1. Introduction Results References Active nuclear transport is disrupted before nuclear envelope breakdown during chemotherapy induced apoptosis in HeLa cells by 5-fluorouracil but not camptothecin and its derivatives Rebecca Anderson, Christina Campbell, Sarah Broskin, Brittany Colossimo, Karen K. Resendes Westminster College, New Wilmington PA Chemotherapeutic agents are used to activate apoptosis to eliminate cancer cells. The stepwise mechanism they activate involves caspase-regulated breakdown of the nuclear envelope to initiate DNA fragmentation. However, there is conflicting evidence on whether nuclear transport is specifically disrupted before the destruction of the nucleus. In this study, we explored the effects of four chemotherapeutic drugs, 5-fluorouracil (5-FU), camptothecin, and its derivatives topotecan and irinotecan, on nuclear transport during apoptosis in HeLa cells. These drugs were selected because they activate different mechanisms of apoptosis (5-FU is extrinsic and the others intrinsic) and because 5-FU is often used in combination with the camptothecin derivatives during chemotherapy. The membrane impermeable DNA dye, propidium iodide (PI), was used to determine the timing of apoptotic nuclear envelope breakdown (NEB), which was confirmed by immunofluorescence of nuclear pore proteins. Based on the PI assays, functioning nuclear transport was monitored prior to NEB via immunofluorescence of Ran, the major directional regulator of nuclear protein transport, which is normally at high concentrations in the nucleus. We found that 5-FU disrupted nuclear transport before NEB, whereas camptothecin and its derivatives only displayed cytoplasmic mislocalization of the Ran concurrent with NEB. This variation in the targeted disruption of nuclear transport between 5-FU and the camptothecin drugs may stem from a variation in intrinsic versus extrinsic apoptosis. Interestingly, combination of 5-FU with camptothecin did not induce an early disruption of nuclear transport, whereas 5-FU with topotecan did in fact disrupt Ran localization before NEB. Future directions will include analysis of the 5-FU and irinotecan combination and investigations into the mechanism behind the variation between targeted disruption of nuclear transport versus maintaining transport until NEB, including profiling the activation of caspases in each scenario. Overall, our results suggest that active nuclear transport may be required in certain mechanisms of apoptosis, whereas its disruption may be needed in others. Because regulation of nuclear transport is altered in many cancer types, this intriguing new variation in the mechanism of apoptosis could possibly be exploited when determining chemotherapeutic agents in relation to the status of nuclear transport in cancer cells. Fulda and Debatin. (2006). Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25, 4798-4811. Gastman, B. R. (2001). Apoptosis and its clinical impact. Head & Neck, 409–425. Available at: www.headandneck.gov. Kauffman, S and Earnshaw, W. (2000). Induction of Apoptosis by Cancer Chemotherapy, Experimental Cell Research, 256, pp. 42-49. Kuersten, S., Ohno, M., & I.W. Mattaj. (2001). Nucleocytoplasmic transport: Ran, beta and beyond. Trends in Cell Biology, 11(12), 497-503. Melchoir, F. (2001). RanGTPase cycle: One mechanism- two functions. Current Biology 11, 257-260. Riemsma, R., Simons, J., Bashir, Z., Gooch, C., and Kleijnen, J. (2010). Systematic review of topotecan (hycamtin) in relapsed small cell lung cancer. BioMed Central Cancer 10. Yasuhara, N., Eguchi, Y., Imamoto, N., Yonedo, Y., and Tsujimoto, Y. (1997). Essential role of active nuclear transport in apoptosis. Genes to cells: devoted to molecular and cellular mechanisms 2, 55-64. Travel grants provided by: CUR ASCB Westminster College Drinkco Center Figure 1: Comparing the rates of apoptosis, ran mislocalization and nuclear envelope breakdown showed 5-Fluorourcil disrupted transport into the nucleus before nuclear envelope break down, whereas Camptothecin and it’s derivatives did not Figure 1: Each chemotherapeutic drug was added to HeLa cells to induce apoptosis at the following concentrations 100μM for 5-Fluorouracil, 20μM for Topotecan, 60μM for Camptothecin, 100μM for Irinotecin. Rate of apoptosis was determined using membrane impermeable propidum iodide. Immunofluorescence for the nuclear transport Ran was used to determine the status of nuclear transport. Nuclear blebbing/nuclear envelope breakdown was assessed via immunofluorescence with mAb 414 which recognizes the FG nucleoporins. Each assay (apoptosis, Ran localization and nuclear envelope breakdown) was performed in triplicate counting 200-500 cells at each time point in each experiment. Error bars represent standard deviation at each time point. Data points without any error bars represent trials with only one replicate. Hour 0Hour 6 Hour 0 Hour 5 mAb414 Ran A B C Figure 2: A combination of 5-Fluorouracil and Camptothecin and it’s derivatives were added to HeLa cells to induce apoptosis at the following concentrations 100 μM for 5-Fluorouracil, 20μM for Topotecan, 60μM for Camptothecin, 100μM for Irinotecin. Rate of apoptosis was determined using membrane impermeable propidum iodide. Immunofluorescence for the nuclear transport Ran was used to determine the status of nuclear transport. Nuclear blebbing/nuclear envelope breakdown was assessed via immunofluorescence with mAb 414 which recognizes the FG nucleoporins. Each assay (apoptosis, Ran localization and nuclear envelope breakdown) was performed in triplicate counting 200-500 cells at each time point in each experiment. Error bars represent standard deviation at each time point. Data points without any error bars represent trials with only one replicate. A Figure 2: Comparing apoptosis to ran mislocalization and nuclear envelope breakdown showed that 5-Fluorouracil in combination with Topotecan disrupted nuclear transport before nuclear envelope breakdown, whereas with Camptothecin and Irinotecan it did not B C Hour 0 Hour 7 Hour 0 Hour 7 mAb414 Ran TreatmentApoptosi s (70% of cells PI positive) Disru pted Nucle ar Trans port Nuclea r envelo pe breakd own ANCO VA analys is of rates (p- value) Conclusions (based on statistical rates, p- value≤ 0.05, then slopes are different) 5-Fluorouracil4-6 hours3-4 hours 4-6 hours 0.113 Rates not significantly different but data suggests that they are, do more replicates Camptothecin7-10 hours 0.369 Ran mislocalization concurrent with nuclear envelope breakdown Topotecan4-5 hours 0.636 Ran mislocalization concurrent with nuclear envelope breakdown Irinotecan8-10 hours 0.112 Ran mislocalization concurrent with nuclear envelope breakdown Camptothecin and 5-fluorouracil 8-9 hours 0.222 Ran mislocalization concurrent with nuclear envelope breakdown Topotecan and 5- fluorouracil 6-8 hours3.5-4 hours 6-8 hours 0.000 Ran mislocalization before nuclear envelope breakdown Irinotecan and 5-fluorouracil 5-7 hours6-7 hours 5-7 hours 0.102 Ran mislocalization concurrent with nuclear envelope breakdown Hour 0Hour 9 Hour 0 Hour 9 mAb414 Ran Hour 0Hour 6 Hour 0 Hour 5 mAb414 Ran D Hour 0Hour 10 Hour 0 Hour 10 mAb414 Ran Hour 0Hour 9 Hour 0 Hour 9 mAb414 Ran Hour 0Hour 7 Hour 0 Hour 4 mAb414 Ran Abstract Conclusions Future Directions -Camptothecin and its derivatives individually displayed disruption of nuclear transport during apoptosis concurrent with nuclear envelope breakdown. -5-FU disrupted nuclear transport during apoptosis prior to nuclear envelope breakdown -Variation in targeted disruption of nuclear transport may stem from the activation of different caspases via the intrinsic and extrinsic pathways of apoptosis -Combination of 5-FU with camptothecin and its derivatives varied in their effects on nuclear transport during apoptosis. -Overall, our results suggest that active nuclear transport could be required for certain mechanisms of apoptosis, where its disruption may be needed in others. -Variation in mechanism of apoptosis could be exploited when determining chemotherapeutic agents in relation to status of nuclear transport in cancer cells. Apoptosis, or programmed cell death, occurs regularly during development of multicellular organisms and is also used to remove infected or abnormal cells. During apoptosis, nuclear transport is involved in relaying signals from the cytoplasm of the cell to the nucleus (Yasuhara et al., 1997). Directionality of nuclear protein transport is mediated by the distribution of the small nuclear GTPase, Ran, which is found predominantly in the nucleus of a cell. Apoptosis-inducing drugs typically disrupt the Ran gradient in the cell, causing RanGTP to mislocalize to the cytoplasm (Gastman, 2001). One mechanism used to eliminate continually proliferating tumor cells is through the introduction of apoptotic-inducing drugs. As with naturally induced apoptosis, chemotherapy will initiate either the intrinsic or extrinsic pathway of cell death (Figure 1). The intrinsic pathway typically begins via DNA damage activating p53, which causes the release of cytochrome C from mitochondria, activating caspases and initiating apoptosis (Fulda, 2006). The extrinsic pathway is initiated when a ligand binds to a death receptor on the cell, causing a signal cascade ending in caspase activation and apoptosis (Fulda, 2006). This extrinsic signal can be produced in an autocrine or paracrine manner. While caspases are activated in each pathway there is some variation in which caspases are produced (Khan et al, 2013). There is currently little literature available on the connection between caspases and direct disruption of nuclear transport factors during apoptosis (Kauffman, 2001). Figure 1. Chemotherapy causes disruption of nuclear transport factors. We were interested in examining the variation in the role of nuclear transport during apoptosis. In order to do so, we assessed how a set of chemotherapeutic agents affect nuclear transport during apoptosis. Specifically, we studied camptothecin a chemotherapeutic agent commonly used in cancer regimens with two derivatives: topotecan and irinotecan (Riemsma et al., 2010). Each of these drugs induces apoptosis via an intrinsic cellular pathway. Additionally we studied 5-fluorouracil (5-FU), a fluoropyrimidine which has been commonly used as a chemotherapeutic agent for over 50 years. For each drug and drug combination we sought to determine the following information: 1) Based on effective drug concentrations in the literature, at what point is apoptosis induced in HeLa cells as determined by DNA staining by membrane impermeable propidium iodide (PI). 2) At what point in the progression of apoptosis did drug treatment lead to disruption of nuclear transport, as assayed by Ran mislocalization to the cytoplasm. 3) At what point the progression of apoptosis are nuclear pores disrupted, as assayed by FG nucleoporin mislocalization from the nuclear envelope. Based on the above data we concluded whether each treatment led to one of the following outcomes: 1) Nuclear transport was targeted/disrupted prior to nuclear envelope breakdown. 2) Nuclear transport disruption coincided with nuclear envelope breakdown.. -Determine the mechanism behind variation in apoptosis between targeted disruption of nuclear transport versus disruption as a result of nuclear envelope breakdown. Goal 1: Profiling activation of caspases for each drug and combination. Goal 2: Use caspase inhibitors to directly connect caspases to altered nuclear transport that occurs before nuclear envelope breakdown. -Determine if particular chemotherapeutic agents are more effective at inducing apoptosis in cancer cells that overexpress nuclear transport factors. Similar to our results, Yasuhara et al, 2003, determined that active nuclear transport is essential for apoptotic signal transduction. Table 1: An ANCOVA was performed to test the regression of the slopes of apoptosis, nuclear transport and nuclear envelope breakdown to determine a difference of the rates (Figure1, Figure2)