Assessment of DNA-damage repair in breast cancer

Background: Current evidence indicates that DNA damage response (DDR) is a highly complex process that involves various pathways working in an orchestrated and interwoven manner in response to different types of damage to DNA. Although specific defects of DDR remain to be deciphered in cancer as a general, there is certainly an undeniable relationship between a particular dysfunction of DDR and the phenotype of tumour [1, 2]. It has been demonstrated that familial forms of breast and ovarian cancer are characterised by defects in one of the main mechanisms of DDR homologous recombination (HR) as a result of germline loss-of-function mutations in one of HR modifying genes, such as BRCA1 and BRCA2 [1, 3, 4]. Defects of genes involved in other DDR pathways are also associated with specific types of cancers; for instance hereditary non polyposis colorectal cancer (HNPCC) is strongly associated with specific mutations in the DNA mismatch repair pathway. Several previous studies have demonstrated that impaired DDR play a fundamental role in the pathogenesis and behaviour of breast cancer (BC). However, characterisation of this complex process, the expression and co-expression of the key proteins involved in the various DDR pathways and their prognostic significance in BC remain to be defined. In BC, it is reported that genes involved in DNA double strand breaks (DSB) repair are the most important. Two main pathways are involved in the repair of DNA-DSB; HR and Non Homologous End Joining (NHEJ) [3]. The common characteristics of global DDR are multiple genes induction directly associated with sensing and repair of DNA, arrest of cell cycle, and cell division inhibition. As a result DDR process does not only include genes activation involved in damage sensing as well as repair but additionally genes involved in control of cell-cycle [5]. Despite the fact that DDR may possibly involve activation of several pathways (such as SUMOylation (SUMO)) [6, 7] and many genes are engaged in different overlapping mechanisms, each pathway is characterised by activation and expression of a unique set of genes. This could allow discovering the active or aberrant pathway in a given tumour [1, 4, 5]. This study explores the hypothesis that investigation of alterations in the different pathways of DNA-DSB, may contribute to the characteristics of BC. Therefore, the aim was to perform a comprehensive profiling of key proteins involved in the different DNA-DSB repair pathways in the different molecular classes of BC. This approach aims to address the inherent problems arising from the complexity of DDR mechanism in BC with the potential of discovering a key pathway that is active or inactive in specific forms of BC that can be helpful to identify DNA repair status in individual BC patients. Method: The study cohort comprises three BC groups: A) Large series of unselected primary sporadic operable invasive tumours (n=1904) in addition to B) 386 cases of oestrogen receptor (ER) negative tumours and C) a well-characterised series of BC from patients with known BRCA1 germline mutations (n=24). The proteins investigated in this study are known to participate in different DNA-DSB repair pathways including, DNA damage sensors (ATM and ATR), HR repair (BRCA1, BARD1, Rad51, γH2AX and SMC6L1), DNA damage checkpoint signalling protein (CHK1 and CHK2), NHEJ repair (KU70/KU80, and DNA-PK), and SUMO (PIAS1, PIAS4, and UBC9). Because subcellular localisation of DDR proteins may affect their function, two markers that have role in nuclear transport in the cell were examined (NPM and KPNA2). The expression of these proteins was assessed using the well-established immunohistochemical technique utilising tissue microarray technology. The expression of proteins was further evaluated in various cell lines; BRCA1 deficient HeLaSilenciX® cells, and control BRCA1 proficient HeLaSilenciX®, MDA-MB-436 (BRCA1 deficient), and MCF-7 (BRCA1 proficient and ER+) using Reverse Phase Protein Microarray (RPPA). Results: Both cytoplasmic and nuclear expression was observed for expression of Rad51, SMC6L1, BRCA1, BARD1; (HR markers), PIAS1, UBC9 (SUMO markers), γH2AX (DNA-DSB marker) and CHK1 (checkpoint signalling protein). In contrast, both NHEJ markers and most of the DNA damage sensors (ATM and ATR), CHK2 and PIAS4 were mainly expressed in the nucleus. Generally, tumours that showed positive cytoplasmic/negative nuclear expression such as CHK1, PIAS1, Rad51, and BRCA1, and positive nuclear NHEJ markers showed an association with a poor outcome and adverse prognostic characteristics including high histologic grade, high mitotic frequency, high nuclear pleomorphism and larger tumour size in addition to ER negativity, and triple negative breast cancer (TNBC). Conversely, nuclear+/cytoplasmic- expression showed an association the better outcome. Interestingly, ATM protein expression showed no association with the expression of the two NHEJ markers, whereas ATR showed an association with cytoplasmic expression of BRCA1 and BARD1 and was positively associated with NHEJ markers. In non-TNBC, tumours showing BRCA1-/KU70/KU80- phenotype had worse breast cancer specific survival (BCSS) than positive expression (P<0.0001), whereas in the TN cohort,complex of KU70/KU80-&DNA-PK+ had the worst BCSS (P=0.001), and both are independent prognostic markers for BC. KPNA2, but not NPM was highly associated with poor BCSS (P<0.0001). At least one of nucleocytoplasmic transport markers (NPM or KPNA2) was significantly associated with the subcellular localisation of the most of the markers that showed cytoplasmic expression including SMC6L1, γH2AX, BRCA1, BARD1, UBC9, PIAS1 ,Rad51 and CHK1. RPPA was used to investigate the protein expression in different cell lines, although the correlation between RPPA and IHC was not significant, the results of RPPA were consistent with that demonstrated by IHC further supporting the finding of the current study. Conclusion: This study highlight the complexity of DDR related proteins and the overlap between different pathways involved in DDR. The finding of this study may help in the classification of BC and therefore, targeting active pathways in the development of drugs would enhance better patients’ outcomes. Major prognostic and predictive variables can be very important in choosing suitable treatment plans, identifying the risk of recurrence and classifying patients for clinical trials. Our results show that the HR- repair marker Rad51, complex of HR and NHEJ repair markers (BRCA1&KU70/KU80) in non-TNBC, and a complex of NHEJ markers (KU70/KU80&DNA-PK) are all independent prognostic markers for BC. In addition to expression, subcellular localisation of DDR proteins appeared to be a major factor in their role. Particularly, HR repair markers (but not NHEJ) showed worse features of cytoplasmic location of expression, whereas nuclear expression was associated with more favourable features. Finally, the results of this study provide further evidence to support combined use of IHC with the parallel analytic capability of protein microarray RPPA to investigate protein alterations in human tumours

Assessment of DNA-damage repair in breast cancer

Background: Current evidence indicates that DNA damage response (DDR) is a highly complex process that involves various pathways working in an orchestrated and interwoven manner in response to different types of damage to DNA. Although specific defects of DDR remain to be deciphered in cancer as a general, there is certainly an undeniable relationship between a particular dysfunction of DDR and the phenotype of tumour [1, 2]. It has been demonstrated that familial forms of breast and ovarian cancer are characterised by defects in one of the main mechanisms of DDR homologous recombination (HR) as a result of germline loss-of-function mutations in one of HR modifying genes, such as BRCA1 and BRCA2 [1, 3, 4]. Defects of genes involved in other DDR pathways are also associated with specific types of cancers; for instance hereditary non polyposis colorectal cancer (HNPCC) is strongly associated with specific mutations in the DNA mismatch repair pathway. Several previous studies have demonstrated that impaired DDR play a fundamental role in the pathogenesis and behaviour of breast cancer (BC). However, characterisation of this complex process, the expression and co-expression of the key proteins involved in the various DDR pathways and their prognostic significance in BC remain to be defined. In BC, it is reported that genes involved in DNA double strand breaks (DSB) repair are the most important. Two main pathways are involved in the repair of DNA-DSB; HR and Non Homologous End Joining (NHEJ) [3]. The common characteristics of global DDR are multiple genes induction directly associated with sensing and repair of DNA, arrest of cell cycle, and cell division inhibition. As a result DDR process does not only include genes activation involved in damage sensing as well as repair but additionally genes involved in control of cell-cycle [5]. Despite the fact that DDR may possibly involve activation of several pathways (such as SUMOylation (SUMO)) [6, 7] and many genes are engaged in different overlapping mechanisms, each pathway is characterised by activation and expression of a unique set of genes. This could allow discovering the active or aberrant pathway in a given tumour [1, 4, 5]. This study explores the hypothesis that investigation of alterations in the different pathways of DNA-DSB, may contribute to the characteristics of BC. Therefore, the aim was to perform a comprehensive profiling of key proteins involved in the different DNA-DSB repair pathways in the different molecular classes of BC. This approach aims to address the inherent problems arising from the complexity of DDR mechanism in BC with the potential of discovering a key pathway that is active or inactive in specific forms of BC that can be helpful to identify DNA repair status in individual BC patients. Method: The study cohort comprises three BC groups: A) Large series of unselected primary sporadic operable invasive tumours (n=1904) in addition to B) 386 cases of oestrogen receptor (ER) negative tumours and C) a well-characterised series of BC from patients with known BRCA1 germline mutations (n=24). The proteins investigated in this study are known to participate in different DNA-DSB repair pathways including, DNA damage sensors (ATM and ATR), HR repair (BRCA1, BARD1, Rad51, γH2AX and SMC6L1), DNA damage checkpoint signalling protein (CHK1 and CHK2), NHEJ repair (KU70/KU80, and DNA-PK), and SUMO (PIAS1, PIAS4, and UBC9). Because subcellular localisation of DDR proteins may affect their function, two markers that have role in nuclear transport in the cell were examined (NPM and KPNA2). The expression of these proteins was assessed using the well-established immunohistochemical technique utilising tissue microarray technology. The expression of proteins was further evaluated in various cell lines; BRCA1 deficient HeLaSilenciX® cells, and control BRCA1 proficient HeLaSilenciX®, MDA-MB-436 (BRCA1 deficient), and MCF-7 (BRCA1 proficient and ER+) using Reverse Phase Protein Microarray (RPPA). Results: Both cytoplasmic and nuclear expression was observed for expression of Rad51, SMC6L1, BRCA1, BARD1; (HR markers), PIAS1, UBC9 (SUMO markers), γH2AX (DNA-DSB marker) and CHK1 (checkpoint signalling protein). In contrast, both NHEJ markers and most of the DNA damage sensors (ATM and ATR), CHK2 and PIAS4 were mainly expressed in the nucleus. Generally, tumours that showed positive cytoplasmic/negative nuclear expression such as CHK1, PIAS1, Rad51, and BRCA1, and positive nuclear NHEJ markers showed an association with a poor outcome and adverse prognostic characteristics including high histologic grade, high mitotic frequency, high nuclear pleomorphism and larger tumour size in addition to ER negativity, and triple negative breast cancer (TNBC). Conversely, nuclear+/cytoplasmic- expression showed an association the better outcome. Interestingly, ATM protein expression showed no association with the expression of the two NHEJ markers, whereas ATR showed an association with cytoplasmic expression of BRCA1 and BARD1 and was positively associated with NHEJ markers. In non-TNBC, tumours showing BRCA1-/KU70/KU80- phenotype had worse breast cancer specific survival (BCSS) than positive expression (P<0.0001), whereas in the TN cohort,complex of KU70/KU80-&DNA-PK+ had the worst BCSS (P=0.001), and both are independent prognostic markers for BC. KPNA2, but not NPM was highly associated with poor BCSS (P<0.0001). At least one of nucleocytoplasmic transport markers (NPM or KPNA2) was significantly associated with the subcellular localisation of the most of the markers that showed cytoplasmic expression including SMC6L1, γH2AX, BRCA1, BARD1, UBC9, PIAS1 ,Rad51 and CHK1. RPPA was used to investigate the protein expression in different cell lines, although the correlation between RPPA and IHC was not significant, the results of RPPA were consistent with that demonstrated by IHC further supporting the finding of the current study. Conclusion: This study highlight the complexity of DDR related proteins and the overlap between different pathways involved in DDR. The finding of this study may help in the classification of BC and therefore, targeting active pathways in the development of drugs would enhance better patients’ outcomes. Major prognostic and predictive variables can be very important in choosing suitable treatment plans, identifying the risk of recurrence and classifying patients for clinical trials. Our results show that the HR- repair marker Rad51, complex of HR and NHEJ repair markers (BRCA1&KU70/KU80) in non-TNBC, and a complex of NHEJ markers (KU70/KU80&DNA-PK) are all independent prognostic markers for BC. In addition to expression, subcellular localisation of DDR proteins appeared to be a major factor in their role. Particularly, HR repair markers (but not NHEJ) showed worse features of cytoplasmic location of expression, whereas nuclear expression was associated with more favourable features. Finally, the results of this study provide further evidence to support combined use of IHC with the parallel analytic capability of protein microarray RPPA to investigate protein alterations in human tumours

A fourth algebraic order explicit trigonometrically- fitted modified Runge-Kutta method for the numerical solution of periodic IVPs

This study has constructed an explicit Trigonometrically-Fitted Modified Runge-Kutta (TFMRK) method for solving first-order differential equations with periodic solutions. The newly developed method was made according to the method of Runge-Kutta Dorm and to fourth algebraic order. Numerical results for the new method were compared with the existing method, showing the potential of the new method over other existing methods

Explicit Runge-Kutta method with trigonometrically-fitted for solving first order ODEs

In this note, an explicit trigonometrically-fitted (RK) method is developed to determine the approximate solution of the first-order IVPs with oscillatory solution. The proposed method solves first order ODEs by first converting the second order ODEs to an equivalent first order; which is based on the RK method of order four. The numerical experiment performed shows the efficacy of our newly developed method

A phase-fitted and amplification-fitted modified Runge-Kutta method of fourth order for periodic initial value problems

A new Runge-Kutta method, with phase-fitted and amplification-fitted is constructed for solving first-order ordinary differential equations with periodic solutions. This new method is based on the Runge-Kutta 3/8 Rule with fourth algebraic order. In the numerical results the new method is compared with the existing method; which show that the new method is more efficient

Human Capital Training Cost and its Impact on Manpower Planning in the Kingdom of Bahrain An Empirical Study (An Empirical Study on a Sample of Commercial bank and the Central bank in the kingdom of Bahrain)

The present paper aims to identify the impact of human capital training cost on manpower planning through in the Ministry of Education and the Ministry of Labour in Kingdom of Bahrain. The population comprised (500) employees from both ministries. a Total of 1,000 employees answered (766) questionnaires: (4) questionnaires were invalid for statistical analysis, so (762) were analyzed. Returns percentage was (76.6%). The results illustrated a positive impact of human capital training cost on manpower planning. Moreover, training cost had a positive impact on both manpower planning indicators of external and internal environment analysis. Furthermore, there was a positive learning impact on the manpower planning in all indicators planning except for the expected demand of the workforce. The study recommends that the Ministry of Education and the Ministry of Labor in Bahrain should increase physical support to evaluate training program, review work procedures in human resources management, appoint new graduates to benefit from their ambitions as well as attempt to qualify them according to the objectives and strategy of the ministry

The Role of Accounting Information Systems in Supporting Privatization Program(An Empirical Study in Bahrain Airport Company, Cars Puplic Transportation Company and AL Hidd Power Company)

The present paper aims to address the role of accounting information systems in supporting the privatization process, highlight the challenges facing the privatization process as well as present recommendations that would support the privatization process. The author formulates the research problem to answer some questions, such as what is the importance of accounting information systems in supporting the privatization process? and What are the obstacles and challenges that may limit the privatization of its goals and how to overcome them? The research was conducted to answer the following question: What is the role of accounting information systems in supporting privatization programs? The population and the sample comprised (20) employees working in three privatized companies in Bahrain. A questionnaire was distributed to them, then data were analyzed. The results demonstrated that the decision-making related to privatization depended on the efficiency and accuracy of the information process provided because it was shown that the most important elements in supporting privatization programs were the efficiency and accuracy of information provided by the accounting system. The study recommends that the applied accounting system in the company should have the ability to control the accounting operations during the preparation work. Moreover, accounting information provided by the accounting system should match the nature of the decisions taken. Furthermore, staff should be trained on the latest accounting systems. In addition, any public facility can be privatized after a deep, objective study that indicates the importance of a privatization process

The Impact of Creative Accounting Methods and the Reliability of Financial Statements - An Analytical Study of a Sample of Employees Working in Auditing Firms and Joint Stock Companies on the Bahrain Stock Exchange

The present paper aims to identify creative accounting methods used by the companies participating in the stock market through analytical study as well as the impact of creative accounting practices on the financial statements of the companies contributing to the stock market. The results showed the importance of the moral dimension of accountants because of the decisions built on the results of creative accounting practices as well as the importance of credibility in the financial statements. The study recommends identifying all the creative accounting practices that help find the appropriate solutions; activating the awareness of the ethical dimension of the accounting profession as it extensively affects the production of the lists of highly credible financial benefit to the economy as well as the relative development and continuation of the companies; activating the powers granted to external auditors and developing the legislations that regulate accounting work. Thus, fraud, manipulation and errors can be detected

The Role of Banks in Monitoring and Detection of Money Laundering Operations (An Empirical Study on a Sample of Commercial Bank and the Central bank in the Kingdom of Bahrain)

The present paper aims to examine the role of banks in controlling and detecting money-laundering operations in Bahrain, define the responsibility of banks in money laundering cases, clarify the concepts for money laundering and risks, and present the recommendations that minimize the phenomenon of money laundering. The sample involved (102) employees from the Central Bank of Bahrain and some commercial banks, (22) members of the Central Bank of Bahrain Bank, and (80) employees from some commercial banks. The results showed a positive relationship between methods of countering money-laundering and understanding the employee’s regulations and legislations on money laundering. Moreover, there was a statistically significant relationship between methods of countering money laundering in commercial banks and the perception of staff regulations and money laundering legislation. Furthermore, there were moral differences in the statistical significance among the methods used to counter money laundering in commercial banks and the Central Bank of Bahrain and the perception of staff regulations and legislation due to some demographic variables. The study recommends that the government should control the availability of equipment to combat money laundering in commercial banks. In addition, all judicial, legislative and executive authorities should cooperate to combat money laundering. Besides, laws should be enacted to combat money laundering, bribery, cronyism and malfeasance at all levels

Clinical and biological significance of RAD51 expression in breast cancer: a key DNA damage response protein

Impaired DNA damage response (DDR) may play a fundamental role in the pathogenesis of breast cancer (BC). RAD51 is a key player in DNA double-strand break repair. In this study, we aimed to assess the biological and clinical significance of RAD51 expression with relevance to different molecular classes of BC and patients’ outcome. The expression of RAD51 was assessed immunohistochemically in a well-characterised annotated series (n = 1184) of early-stage invasive BC with long-term follow-up. A subset of cases of BC from patients with known BRCA1 germline mutations was included as a control group. The results were correlated with clinicopathological and molecular parameters and patients’ outcome. RAD51 protein expression level was also assayed in a panel of cell lines using reverse phase protein array (RPPA). RAD51 was expressed in the nuclei (N) and cytoplasm (C) of malignant cells. Subcellular colocalisation phenotypes of RAD51 were significantly associated with clinicopathological features and patient outcome. Cytoplasmic expression (RAD51C+) and lack of nuclear expression (RAD51 N-) were associated with features of aggressive behaviour, including larger tumour size, high grade, lymph nodal metastasis, basal-like, and triple-negative phenotypes, together with aberrant expression of key DDR biomarkers including BRCA1. All BRCA1-mutated tumours had RAD51C+/N- phenotype. RPPA confirmed IHC results and showed differential expression of RAD51 in cell lines based on ER expression and BRCA1 status. RAD51 N+ and RAD51C+ tumours were associated with longer and shorter breast cancer-specific survival (BCSS), respectively. The RAD51 N+ was an independent predictor of longer BCSS (P<0.0001). Lack of RAD51 nuclear expression is associated with poor prognostic parameters and shorter survival in invasive BC patients. The significant associations between RAD51 subcellular localisation and clinicopathological features, molecular subtype and patients’ outcome suggest that the trafficking of DDR proteins between the nucleus and cytoplasm might play a role in the development and progression of BC