DSpace Academic/Research Unit: Histopathology & Morbid Anatomy

Epitope presentation is an important determinant of the utility of antigens identified from protein arrays in the development of autoantibody diagnostic assays

2012-06-19T14:10:08Z

Title: Epitope presentation is an important determinant of the utility of antigens identified from protein arrays in the development of autoantibody diagnostic assays Author: MARTIN, CARA; O'LEARY, JOHN JAMES; SHEILS, ORLA; MURPHY, MAIREAD; O'TOOLE, SHARON Abstract: Autoantibodies represent an attractive biomarker for diagnostic assays principally due to the stability of immunoglobulin in patient serum facilitating measurement with conventional assays. Immune responses to tumorigenesis may facilitate detection of ovarian cancer in the early stages of the disease with identification of a panel of tumour specific autoantibodies. Despite the reporting of many tumour associated autoantibodies using arrays of tumour antigens, this has not led to the advance in diagnostic capability as rapidly as was initially expected. Here we examine the potential diagnostic utility of candidate autoantibody biomarkers identified via screening of serum samples on a high content human protein array from a unique cohort of early stage and late stage ovarian cancer patients. We analyse the performance of autoantibodies to the tumour suppressor protein p53 and the novel autoantigens alpha adducin and endosulfine alpha identified in our array screen. Each antigen has different performance characteristics using conventional ELISA format and Western blot immunoassay. The high attrition rate of promising autoantigens identified by array screening can in part be explained by the presentation of the epitope of the antigen in the subsequent method of validation and this study provides directions on maximising the potential of candidate biomarkers. Description: IN_PRESS

Oxaliplatin induces drug resistance more rapidly than cisplatin in H69 small cell lung cancer cells

2012-06-15T09:37:49Z

Title: Oxaliplatin induces drug resistance more rapidly than cisplatin in H69 small cell lung cancer cells Author: STORDAL, BRITTA KRISTINA Abstract: Cisplatin produces good responses in solid tumours including small cell lung cancer (SCLC) but this is limited by the development of resistance. Oxaliplatin is reported to show activity against some cisplatin-resistant cancers but there is little known about oxaliplatin in SCLC and there are no reports of oxaliplatin resistant SCLC cell lines. Studies of drug resistance mainly focus on the cellular resistance mechanisms rather than how the cells develop resistance. This study examines the development of cisplatin and oxaliplatin resistance in H69 human SCLC cells in response to repeated treatment with clinically relevant doses of cisplatin or oxaliplatin for either 4 days or 2h. Treatments with 200ng/ml cisplatin or 400ng/ml oxaliplatin for 4 days produced sublines (H69CIS200 and H69OX400 respectively) that showed low level (approximately 2-fold) resistance after 8 treatments. Treatments with 1000ng/ml cisplatin or 2000ng/ml oxaliplatin for 2h also produced sublines, however these were not stably resistant suggesting shorter treatment pulses of drug may be more effective. Cells survived the first five treatments without any increase in resistance, by arresting their growth for a period and then regrowing. The period of growth arrest was reduced after the sixth treatment and the H69CIS200 and H69OX400 sublines showed a reduced growth arrest in response to cisplatin and oxaliplatin treatment suggesting that "regrowth resistance" initially protected against drug treatment and this was further upregulated and became part of the resistance phenotype of these sublines. Oxaliplatin dose escalation produced more surviving sublines than cisplatin dose escalation but neither set of sublines were associated with increased resistance as determined by 5-day cytotoxicity assays, also suggesting the involvement of regrowth resistance. The resistant sublines showed no change in platinum accumulation or glutathione levels even though the H69OX400 subline was more sensitive to buthionine sulfoximine treatment. The H69CIS200 cells were cross-resistant to oxaliplatin demonstrating that oxaliplatin does not have activity against low level cisplatin resistance. Relative to the H69 cells, the H69CIS200 and H69OX400 sublines were more sensitive to paclitaxel and taxotere suggests the taxanes may be useful in the treatment of platinum resistant SCLC. These novel cellular models of cisplatin and oxaliplatin resistant SCLC will be useful in developing strategies to treat platinum-resistant SCLC. Description: PUBLISHED

Oxaliplatin for the treatment of cisplatin-resistant cancer: A systematic review.

2012-06-15T09:30:41Z

Title: Oxaliplatin for the treatment of cisplatin-resistant cancer: A systematic review. Author: STORDAL, BRITTA KRISTINA Abstract: Oxaliplatin is widely regarded as being active in cisplatin-resistant cancer. We undertook a systematic review of the literature to identify, describe and critique the clinical and pre-clinical evidence for the use of oxaliplatin in patients with “cisplatinresistant” cancer. We identified 25 pre-clinical cell models of platinum resistance and 24 clinical trials reporting oxaliplatin based salvage therapy for cisplatin-resistant cancer. The pre-clinical data suggests that there is cross-resistance between cisplatin and oxaliplatin in low-level resistance models. In models with high level resistance (>10 fold) there is less cross resistance between cisplatin and oxaliplatin, which may be a reason why oxaliplatin is thought to be active in cisplatin-resistant cancer. In clinical trials where oxaliplatin has been used as part of salvage therapy for patients who have failed cisplatin or carboplatin combination chemotherapy, there was a much lower response rate in patients with platinum-refractory or resistant cancers compared to platinum-sensitive cancers. This suggests that there may be cross-resistance between cisplatin and oxaliplatin in the clinic. Oxaliplatin as a single agent had a poor response rate in cisplatin refractory and resistant cancer. Oxaliplatin performed better in combination with other agents for the treatment of platinum resistant/refractory cancer suggesting that the benefit of oxaliplatin may lie in its more favourable toxicity and ability to be combined with other drugs rather than an underlying activity in cisplatin resistance. Oxaliplatin therefore should not be considered broadly active in cisplatin-resistant cancer. Description: PUBLISHED

Understanding cisplatin resistance using cellular models.

2012-06-15T09:26:29Z

Title: Understanding cisplatin resistance using cellular models. Author: STORDAL, BRITTA KRISTINA Abstract: Many mechanisms of cisplatin resistance have been proposed from studies of cellular models of resistance including changes in cellular drug accumulation, detoxification of the drug, inhibition of apoptosis and repair of the DNA adducts. A series of resistant models were developed from CCRF-CEM leukaemia cells with increasing doses of cisplatin from 100 ng/ml. This produced increasing resistance up to 7-fold with a treatment dose of 1.6 μg/ml. Cisplatin resistance in these cells correlated with increases in the antioxidant glutathione, yet treatment with buthionine sulphoximine, an inhibitor of glutathione synthesis, had no effect on resistance, suggesting that the increase in glutathione was not directly involved in cisplatin resistance. Two models were developed from H69 SCLC cells, H69-CP and H69CIS200 using 100 ng/ml or 200 ng/ml cisplatin respectively. Both cell models were 2-4 fold resistant to cisplatin, and have decreased expression of p21 which may increase the cell’s ability to progress through the cell cycle in the presence of DNA damage. Both the H69-CP and H69CIS200 cells showed no decrease in cellular cisplatin accumulation. However, the H69-CP cells have increased levels of cellular glutathione and are cross resistant to radiation whereas the H69CIS200 cells have neither of these changes. This suggests that increases in glutathione may contribute to cross-resistance to other drugs and radiation, but not directly to cisplatin resistance. There are multiple resistance mechanisms induced by cisplatin treatment, even in the same cell type. How then should cisplatin-resistant cancers be treated? Cisplatin resistant cell lines are often more sensitive to another chemotherapeutic drug paclitaxel(H69CIS200), or are able to be sensitised to cisplatin with paclitaxel pre-treatment (H69-CP). The understanding of this sensitisation by paclitaxel using cell models of cisplatin resistance will lead to improvements in the clinical treatment of cisplatin resistant tumours. Description: PUBLISHED

A systematic review of platinum and taxane resitance from bench to clinic: An inverse relationship.

2012-06-15T09:15:03Z

Title: A systematic review of platinum and taxane resitance from bench to clinic: An inverse relationship. Author: STORDAL, BRITTA KRISTINA Abstract: We undertook a systematic review of the pre-clinical and clinical literature for studies investigating the relationship between platinum and taxane resistance. Medline was searched for 1) cell models of acquired drug resistance reporting platinum and taxane sensitivities and 2) clinical trials of platinum or taxane salvage therapy in ovarian cancer. 137 models of acquired drug resistance were identified. 68.1% of cisplatin-resistant cells were sensitive to paclitaxel and 66.7% of paclitaxel-resistant cells were sensitive to cisplatin. A similar inverse pattern was observed for cisplatin vs docetaxel, carboplatin vs paclitaxel and carboplatin vs docetaxel. These associations were independent of cancer type, agents used to develop resistance and reported mechanisms of resistance. 65 eligible clinical trials of paclitaxel-based salvage after platinum therapy were identified. Studies of single agent paclitaxel in platinum-resistant ovarian cancer where patients had previously recieved paclitaxel had a pooled response rate of 35.3% n=232, compared to 22% in paclitaxel naïve patients n=1918 (p<0.01 Chi-squared). Suggesting that pretreatment with paclitaxel may improve the response of salvage paclitaxel therapy. The response rate to paclitaxel/platinum combination regimens in platinum-sensitive ovarian cancer was 79.5% n=88 compared to 49.4% n=85 for paclitaxel combined with other agents (p<0.001 Chi-squared), suggesting a positive interaction between taxanes and platinum. Therefore the inverse relationship between platinum and taxanes resistance seen in cell models is mirrored in the clinical response to these agents in ovarian cancer. An understanding of the cellular and molecular mechanisms responsible would be valuable in predicting response to salvage chemotherapy and may identify new therapeutic targets. Description: PUBLISHED

A 39kDa fragment of endogenous ASK1 suggests specific cleavage not degradation by the proteasome.

2012-06-15T09:08:25Z

Title: A 39kDa fragment of endogenous ASK1 suggests specific cleavage not degradation by the proteasome. Author: STORDAL, BRITTA KRISTINA Abstract: Transfected human ASK1 produces a 150kDa protein. However, we have detected endogenous ASK1 predominantly as 39kDa and 50kDa C-terminal and 75kDa and 110kDa N-terminal fragments in a panel of non-transfected cancer cell lines and HUVEC endothelial cells. This suggests that in non-apoptotic cells, endogenous ASK1 protein is normally cleaved at a number of specific sites, some of which are in the kinase domain. Transfected ASK1 protein is known to be degraded by the proteasome. In contrast, the cleavage of endogenous ASK1 is independent of the proteasome as treatment with the proteasome inhibitor, lactacystin did not inhibit cleavage. Cisplatin treatment decreased the amount of 39kDa C-terminal ASK1 fragment in mutant p53 cell lines suggesting a decrease in cleavage associated with apoptosis. Transfected ASK1 may therefore not accurately reflect the role of endogenous ASK1 Description: PUBLISHED

ERCC1 expression and RAD51B activity correlate with cell cycle response to platinum drug treatment not DNA repair.

2012-06-15T08:56:52Z

Title: ERCC1 expression and RAD51B activity correlate with cell cycle response to platinum drug treatment not DNA repair. Author: STORDAL, BRITTA KRISTINA Abstract: Background: The H69CIS200 and H69OX400 cell lines are novel models of low- level platinum-drug resistance. Resistance was not associated with increased cellular glutathione or decreased accumulation of platinum, rather the resistant cell lines have a cell cycle alteration allowing them to rapidly proliferate post drug treatment. Results: A decrease in ERCC1 protein expression and an increase in RAD51B foci activity was observed in association with the platinum induced cell cycle arrest but these changes did not correlate with resistance or altered DNA repair capacity. The H69 cells and resistant cell lines have a p53 mutation and consequently decrease expression of p21 in response to platinum drug treatment, promoting progression of the cell cycle instead of increasing p21 to maintain the arrest. Conclusion: Decreased ERCC1 protein and increased RAD51B foci may in part be mediating the maintenance of the cell cycle arrest in the sensitive cells. Resistance in the H69CIS200 and H69OX400 cells may therefore involve the regulation of ERCC1 and RAD51B independent of their roles in DNA repair. The novel mechanism of platinum resistance in the H69CIS200 and H69OX400 cells demonstrates the multifactorial nature of platinum resistance which can occur independently of alterations in DNA repair capacity and changes in ERCC1. Description: PUBLISHED

A systematic review of genes involved in the inverse resistance relationship between cisplatin and paclitaxel chemotherapy: Role of BRCA1.

2012-06-15T08:48:44Z

Title: A systematic review of genes involved in the inverse resistance relationship between cisplatin and paclitaxel chemotherapy: Role of BRCA1. Author: STORDAL, BRITTA KRISTINA Abstract: A systematic review of cell models of acquired drug resistance not involving genetic manipulation showed that 80% of cell models had an inverse resistance relationship between cisplatin and paclitaxel[1]. Here we systematically review genetically modified cell lines in which the inverse cisplatin/paclitaxel resistance phenotype has resulted. This will form a short list of genes which may play a role in the mechanism of the inverse resistance relationship as well as potential markers for monitoring the development of resistance in the clinical treatment of cancer. The literature search revealed 91 genetically modified cell lines which report toxicity or viability/apoptosis data for cisplatin and paclitaxel relative to their parental cell lines. This resulted in 26 genes being associated with the inverse cisplatin/paclitaxel phenotype. The gene with the highest number of genetically modified cell lines associated with the inverse resistance relationship was BRCA1 and this gene is discussed in detail with reference to chemotherapy response in cell lines and in the clinical treatment of breast, ovarian and lung cancer. Other genes associated with the inverse resistance phenotype included dihydrodiol dehydrogenase (DDH) and P-glycoprotein. Genes which caused cross resistance or cross sensitivity between cisplatin and paclitaxel were also examined, the majority of these genes were apoptosis associated genes which may be useful for predicting cross resistance. We propose that BRCA1 should be the first of a panel of cellular markers to predict the inverse cisplatin/paclitaxel resistance phenotype. Description: PUBLISHED

Correlation of p16(INK4A) expression and HPV copy number with cellular FTIR spectroscopic signatures of cervical cancer cells.

2012-01-10T17:31:29Z

Title: Correlation of p16(INK4A) expression and HPV copy number with cellular FTIR spectroscopic signatures of cervical cancer cells. Author: MARTIN, CARA Abstract: Cervical cancer, a potentially preventable disease, has its main aetiology in infection by high risk human papillomavirus (HR-HPV). Approaches to improving cervical cancer screening and diagnostic methodologies include molecular biological analysis, targeting of biomarker proteins, but also exploration and implementation of new techniques such as vibrational spectroscopy. This study correlates the biomarker protein p16(INK4A) expression levels dependent on HPV copy number with the infrared absorption spectral signatures of the cervical cancer cell lines, HPV negative C33A, HPV-16 positive SiHa and CaSki and HPV-18 positive HeLa. Confocal fluorescence microscopy demonstrated that p16(INK4A) is expressed in all investigated cell lines in both nuclear and cytoplasmic regions, although predominantly in the cytoplasm. Flow cytometry was used to quantify the p16(INK4A) expression levels and demonstrated a correlation, albeit nonlinear, between the reported number of integrated HPV copies and p16(INK4A) expression levels. CaSki cells were found to have the highest level of expression, HeLa intermediate levels, and SiHa and C33A the lowest levels. FTIR spectra revealed differences in nucleic acid, lipid and protein signatures between the cell lines with varying HPV copy number. Peak intensities exhibited increasing tendency in nucleic acid levels and decreasing tendency in lipid levels with increasing HPV copy number, and although they were found to be nonlinearly correlated with the HPV copy number, their dependence on p16(INK4A) levels was found to be close to linear. Principal Component Analysis (PCA) of the infrared absorption spectra revealed differences between nuclear and cytoplasmic spectroscopic signatures for all cell lines, and furthermore clearly differentiated the groups of spectra representing each cell line. Finally, Partial Least Squares (PLS) analysis was employed to construct a model which can predict the p16(INK4A) expression level based on a spectral fingerprint of a cell line, demonstrating the diagnostic potential of spectroscopic techniques. Description: PUBLISHED

The HIF-1alpha C1772T polymorphism may be associated with susceptibility to clinically localised prostate cancer but not with elevated expression of hypoxic biomarkers.

2013-03-29T20:46:28Z

Title: The HIF-1alpha C1772T polymorphism may be associated with susceptibility to clinically localised prostate cancer but not with elevated expression of hypoxic biomarkers. Author: DUNNE, BARBARA; LYNCH, THOMAS H; PERRY, ANTOINETTE; MARIGNOL, LAURE; HOLLYWOOD, DONAL; LAWLER, MARK; LOFTUS, BARBARA MARY; TEWARI, PRERNA Abstract: We investigated the role of the C1772T polymorphisms in exon 12 of the Hypoxia-inducible factor-1 alpha (HIF-1alpha) gene C1772T genotype in prostate cancer (PCa) and amplification of the hypoxic response. We identified the heterozygous germline CT genotype as an increased risk factor for clinically localised prostate cancer (Odds ratio = 6.2; p < 0.0001). While immunostaining intensity for HIF-1alpha and VEGF was significantly enhanced in 75% of PCa specimens when compared to matched benign specimens (p < 0.0001), the CT genotype did not modulate the kinetics of HIF-1alpha protein expression in hypoxia in vitro, and was not associated with enhanced expression of hypoxic biomarkers. This study provides the first evidence of an increased risk for clinically localised prostate cancer in men carrying the C1772T HIF-1alpha gene polymorphism. Although our results did not suggest an association between expression of hypoxic biomarkers and genotype status, the correlation may merit further investigation. Description: PUBLISHED