http://hdl.handle.net/2123/3947 2013-05-19T11:44:01Z http://hdl.handle.net/2123/5687 Title: Regrowth resistance: low-level platinum resistance mediated by rapid recovery from platinum-induced cell-cycle arrest Authors: Stordal, Britta; Davey, Ross Abstract: The H69CIS200 and H69OX400 cell lines are novel models of low-level platinum drug resistance developed from H69 human small-cell lung cancer cells with eight 4-day treatments of 200 ng/ml cisplatin and 400 ng/ml oxaliplatin, respectively. A recovery period was given between treatments to emulate the cycles of chemotherapy given in the clinic. The resistant cell lines were approximately twofold resistant to cisplatin and oxaliplatin, and were cross-resistant to both drugs. Platinum resistance was not associated with increased cellular glutathione, decreased accumulation of platinum or increased DNA repair capacity. The H69 platinum sensitive cells entered a lengthy 3-week growth arrest in response to low-level cisplatin or oxaliplatin treatment. This is an example of the coordinated response between the cell cycle and DNA repair. In contrast, the H69CIS200 and H69OX400 cells have an alteration in the cell cycle allowing them to rapidly proliferate post drug treatment. The resistant cell lines also have many chromosomal rearrangements most of which are not associated with the resistant phenotype, suggesting an increase in the genomic instability in the resistant cell lines. We hypothesized that there was a lack of coordination between the cell cycle and DNA repair in the resistant cell lines allowing proliferation in the presence of DNA damage which has created an increase in genomic instability. The H69 cells and resistant cell lines have mutant p53 and consequently decrease the expression of p21 in response to platinum drug treatment; promoting progression of the cell cycle instead of increasing p21 to maintain the arrest. A decrease in ERCC1 protein expression and an increase in RAD51B foci activity were observed with the platinum-induced cell-cycle arrest and did not correlate with resistance or altered DNA repair capacity. These changes may, in part, be mediating and maintaining the cell-cycle arrest in place of p21.The rapidly proliferating resistant cells have restored the levels of both these proteins to their levels in untreated cells. We use the term “regrowth resistance” to describe this low-level platinum resistance where cells survive treatment through increased proliferation. Regrowth resistance may play a role in the onset of clinical resistance. 2009-01-01T00:00:00Z http://hdl.handle.net/2123/5686 Title: Treating cisplatin-resistant cancer: a systematic analysis of oxaliplatin or paclitaxel salvage chemotherapy. Authors: Stordal, Britta; Pavlakis, Nick; Davey, Ross Abstract: Objective: To examine the pre-clinical and clinical evidence for the use of oxaliplatin or paclitaxel salvage chemotherapy in patients with cisplatin-resistant cancer. Methods: Medline was searched for 1) Cell models of acquired resistance reporting cisplatin, oxaliplatin and paclitaxel sensitivities and 2) Clinical trials of single agent oxaliplatin or paclitaxel salvage therapy for cisplatin/carboplatin-resistant ovarian cancer. Results: Oxaliplatin - Oxaliplatin is widely regarded as being active in cisplatin-resistant cancer. In contrast, data in cell models suggests that there is crossresistance between cisplatin and oxaliplatin in cellular models with resistance levels which reflect clinical resistance (<10 fold). Oxaliplatin as a single agent had a poor response rate in patients with cisplatin-resistant ovarian cancer (8%, n=91). Oxaliplatin performed better in combination with other agents for the treatment of platinum-resistant 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. Paclitaxel – Cellular data 2 suggests that paclitaxel is active in cisplatin-resistant cancer. 68.1% of cisplatinresistant cells were sensitive to paclitaxel. Paclitaxel as a single agent had a response rate of 22% in patients with platinum-resistant ovarian cancer (n = 1918), a significant increase from the response of oxaliplatin (p<0.01). Paclitaxel-resistant cells were also sensitive to cisplatin, suggesting that alternating between agents may be beneficial. Studies of single agent paclitaxel in platinum-resistant ovarian cancer where patients had previously received paclitaxel had an improved response rate of 35.3% n=232 (p<0.01), suggesting that pre-treatment with paclitaxel improves the response of salvage paclitaxel therapy. Conclusions: Cellular models reflect the resistance observed in the clinic as the cross resistant agent oxaliplatin has a lower response rate compared to the non-cross resistant agent paclitaxel in cisplatin-resistant ovarian cancer. Alternating therapy with cisplatin and paclitaxel may therefore lead to an improved response rate in ovarian cancer. 2009-01-01T00:00:00Z http://hdl.handle.net/2123/5685 Title: ERCC1 expression and RAD51B activity correlate with cell cycle response to platinum drug treatment not DNA repair Authors: Stordal, Britta; Davey, Ross 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. 2008-06-25T00:00:00Z http://hdl.handle.net/2123/5684 Title: A 39 kDa fragment of endogenous ASK1 suggests specific cleavage not degradation by the proteasome Authors: Stordal, Britta; Davey, Ross 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. 2008-02-19T00:00:00Z http://hdl.handle.net/2123/4068 Title: A systematic review of platinum and taxane resistance from bench to clinic: an inverse relationship Authors: Stordal, Britta; Pavlakis, Nick; Davey, Ross 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 pre-treatment 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. 2007-01-01T00:00:00Z http://hdl.handle.net/2123/4067 Title: Understanding cisplatin resistance using cellular models Authors: Stordal, Britta; Davey, Mary 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. 2007-01-01T00:00:00Z http://hdl.handle.net/2123/4066 Title: Oxaliplatin for the treatment of cisplatin-resistant cancer: a systematic review Authors: Stordal, Britta; Pavlakis, Nick; Davey, Ross 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 “cisplatin-resistant” 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. 2007-01-01T00:00:00Z http://hdl.handle.net/2123/4065 Title: Oxaliplatin induces drug resistance more rapidly than cisplatin in H69 small cell lung cancer cells Authors: Stordal, Britta K; Davey, Mary W; Davey, Ross A 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. 2006-01-01T00:00:00Z