Stressing out-carp edema virus induces stress and modulates immune response in common carp.

Introduction: Carp edema virus (CEV) is a fish poxvirus that primarily infects the gills of common carp. CEV causes koi sleepy disease (KSD), which is highly contagious and can result in mortality of up to 100%. Methods: In the present study, we analyzed the stress and immune responses during KSD in two strains of common carp with different resistance to CEV: susceptible koi and resistant Amur sazan. Experiments were performed at two temperatures: 12°C and 18°C. In the case of koi carp, we also analyzed the effect of supplementation of 0.6% NaCl into tank water, which prevents mortality of the CEV-infected fish (salt rescue model). Results: We found that CEV-infected koi kept at 18°C had the highest viral load, which correlated with the most severe histopathological changes in the gills. CEV infection resulted in the activation of stress response reflected by the upregulated expression of genes involved in stress response in the stress axis organs and increased levels of cortisol and glucose in the blood plasma. These changes were the most pronounced in CEV-infected koi kept at 18°C. At both temperatures, the activation of antiviral immune response was observed in koi kept under freshwater and NaCl conditions upon CEV infection. Interestingly, a clear downregulation of the expression of adaptive immune genes was observed in CEV-infected koi kept under freshwater at 18°C. Conclusion: CEV induces a stress response and modulates adaptive immune response in koi, and this is correlated with the level of viral load and disease development.

RAD51 and XRCC3 Polymorphisms Are Associated with Increased Risk of Prostate Cancer.

Genetic polymorphisms in DNA repair genes may affect DNA repair efficiency and may contribute to the risk of developing cancer. The aim of our study was to investigate single nucleotide polymorphisms (SNPs) in RAD51 (rs2619679, rs2928140, and rs5030789) and XRCC3 (rs1799796) involved in DNA double-strand break repair and their relationship to prostate cancer. The study group included 99 men diagnosed with prostate cancer and 205 cancer-free controls. SNP genotyping was performed using the PCR-RFLP method. A significant association was detected between RAD51 rs5030789 polymorphism and XRCC3 rs1799796 polymorphism and an increased risk of prostate cancer. Our results indicate that RAD51 and XRCC3 polymorphism may contribute to prostate cancer.

DNA methylation and histone modifications as epigenetic regulation in prostate cancer (Review).

Prostate cancer is the second most commonly diagnosed cancer in men in Poland after lung cancer and the third leading cause of cancer-related mortality after lung and colon cancer. The etiology of most cases of prostate cancer are not fully known, and therefore it is essential to search for the molecular basis of prostate cancer and markers for the early diagnosis of this type of cancer. Epigenetics deals with changes in gene expression that are not determined by changes in the DNA sequence. Epigenetic changes refer to changes in the structure of DNA, which are the result of DNA modification after replication and/or post-translational modification of proteins associated with DNA. In contrast to mutations, epigenetic changes are reversible and occur very rapidly. The major epigenetic mechanisms include DNA methylation, modification of histone proteins, chemical modification and chromatin remodeling changes in gene expression caused by microRNAs (miRNAs). Epigenetic changes play an important role in malignant transformation and can be specific to types of cancers including prostate cancer.

The c.*229C > T gene polymorphism in 3'UTR region of the topoisomerase IIß binding protein 1 gene and LOH in BRCA1/2 regions and their effect on the risk and progression of human laryngeal carcinoma.

Topoisomerase IIß binding protein 1 (TopBP1), a multiple-BRCT-domain, protein plays crucial roles in chromosome replication, DNA damage repair, apoptosis, and cell cycle checkpoint signalling. The aim of this study was to identify five SNPs at loci potentially located in the 3'UTR region of the TopBP1 gene (rs185903567, rs116645643, rs115160714, rs116195487, rs112843513), their relationship with the risk of squamous cell laryngeal cancer (SCLC), tumor invasiveness, and prognosis. Genotyping was performed in 323 genetically unrelated individuals with SCLC and 418 randomly selected healthy volunteers. Allele-specific TopBP1 mRNA and protein expressions were determined by using real-time PCR and Western blotting techniques, respectively. LOH in BRCA1/BRCA2 was determined by using microsatellite markers. Compared to homozygous common allele carriers, heterozygosity for the T variant was associated with increased risk of SCLC (adjusted odds ratio [OR] = 9.83, 95 % confidence interval [CI]: 3.12-22.16, p dominant < 0.0001). The presence of risk allele at rs115160714 TopBP1 determined a higher incidence of nodal metastases (OR = 7.98, 95 % CI: 3.94-16.00, p = 0.001) and higher tumor grade (OR = 6.48, 95 % CI: 0.86-48.01, p = 0.03). The heterozygotes displayed diffuse tumor growth with no distinct borderline (OR = 3.10, 95 % Cl: 0.92-10.62, p = 0.049) and higher depth of invasion (OR = 2.66, 95 % Cl: 0.78-9.03, p = 0.04). Relationships were also identified between TopBP1 mRNA/protein expression and overall survival (p < 0.0001). The incidence of LOH in BRCA1/BRCA2 was significantly related to higher tumor grade and TFG (p < 0.05). The results of this study suggest that rs115160714 TopBP1 may be a genetic marker of etiology and progression in laryngeal cancer.

Significance of TGFBR3 allelic loss in the deregulation of TGFß signaling in primary human endometrial carcinomas.

Downregulation of betaglycan (ß-glycan) [transforming growth factor ß receptor type III (TGFßR3)], which belongs to co-receptors of the TGFß pathway, occurs in a broad spectrum of primary human malignancies. However, in the case of endometrial cancer (EC), the mechanisms responsible for genetic alterations are still unknown. Therefore, we investigated allelic imbalance at the TGFBR3 locus (1p33­p32) in the context of ß-glycan mRNA and protein expression, as a possible genetic event determining ß-glycan deregulation in EC patients. Study of ß-glycan allelic imbalance in 48 primary human ECs was performed with the use of three different microsatellite markers, spanned within or in direct proximity to the TGFBR3 locus. Real­time PCR and western blotting were used for ß-glycan mRNA and protein quantification methods, respectively. Altogether, 25 of 39 (64%) informative cases and 25 of 48 (52%) of all specimens showed allelic imbalance in at least one microsatellite marker, concomitantly with decrease at both the ß-glycan transcript and protein levels. Interestingly, 54% (15/28), 36% (8/22) and 35% (7/20) of informative ECs displayed allelic loss in D1S188, D1S435 and D1S1588 microsatellite markers, respectively. It is worth pointing out that 5 out of 39 (13%) informative cases showed loss of heterozygosity (LOH) at two microsatellite markers. Microsatellite instability (MSI) was found in two markers, but to a very strictly limited extent. None of the clinicoprognostic features was found to be of significance. Our results suggest that LOH in the TGFBR3 locus may be one of the mechanisms responsible for loss of ß-glycan expression. No correlation of LOH at the TGFBR3 locus with clinicopathological parameters suggests that allelic imbalance may be an early genetic event during neoplastic transformation of human endometrium.

Loss of heterozygosity for chromosomal regions 15q14-21.1, 17q21.31, and 13q12.3-13.1 and its relevance for prostate cancer.

Although prostate cancer is one of the most common cancers in men, the genetic defects underlying its pathogenesis remain poorly understood. DNA damage repair mechanisms have been implicated in human cancer. Accumulating evidence indicates that the fidelity of the response to DNA double-strand breaks is critical for maintaining genome integrity. RAD51 is a central player in double-strand break repair via homologous recombination, and its alterations may confer and increase the risk of cancer. RAD51 functioning depends on the indirect or direct interactions with BRCA1 and BRCA2. To evaluate the contribution of RAD51 to sporadic prostate cancer, loss of heterozygosity (LOH) for chromosomal region 15q14-21.1 (RAD51 locus) was determined and compared to LOH in 17q21.31 (BRCA1 locus) and 13q12.3-13.1 (BRCA2 region). DNA was isolated from prostate biopsies and matched peripheral blood of 50 patients. The regions 15q14-21.1, 17q21.31, and 13q12.3-13.1 were examined using microsatellite markers on chromosome 15 (D15S118, D15S214, D15S1006), chromosome 17 (D17S855, D17S1323), and chromosome 13 (D13S260, D13S290), respectively. The LOH in tumors was analyzed by PCR with fluorescently labeled primers and an ABI PRISM 377 DNA Sequencer. Allele sizing was determined by GeneScan version 3.1.2 and Genotyper version 2.5 software (Applied Biosystems, USA). LOH was identified in 57.5, 23, and 40 % for chromosomal regions 15q14-21.1, 17q21.31, and 13q12.3-13.1, respectively. Twenty-six percent of studied cases manifested LOH for at least one marker in 15q14-21.1 exclusively. A significant correlation was found between LOH for studied region and PSAD (prostate-specific antigen density). The findings suggest that RAD51 may be considered as a prostate cancer susceptibility gene.

Polymorphisms of homologous recombination RAD51, RAD51B, XRCC2, and XRCC3 genes and the risk of prostate cancer.

Genetic polymorphisms in DNA repair genes may induce individual variations in DNA repair capacity, which may in turn contribute to the risk of cancer developing. Homologous recombination repair (HRR) plays a critical role in maintaining chromosomal integrity and protecting against carcinogenic factors. The aim of the present study was to evaluate the relationship between prostate cancer risk and the presence of single nucleotide polymorphisms (SNPs) in the genes involved in HRR, that is, RAD51 (rs1801320 and rs1801321), RAD51B (rs10483813 and rs3784099), XRCC2 (rs3218536), and XRCC3 (rs861539). Polymorphisms were analyzed by PCR-RFLP and Real-Time PCR in 101 patients with prostate adenocarcinoma and 216 age- and sex-matched controls. A significant relationship was detected between the RAD51 gene rs1801320 polymorphism and increased prostate cancer risk. Our results indicate that the RAD51 gene rs1801320 polymorphism may contribute to prostate cancer susceptibility in Poland.

[Triple-negative breast cancer: molecular characteristics and potential therapeutic approaches]. / Potrójnie negatywny rak piersi: molekularna charakterystyka i potencjalne strategie terapeutyczne.

The term triple-negative breast cancer (TNBC) defines breast tumors that do not express estrogen receptors, progesterone receptor or epidermal growth factor receptor HER2 on immunohistochemical analysis. TNBC accounts for 12-17% of all types of breast cancer. Molecular profiling indicated that triple-negative breast cancer represents a heterogeneous subgroup of breast cancer. Triple-negative breast cancer shares histological and genetic abnormalities with basal-like subtype of breast cancer; however, this overlap is incomplete. Breast cancer found in BRCA1 mutation carriers is also frequently TNBC. Triple-negative breast cancer does not benefit from hormonal therapies or treatments targeted against HER2. The results of ongoing studies as well as the results of clinical trials suggest that poly(ADP-ribose) polymerase inhibitors, EGFR, Hedgehog, Notch, Wnt/ß-catenin, VEGF signaling inhibitors, and mTOR, Src, and Bcr/Abl kinase inhibitors used alone or in combination with other anticancer drugs might be effective in triple-negative breast cancer treatment. In this review, current knowledge on molecular characteristics of triple-negative breast cancer and its subtypes' treatment options is presented.

Expression of endoglin in primary endometrial cancer.

OBJECTIVE: Alterations in the transforming growth factor-ß (TGF-ß) signaling cascade are engaged in the development of human neoplasms through the deregulation of proliferation, differentiation and migration. However, in endometrial cancer, the role of endoglin, which acts as an accessory receptor in the TGF-ß pathway, is still unknown. The aim of our study was the evaluation of endoglin mRNA and protein expression levels in endometrial cancer as compared to normal endometrium. TGF-ß(1) and TGF-ß type II receptor were involved in the investigation since they directly cooperate with endoglin during signal propagation. Obtained results were correlated with clinicopathological parameters of studied material to determine endoglin contribution to tumor development and progression. METHODS: mRNA level assessment was performed using real-time technique, whereas protein expression was determined by ELISA assay. RESULTS: The endoglin mRNA level was not significantly altered in cancerous samples as compared to normal tissue, whereas its protein level demonstrated significant upregulation (p < 0.001) associated with increased tumor malignancy, assessed by histological grade and myometrium infiltration. CONCLUSIONS: An increase in endoglin protein expression level may interfere with the oncogenic potential of TGF-ß(1) and TGF-ß type II receptor in endometrial cancer. Correlation of the endoglin level with pronounced cancer malignancy suggests that it may be regarded as a potential prognostic marker of primary endometrial cancer.

Loss of heterozygosity in the RAD51 and BRCA2 regions in breast cancer.

BACKGROUND: Loss of heterozygosity (LOH) in the 15q14-21 and 13q12-13 regions can contribute to the inactivation of RAD51 and BRCA2 genes implicated in the pathogenesis of breast cancer. We investigated allelic losses in microsatellites in the RAD51 and BRCA2 regions, and their association with clinicopathological parameters in breast cancer. METHODS: The LOH analysis was performed by amplifying DNA by PCR, using D15S118, D15S214, D15S1006 polymorphic markers in the 15q14-21 region and D13S260, D13S290 polymorphic markers in the 13q12-13 region in 36 sporadic breast cancer cases. RESULTS: LOH in the RAD51 region ranged from 29% to 46% and in the BRCA2 region from 38% to 43% of informative cases. Eleven percent of the breast cancer cases displayed LOH for at least one studied marker in the RAD51 region exclusively. On the other hand, 44% of cases manifested statistically significant LOH for at least one microsatellite marker concomitantly in the RAD51 and BRCA2 regions. LOH in the RAD51 region similarly as in the BRCA2 region appeared to correlate with steroid receptors content and lymph node status. DISCUSSION: The obtained results indicate that alteration in RAD51 region may contribute to the disturbances of DNA repair involving RAD51 and/or BRCA2 penetration and thus enhance the risk of breast cancer development.

Dinucleotide repeat polymorphisms of RAD51, BRCA1, BRCA2 gene regions in breast cancer.

Recent studies suggest that genetic polymorphisms of the DNA repair genes have been implicated in breast cancer risk. BRCA1 and BRCA2, two breast cancer susceptibility genes, are essential to maintain chromosomal integrity. This is mediated via regulation of RAD51 during homologous recombination. Dinucleotide polymorphism repeats in the 15q14-21, 17q21 and 13q12-13 regions, where the RAD51, BRCA1 and BRCA2 genes are located, respectively, have been evaluated. The polymorphism was determined using the following microsatellite markers: D15S118, D15S214, D15S1006, D17S855, D17S1323, D13S260 and D13S290. Genotypes containing the (CA)(17) or (CA)(19) alleles in the RAD51 region were found to be associated with a decreased breast cancer risk. Genotype containing the (CA)(17) allele in the 13q12-13 region was found to be associated with an increased breast cancer risk. The results indicate that dinucleotide CA repeat polymorphism at RAD51 and BRCA2 gene regions might be associated with genetic susceptibility to breast cancer.

Loss of heterozygosity in the RAD54B region is not predictive for breast carcinomas.

This study was carried out to evaluate the loss of heterozygosity (LOH) in the 8q12-q24.1 chromosomal region, containing RAD54B gene in breast cancer. Polymorphic markers D8S539 and D8S543 were used. For alleles frequency estimation 100 primary breast cancers were tested. DNA was isolated from paraffin-embedded tissues and their matched blood samples. Polymerase chain reaction amplified products of normal and tumor DNA pairs were compared in ABI PRISM 377 DNA sequencer. In analyzed cases LOH was found in 1% and 2% of informative cases for microsatellite markers D8S539 and D8S543, respectively. This date indicate, that LOH isn't predictive for breast cancer.

Genetic instability in the RAD51 and BRCA1 regions in breast cancer.

Breast cancer is the most prevalent cancer type in women. Accumulating evidence indicates that the fidelity of double-strand break repair in response to DNA damage is an important step in mammary neoplasias. The RAD51 and BRCA1 proteins are involved in the repair of double-strand DNA breaks by homologous recombination. In this study, we evaluated loss of heterozygosity (LOH) in the RAD51 and BRCA1 regions, and their association with breast cancer. The polymorphic markers D15S118, D15S214 and D15S1006 were the focus for RAD51, and D17S855 and D17S1323 for BRCA1. Genomic deletion detected by allelic loss varied according to the regions tested, and ranged from 29 to 46% of informative cases for the RAD51 region and from 38 to 42% of informative cases for the BRCA1 region. 25% of breast cancer cases displayed LOH for at least one studied marker in the RAD51 region exclusively. On the other hand, 31% of breast cancer cases manifested LOH for at least one microsatellite marker concomitantly in the RAD51 and BRCA1 regions. LOH in the RAD51 region, similarly as in the BRCA1 region, appeared to correlate with steroid receptor status. The obtained results indicate that alteration in the RAD51 region may contribute to the disturbances of DNA repair involving RAD51 and BRCA1 and thus enhance the risk of breast cancer development.

Loss of heterozygosity and microsatellite instability at RAD52 and RAD54 loci in breast cancer.

This study was carried out to evaluate the loss of heterozygosity (LOH) and microsatellite instability (MSI) in breast cancer, in the 12p13.3 and 1p32 chromosomal regions where RAD52 and RAD54 genes are localized. Polymorphic markers D12S98, D12S1698 for RAD52 and D1S209, D1S411 for RAD54 were used. Relationships between LOH and clinicopathological parameters, i.e. tumor type and grade, patient's age, steroid receptors status and lymph node and distal metastases were assessed. For alleles frequency estimation 100 primary breast cancers were tested. DNA isolated from paraffin-embedded tissues and their matched blood samples were analyzed for PCR-based LOH and MSI by fluorescence-based DNA sequencing technology. In analyzed cases LOH was found in 14% and 11% of informative cases for D12S98 and D12S1698 markers, respectively and in 18% and 17% of informative cases for D1S209 and D1S411 markers, respectively. The highest frequency of MSI was identified at loci D12S98 (10%) and D1S209 (11%). Significant correlations between RAD52 and RAD54 regions with concomitant LOH and histological type and progesterone receptor status were observed. In the case of RAD54 further correlations with respect to tumor grade and the presence of distal metastases were noticed.