgroup, rs699947 predicted local, nodal, and loco-regional recurrence-free survival, whereas VEGFR2 rs2071559 showed an association with nodal recurrence-free survival. However, these associations were not statistically significant after multiple testing correction. Moreover, a strong cumulative effect of SNPs was observed that survived this adjustment. These SNPs and their combinations were independent risk factors for specific endpoints. Our data suggest that certain germline variants in ANGPT2/TEK and VEGF/VEGFR2 axes may have predictive and prognostic potential in HNSCC treated with radiation or chemoradiation.

Synergistic activation of p53 by actinomycin D and nutlin-3a is associated with the upregulation of crucial regulators and effectors of innate immunity.

Actinomycin D and nutlin-3a (A + N) activate p53, partly through induction of phosphorylation on Ser392. The death of A549 cells induced by A + N morphologically resembles inflammation-inducing pyroptosis - cell destruction triggered by activated caspase-1. The treatment with A + N (or camptothecin) strongly upregulated caspase-1 and its two activators: IFI16 and NLRP1, however, caspase-1 activation was not detected. A549 cells may have been primed for pyroptosis, with the absence of a crucial trigger. The investigation of additional innate immunity elements revealed that A + N (or camptothecin) stimulated the expression of NLRX1, STING (stimulator of interferon genes) and two antiviral proteins, IFIT1 and IFIT3. IFI16 and caspase-1 are coded by p53-regulated genes which led us to investigate regulation of NLRP1, NLRX1, STING, IFIT1 and IFIT3 in p53-dependent mode. The upregulation of NLRP1, NLRX1 and STING was attenuated in p53 knockdown cells. The upsurge of the examined genes, and activation of p53, was inhibited by C16, an inhibitor of PKR kinase. PKR was tested due to its ability to phosphorylate p53 on Ser392. Surprisingly, C16 was active even in PKR knockdown cells. The ability of C16 to prevent activation of p53 and expression of innate immunity genes may be the source of its strong anti-inflammatory action. Moreover, cells exposed to A + N can influence neighboring cells in paracrine fashion, for instance, they shed ectodomain of COL17A1 protein and induce, in p53-dependent mode, the expression of gene for interleukin-7. Further, the activation of p53 also spurred the expression of SOCS1, an inhibitor of interferon triggered STAT1-dependent signaling. We conclude that, stimulation of p53 primes cells for the production of interferons (through upregulation of STING), and may activate negative-feedback within this signaling system by enhancing the production of SOCS1.

PIM2 survival kinase is upregulated in a p53-dependent manner in cells treated with camptothecin or co-treated with actinomycin D and nutlin-3a.

The p53 protein is an inducer of apoptosis, acting as a transcriptional regulator of apoptotic genes. In a previous study, we found that actinomycin D and nutlin-3a (A + N) synergistically activate p53. To better understand the molecular consequences of this synergism, we incubated arrays of antibodies against apoptotic proteins with extracts of A549 cells in which p53 had been activated. We found that strong activation of p53, marked by serine 46 and 392 phosphorylation, was associated with inactivating phosphorylation of proapoptotic BAD protein on serine 136. Investigation of the source of this phosphorylation revealed that activation of p53 was associated with accumulation of PIM2, a survival kinase. The accumulation of PIM2 following treatment with A + N was suppressed in p53-knockdown cells. Others discovered that PIM2 was activated by cooperatively acting p53 molecules. Our results are consistent with this finding. Moreover, we found that in A549 cells, the treatment with A + N stimulated in p53-dependent fashion the expression of other high cooperativity p53 target genes, DRAXIN and H19. Activation of antiapoptotic H19 can mechanistically explain relatively low rate of apoptosis of A549 cells exposed to A + N. We conclude that PIM2, DRAXIN and H19 are efficiently stimulated by strongly activated p53 molecules, probably acting cooperatively.

The Alzheimer's disease-associated TREM2 gene is regulated by p53 tumor suppressor protein.

TREM2 mutations evoke neurodegenerative disorders, and recently genetic variants of this gene were correlated to increased risk of Alzheimer's disease. The signaling cascade originating from the TREM2 membrane receptor includes its binding partner TYROBP, BLNK adapter protein, and SYK kinase, which can be activated by p53. Moreover, in silico identification of a putative p53 response element (RE) at the TREM2 promoter led us to hypothesize that TREM2 and other pathway elements may be regulated in p53-dependent manner. To stimulate p53 in synergistic fashion, we exposed A549 lung cancer cells to actinomycin D and nutlin-3a (A + N). In these cells, exposure to A + N triggered expression of TREM2, TYROBP, SYK and BLNK in p53-dependent manner. TREM2 was also activated by A + N in U-2 OS osteosarcoma and A375 melanoma cell lines. Interestingly, nutlin-3a, a specific activator of p53, acting alone stimulated TREM2 in U-2 OS cells. Using in vitro mutagenesis, chromatin immunoprecipitation, and luciferase reporter assays, we confirmed the presence of the p53 RE in TREM2 promoter. Furthermore, activation of TREM2 and TYROBP by p53 was strongly inhibited by CHIR-98014, a potent and specific inhibitor of glycogen synthase kinase-3 (GSK-3). We conclude that TREM2 is a direct p53-target gene, and that activation of TREM2 by A + N or nutlin-3a may be critically dependent on GSK-3 function.

Identification of serum proteome signatures of locally advanced and metastatic gastric cancer: a pilot study.

BACKGROUND: The gastric cancer is one of the most common and mortal cancer worldwide. The initial asymptomatic development and further nonspecific symptoms result in diagnosis at the advanced stage with poor prognosis. Yet, no clinically useful biomarkers are available for this malignancy, and invasive gastrointestinal endoscopy remains the only reliable option at the moment. Hence, there is a need for discovery of clinically useful noninvasive diagnostic and/or prognostic tool as an alternative (or complement) for current diagnostic tools. Here we aimed to search for serum proteins characteristic for local and invasive gastric cancer. METHODS: Pre-treatment blood samples were collected from patients with diagnosed gastric adenocarcinoma at the different stage of disease: 35 patients with locally advanced cancer and 18 patients with metastatic cancer; 50 healthy donors were also included as a control group. The low-molecular-weight fraction of serum proteome (i.e., endogenous peptidome) was profiled by the MALDI-ToF mass spectrometry, and the whole proteome components were identified and quantified by the LC-MS/MS shotgun approach. RESULTS: Multicomponent peptidome signatures were revealed that allowed good discrimination between healthy controls and cancer patients, as well as between patients with locally advanced and metastatic cancer. Moreover, a LC-MS/MS approach revealed 49 serum proteins with different abundances between healthy donors and cancer patients (predominantly proteins associated with inflammation and acute phase response). Furthermore, 19 serum proteins with different abundances between patients with locally advanced and metastatic cancer were identified (including proteins associated with cytokine/chemokine response and metabolism of nucleic acids). However, neither peptidome profiling nor shotgun proteomics approach allowed detecting serum components discriminating between two subgroups of patients with local disease who either developed or did not develop metastases during follow-up. CONCLUSIONS: The molecular differences between locally advanced and metastatic gastric cancer, as well as more obvious differences between healthy individuals and cancer patients, have marked reflection at the level of serum proteome. However, we have no evidence that features of pre-treatment serum proteome could predict a risk of cancer dissemination in patients treated due to local disease. Nevertheless, presented data confirmed potential applicability of a serum proteome signature-based biomarker in diagnostics of gastric cancer.

Actinomycin D and nutlin-3a synergistically promote phosphorylation of p53 on serine 46 in cancer cell lines of different origin.

The p53 tumor suppressor protein is a transcription factor activated by phosphorylation of its N-terminus. MDM2, encoded by a p53-activated gene, acts as a negative-feedback regulator of p53 by promoting p53 degradation. Moreover, MDM2 inhibits p53 by binding to and concealing its N-terminal transcription-activating domain. p53 can be activated by nutlin-3a, a molecule designed to bind MDM2 and prevent its interaction with p53. Actinomycin D promotes phosphorylation and accumulation of p53 via a mechanism that involves high expression of MDM2. We hypothesized that co-treatment of cells with actinomycin D and nutlin-3a would lead to synergistic activation of p53 by stimulating kinases and preventing accumulated MDM2 from binding to p53. Indeed, co-treatment of various cell lines with actinomycin D and nutlin-3a resulted in a synergistic increase of p53 phosphorylation on serine 46. We focused on this residue because it is a marker of the highest level of p53 activation. Co-treatment was associated with conspicuous decrease in a marker of mTOR activity in NCI-H28 cells and very strong activation of p53 targets, including CDKN1A and PML, in A549 cells. Other p53 target genes (SESN1, SESN2, TIGAR, DRAM1) were also efficiently upregulated; however, a marker of apoptosis (active caspase-3) appeared only in some cancer cell lines (e.g., A375 and other cell lines derived from melanoma) indicating that phosphorylation of p53 on serine 46 is not straightforwardly associated with induction of apoptosis. Moreover, our data suggest that melanoma may be a suitable target for drug combination used in this study.

The VEGFR2, COX-2 and MMP-2 polymorphisms are associated with clinical outcome of patients with inoperable non-small cell lung cancer.

Certain common inherited variations in genes involved in tumor angiogenesis, progression and metastasis may contribute to cancer therapy outcome and prognosis by altering the gene expression and protein activity. In this report, we examined the effect of functional polymorphisms in MMP-1, MMP-2, MMP-3, VEGF, VEGFR2, FGFR4 and COX-2 genes on overall (OS) and progression-free survival (PFS) of 350 Caucasian patients with inoperable non-small cell lung cancer (NSCLC). The results of multivariate analysis indicated that VEGFR2 -906C and COX-2 -1195G alleles were strongly associated with poor OS and PFS (p = 0.002 and 0.015, respectively, for OS; p = 0.009 and 0.015, respectively, for PFS), while MMP-2 -1306 T allele carriers had significantly reduced PFS (p = 0.010). Moreover, an increased risk of death and progression was significantly associated with the number of adverse alleles for VEGFR2/COX-2 (p = 0.0005 for OS and 0.0006 for PFS in >1 adverse allele carriers) and VEGFR2/COX-2/MMP-2 combinations (p = 0.0003 for OS and 0.0001 for PFS in patients with >2 adverse alleles). Finally, VEGFR2 TC/CC, COX-2 AG/GG and MMP-2 CT/TT genotypes as well as "at risk" allele combinations were identified as independent predictors of unfavorable OS and PFS in the group. In conclusion, the data suggest that selected VEGFR2, COX-2 and MMP-2 polymorphisms may be potential prognostic markers in unresectable NSCLC treated with radiotherapy with or without chemotherapy, although further validation studies are warranted to confirm our observations.

The SIPA1 -313A>G polymorphism is associated with prognosis in inoperable non-small cell lung cancer.

Polymorphism in signal-induced proliferation-associated 1 (SIPA1) gene may contribute to the development of metastasis in human cancers. In this preliminary study, we examined the association of the SIPA1 -313A>G (rs931127) polymorphism with overall survival (OS) and progression-free survival (PFS) in 351 inoperable patients with non-small cell lung cancer (NSCLC) treated with radiotherapy or radiochemotherapy (curative or palliative). The GG homozygotes had significantly shorter PFS under codominant and recessive models in all patients (hazard ratio (HR) 1.47, p = 0.035, and HR 1.47, p = 0.022, respectively) and in advanced stage subgroup (HR 1.49, p = 0.037, and HR 1.48, p = 0.023, respectively). The GG genotype was also associated with reduced OS and PFS (codominant model: HR 2.41, p = 0.020, and HR 2.34, p = 0.020, respectively; recessive model: HR 2.16, p = 0.026, and HR 2.18, p = 0.022, respectively) in radiotherapy alone subgroup. Moreover, the SIPA1 -313GG was identified as an independent adverse prognostic factor for PFS in the cohort. Our results indicate, for the first time, that the SIPA1 -313A>G may have a prognostic role in unresected NSCLC making it a potential predictor of poor survival due to earlier progression.

Identification of serum proteome components associated with progression of non-small cell lung cancer.

The aim of the present study was to perform comparative analysis of serum from patients with different stages of non-small cell lung cancer (NSCLC) using the three complementary proteomic approaches to identify proteome components associated with the progression of cancer. Serum samples were collected before any treatment from 200 patients with NSCLC, including 103 early stage, 64 locally advanced and 33 metastatic cancer samples, and from 200 donors without malignancy. The low-molecular-weight fraction of serum proteome was MALDI-profiled in all samples. Serum proteins were characterized using 2D-PAGE and LC-MS/MS approaches in a representative group of 30 donors. Several significant differences were detected between serum samples collected from patients with early stage cancer and patients with locally advanced cancer, as well as between patients with metastatic cancer and patients with local disease. Of note, serum components discriminating samples from early stage cancer and healthy persons were also detected. In general, about 70 differentiating serum proteins were identified, including inflammatory and acute phase proteins already reported to be associated with the progression of lung cancer (serum amyloid A or haptoglobin). Several differentiating proteins, including apolipoprotein H or apolipoprotein A1, were not previously associated with NSCLC. No significant differences in patterns of serum proteome components were detected between patients with adenocarcinoma and squamous cell carcinoma. In conclusion, we identified the biomarker candidates with potential importance for molecular proteomic staging of NSCLC. Additionally, several serum proteome components revealed their potential applicability in early detection of the lung cancer.

Radiotherapy-related changes in serum proteome patterns of head and neck cancer patients; the effect of low and medium doses of radiation delivered to large volumes of normal tissue.

BACKGROUND: Conformal intensity-modulated radiation therapy (IMRT) involves irradiation of large volume of normal tissue with low and medium doses, biological relevance of which is not clear yet. Serum proteome features were used here to study the dose-volume effects in patients irradiated with IMRT due to head and neck cancer. METHODS: Blood samples were collected before and during RT, and also about one month and one year after the end of RT in a group of 72 patients who received definitive treatment. Serum proteome profiles were analyzed using MALDI-ToF mass spectrometry in 800-14,000 Da range. RESULTS: Major changes in serum proteome profiles were observed between pre-treatment samples and samples collected one month after RT. Radiation-related changes in serum proteome features were affected by low-to-medium doses delivered to a large fraction of body mass. Proteome changes were associated with intensity of acute radiation toxicity, indicating collectively that RT-related features of serum proteome reflected general response of patient's organism to irradiation. However, short-term dose-related changes in serum proteome features were not associated significantly with the long-term efficacy of the treatment. CONCLUSIONS: The effects of low and medium doses of radiation have been documented at the level of serum proteome, which is a reflection of the patient's whole body response.

The influence of XPD, APE1, XRCC1, and NBS1 polymorphic variants on DNA repair in cells exposed to X-rays.

Polymorphism of genes coding for proteins which participate in DNA repair may predispose to or protect against development of cancer. Here we studied how common polymorphisms of the genes XPD (Asp312Asn and Lys751Gln), APE1 (Asp148Glu), XRCC1 (Arg399Gln), and NBS1 (Gln185Glu) influence DNA repair and other responses after X-irradiation of lymphocytes from colon carcinoma patients. Genotypes with polymorphic Asp148Glu APE1 and Asp312Asn XPD showed a significantly higher level of DNA incisions immediately after irradiation (p=0.049 and p=0.047 respectively) and Asp312Asn XPD showed a significantly increased capacity to repair of DNA strand breaks as measured 180min after irradiation by comet assays (p=0.004). In contrast, it was the wild type XRCC1 genotype which was associated with a lower level of DNA breaks after irradiation (p=0.014, at 180min after irradiation) and polymorphism of NBS1 did not correlate with any changes in DNA breaks or repair capacity. To confirm the influence of XPD polymorphism on repair, we established stably-transfected HCT116 (colon carcinoma) cells which over-expressed the wild-type or variant XPD protein. Cells over-expressing Asp312Asn XPD showed a higher level of DNA breaks shortly after irradiation and more efficient repair than cells over-expressing the wild-type gene XPD312Asp, and an earlier inhibition of cell cycle transit but faster recovery from this inhibition. Polymorphisms in DNA repair genes therefore influence not only DNA repair capacity but also cell proliferation, and may serve as markers of individual repair capacity and susceptibility to environmental and occupational carcinogens.