Image-Based Network Analysis of DNp73 Expression by Immunohistochemistry in Rectal Cancer Patients.

Background: Rectal cancer is a disease characterized with tumor heterogeneity. The combination of surgery, radiotherapy, and chemotherapy can reduce the risk of local recurrence. However, there is a significant difference in the response to radiotherapy among rectal cancer patients even they have the same tumor stage. Despite rapid advances in knowledge of cellular functions affecting radiosensitivity, there is still a lack of predictive factors for local recurrence and normal tissue damage. The tumor protein DNp73 is thought as a biomarker in colorectal cancer, but its clinical significance is still not sufficiently investigated, mainly due to the limitation of human-based pathology analysis. In this study, we investigated the predictive value of DNp73 in patients with rectal adenocarcinoma using image-based network analysis. Methods: The fuzzy weighted recurrence network of time series was extended to handle multi-channel image data, and applied to the analysis of immunohistochemistry images of DNp73 expression obtained from a cohort of 25 rectal cancer patients who underwent radiotherapy before surgery. Two mathematical weighted network properties, which are the clustering coefficient and characteristic path length, were computed for the image-based networks of the primary tumor (obtained after operation) and biopsy (obtained before operation) of each cancer patient. Results: The ratios of two weighted recurrence network properties of the primary tumors to biopsies reveal the correlation of DNp73 expression and long survival time, and discover the non-effective radiotherapy to a cohort of rectal cancer patients who had short survival time. Conclusion: Our work contributes to the elucidation of the predictive value of DNp73 expression in rectal cancer patients who were given preoperative radiotherapy. Mathematical properties of fuzzy weighted recurrence networks of immunohistochemistry images are not only able to show the predictive factor of DNp73 expression in the patients, but also reveal the identification of non-effective application of radiotherapy to those who had poor overall survival outcome.

Effects of Low-Dose Bisphenol A on DNA Damage and Proliferation of Breast Cells: The Role of c-Myc.

BACKGROUND: Humans are exposed to low-dose bisphenol A (BPA) through plastic consumer products and dental sealants containing BPA. Although a number of studies have investigated the mammary gland effects after high-dose BPA exposure, the study findings differ. Furthermore, there has been a lack of mechanistic studies. OBJECTIVE: The objective of this study was to investigate the effect and the mechanism of low-dose BPA in mammary gland cells. METHODS: We evaluated DNA damage following BPA exposure using the comet assay and immunofluorescence staining, and used cell counting and three-dimensional cultures to evaluate effects on proliferation. We examined the expressions of markers of DNA damage and cell-cycle regulators by immunoblotting and performed siRNA-mediated gene silencing to determine the role of c-Myc in regulating BPA's effects. RESULTS: Low-dose BPA significantly promoted DNA damage, up-regulated c-Myc and other cell-cycle regulatory proteins, and induced proliferation in parallel in estrogen receptor-α (ERα)-negative mammary cells. Silencing c-Myc diminished these BPA-induced cellular events, suggesting that c-Myc is essential for regulating effects of BPA on DNA damage and proliferation in mammary cells. CONCLUSIONS: Low-dose BPA exerted c-Myc-dependent genotoxic and mitogenic effects on ERα-negative mammary cells. These findings provide significant evidence of adverse effects of low-dose BPA on mammary cells.

Effects of ΔNp73ß on cisplatin treatment in colon cancer cells.

p73 can activate transcription of p53-responsive genes, thereby inhibiting cell growth. An alternative promoter in the TP73 gene gives rise to an N-terminally truncated isoform of p73, ΔNp73, which lacks the transactivation domain of the full length TAp73 protein. TAp73 is considered pro-apoptotic, and ΔNp73 anti-apoptotic. In this study, we overexpressed ΔNp73ß in p53 wild type and p53 mutant colon cancer cell lines and further exposed the cells to cancer therapeutic drug cisplatin. The results showed that cisplatin decreased the protein expression levels of ΔNp73ß in a dose-dependent manner, and both TAp73 and p53 were upregulated after cisplatin treatment. Further, clonogenic potential and cell viability were decreased, and apoptotic cells increased, in p53 mutant and in p53 wild type cells. Cellular viability was significantly higher in ΔNp73ß-cells than mock-transfected cells. However, ΔNp73ß overexpression did not affect the cellular susceptibility to cisplatin. In conclusion, the overexpression of ΔNp73ß increases viability in p53 wild type and p53 mutant colon cancer cells, and cisplatin induces the degradation of ΔNp73ß in a dose-dependent manner.

Significance of an exon 2 G4C14-to-A4T14 polymorphism in the p73 gene on survival in rectal cancer patients with or without preoperative radiotherapy.

BACKGROUND AND PURPOSE: An exon 2 G4C14-->A4T14 polymorphism in the p73 gene was shown to be related to survival in several types of cancers, including colorectal cancer. The purpose was to investigate if this polymorphism was related to survival in rectal cancer patients with or without preoperative radiotherapy. MATERIALS AND METHODS: DNA extracted from tissue of 138 rectal cancer patients that received preoperative radiotherapy or had surgery alone was typed for the polymorphism by PCR using confronting two-pair primers. RESULTS: Among patients, 69% had GC/GC genotype, 27% had GC/AT and 4% had AT/AT. In the radiotherapy group, patients carrying the AT (GC/AT+AT/AT) allele had stronger expression of p53 (p=0.001) and survivin protein (p=0.03) than those carrying the GC/GC genotype. Further, among patients receiving preoperative radiotherapy the GC/GC genotype tended to be related to better survival (p=0.20). Patients with GC/GC genotype, along with negative p53 and weak survivin expression showed better survival than the other patients (p=0.03), even after adjusting for TNM stage and tumor differentiation (p=0.01, RR, 7.63, 95% CI, 1.50-38.74). In the non-radiotherapy group, the polymorphism was not related to survival (p=0.74). CONCLUSIONS: Results suggest that the p73 G4C14-->A4T14 polymorphism could be one factor influencing outcome of preoperative radiotherapy in rectal cancer patients.

Protein expression following gamma-irradiation relevant to growth arrest and apoptosis in colon cancer cells.

PURPOSE: To study expression of proteins previously connected to radiotherapy response in rectal cancer patients, namely, p53, TAp73, DeltaNp73, survivin and PRL-3, after irradiation in colon cancer cells to gain standing ground for further studies of pathways and mechanisms. METHODS: Three colon cancer cell lines (KM12C, KM12SM and KM12L4a) with one origin were radiated with gamma-radiation. Radiosensitivity was determined with cell cycle, survival fraction at 5 Gy (SF5) and apoptosis analysis and protein expression by Western blot. RESULTS: Following irradiation, KM12C showed no cell cycle arrest, and low SF5 and apoptosis, whilst KM12L4a showed high SF5 and apoptosis. KM12SM had moderate radiosensitivity. After irradiation, the anti-apoptotic DeltaNp73 and mitosis-factor PRL-3 increased in KM12C and the radioresistance factor survivin increased in KM12L4a. CONCLUSIONS: The cell lines seem to have evolved different protein patterns regarding the studied proteins and partly therefore developed different resistance mechanisms, less apoptosis for KM12C and continued proliferation for KM12L4a, after gamma-irradiation.

Association of NFKBIA polymorphism with colorectal cancer risk and prognosis in Swedish and Chinese populations.

OBJECTIVE: The inhibitory proteins, IkappaBs, regulate the activity of nuclear factor kappa-beta (NF-kappaB), which is implicated in tumorigenesis by regulating expression of a variety of genes involved in cellular transformation, proliferation, invasion, angiogenesis and metastasis. Variants in the genes encoding IkappaBs may be involved in cancer development through the activation of NF-kappaB. The objective of this study was to investigate the susceptibility of an A to G variation (rs696) in the 3' UTR of NFKBIA (encoding IkappaBalpha) to colorectal cancer (CRC) and the association of this polymorphism with clinicopathologic variables in CRC patients. MATERIAL AND METHODS: A case-control study was carried out on a Swedish (155 CRCs, 438 controls) and a Chinese population (199 CRCs, 577 controls). The genotype of NFKBIA was determined by PCR-restriction fragment length polymorphism. RESULTS: The frequency of the AG genotype was increased in the Chinese patients >or=50 years of age compared with the Chinese controls (odds ratio (OR)=3.06, 95% confidence interval (CI)=1.55-6.02, p=0.001), even when adjusted for age (OR=3.20, 95% CI=1.61-6.38, p=0.001). The GG genotype of NFKBIA was related to a poorer survival rate in the Swedish patients, independent of gender, age, tumour location, Dukes' stage and differentiation (hazard ratio = 3.10, 95% Cl=1.28-7.60, p=0.01). CONCLUSIONS: Chinese individuals >or=50 years of age carrying the AG genotype of NFKBIA may be at an increased risk of developing CRC, and the GG genotype of NFKBIA may be considered as a prognostic factor for Swedish CRC patients.

Expression of the p73 protein in rectal cancers with or without preoperative radiotherapy.

PURPOSE: To investigate p73 expression in normal mucosa, primary tumor, and metastasis in relation to radiotherapy (RT) response and clinicopathologic/biologic variables in rectal cancers. METHODS AND MATERIALS: p73 was immunohistochemically examined on biopsies (unirradiated, n=102), distant (from the large bowel, n=82), and adjacent (adjacent to primary tumor, n=89) normal mucosa samples, primary tumors (n=131), and lymph node metastasis (n=32) from rectal cancer patients participating in a clinical trial of preoperative RT. Seventy-four patients received surgery alone and 57 received additional RT. RESULTS: Cytoplasmic p73 was increased in the primary tumor compared with the distant or adjacent mucosa (p

Polymorphism of the p73 gene in relation to colorectal cancer risk and survival.

The results regarding a GC/AT polymorphism in the p73 gene in relation to cancer risk are inconsistent, and the significance of loss of heterozygosity (LOH) of the gene is unclear. In the present study, we investigated whether this polymorphism was related to the risk of colorectal cancer, and whether there were relationships between the polymorphism and LOH, protein expression or clinicopathological variables. 179 patients with colorectal cancer and 260 healthy controls were genotyped for the polymorphism by PCR-restriction fragment length polymorphism (RFLP). Fifty informative cases were examined for LOH in tumours. Immunohistochemistry was performed on distant (n = 42) and adjacent normal mucosa (n = 33), primary tumour (n = 6 9) and lymph node metastasis (n = 12). The frequencies of the genotypes were 63% for wild-type (GC/GC), 30% for heterozygotes (GC/AT) and 7% for variants (AT/AT) in patients, and 62, 36 and 2% in controls, respectively. The frequencies of the genotypes in the patients and controls were significantly different (P = 0.02). The patients carrying the AT allele had a better prognosis than those with the GC/GC genotype (OR = 0.42, 95% CI = 1.15-5.02, P = 0.02). No LOH was observed at the p73 locus. Expression of p73 protein was increased from normal mucosa to primary tumours (P = 0.02), but was not significantly changed between primary tumours and metastases (P = 1.0). In conclusion, the AT/AT homozygotes may have a greater risk of developing colorectal cancer, while the patients who carried the AT allele had a better prognosis.