Bergeron, S., Beauchemin, M., and Bertrand, R. (2004). Camptothecin- and etoposide-induced apoptosis in human leukemia cells is independent of cell death receptor-3 and -4 aggregation but accelerates tumor necrosis factor-related apoptosis-inducing ligand-mediated cell death. Mol Cancer Ther 3, 1659-1669.

Cunha, K.S., Reguly, M.L., Graf, U., and Rodrigues de Andrade, H.H. (2002). Comparison of camptothecin derivatives presently in clinical trials: genotoxic potency and mitotic recombination. Mutagenesis 17, 141-147.

Gasiorowski, J.Z., and Dean, D.A. (2003). Mechanisms of nuclear transport and interventions. Adv Drug Deliv Rev 55, 703-716.

Ghobrial, I.M., Witzig, T.E., and Adjei, A.A. (2005). Targeting apoptosis pathways in cancer therapy. CA Cancer J Clin 55, 178-194.

Hsiang, Y.H., Hertzberg, R., Hecht, S., and Liu, L.F. (1985). Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 260, 14873-14878.

Johnstone, R.W., Ruefli, A.A., and Lowe, S.W. (2002). Apoptosis: a link between cancer genetics and chemotherapy. Cell 108, 153-164.

Saleh, E.M., El-Awady, R.A., and Anis, N. (2013). Predictive markers for the response to 5-fluorouracil therapy in cancer cells: Constant-field gel electrophoresis as a tool for prediction of response to 5-fluorouracil-based chemotherapy. Oncol Lett 5, 321-327.

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A. B. Dapi PI

Figure 1: A.500 HeLa cells were counted for each time point. Cells with propidium iodide staining were considered apoptotic. Totals were averaged over three trials. Error bars represent standard error. B. Representative images are shown for control with no treatment and at 9 hours with 100 μL 5-FU and 60 μL camptothecin treatment. A student t-test was done comparing the later hours of apoptosis to that of hour zero, the p-value (one-tailed)=0.004, meaning that they were significantly different.

Figure 2. The effect of 5-fluorouracil and camptothecin on nuclear RanGTP and nuclear envelope integrity

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Figure 2: A RanGTP localization: 500 HeLa cells were counted for each time point and categorized into normal and mislocalized if staining was dispersed and outside the nucleus for mislocalization, and there were three trials. Error bars represent standard error. A student t-test was done comparing the control to the last time point, P(one tail)=0.0023 which indicates that they are significantly different. B. Nuclear integrity: 500 HeLa cells were counted for each time point and categorized into normal nuclear structure and nuclear blebbing, this was determined by the distinct ring showing the nuclear membrane for normal nuclear integrity. The cells were averaged over three trials. The error bars represent standard error. A student t-test was done comparing the control to the last time point, P(one tail)=0.01 indicating that they are significantly different. C Ran staining: representative images at each indicated hour for nuclear RanGTP staining. D Nuclear integrity: representative images at each indicated time point for nuclear envelope integrity which was visualized by nuclear pore staining, mAb414 a monoclonal mouse anti-body.

5-fluorouracil and camptothecin are common chemotherapy drugs that are used in combination treatments in many cancer types. In combination, these two drugs effectively induced apoptosis in at most a ten hour time span (Figure 1A). When active nuclear transport and nuclear envelope breakdown are assayed during apoptosis induced by each drug individually there is a variation in their mechanism of action. Specifically, 5-fluorouracil disrupts nuclear transport independent of nuclear envelope breakdown, whereas camptothecin does not.

During the progression of apoptosis induced by camptothecin and 5-fluorouracil together the combination of these drugs caused nuclear envelope breakdown (Figure 2B) with disruption of nuclear transport coincides with nuclear envelope breakdown, rather than occurring at an earlier time point. This result indicates that in combination these drugs display the mechanism of camptothecin rather than 5-fluorouracil induced apoptosis in regards to disruption of nuclear transport. This result is consistent with camptothecin and it’s derivatives, which have all been shown to have the same mechanisms and have transport disrupted at the same time as nuclear envelope breakdown (Bergeron et al., 2004, Resendes lab, personal communication). However, other apoptosis inducing agents disrupt nuclear transport before nuclear envelope breakdown (Kihlmark et al., 2004). Having nuclear transport active until complete apoptosis could be beneficial in certain chemotherapies that require transport into the nucleus. This could be a differentiation of mechanisms used to achieve apoptosis. Comparing other chemotherapeutic agents that use different mechanisms of apoptosis, particularly looking at which caspases are active would indicate which pathway of apoptosis these combinations go through. This would create a deeper understanding of how nuclear transport could be needed all throughout apoptosis until complete degradation of the cell. 

Effects of 5-fluorouracil and camptothecin on active nuclear transport and nuclear envelope breakdown during apoptosis in HeLa cells

Rebecca Anderson & Dr. Karen K. Resendes, Westminster College, New Wilmington

AcknowledgementsI would like to thank Dr. Karen K. Resendes for all of the help, insight and support she has given to this project.

Literature Cited

Apoptosis, or programmed cell death, normally regulates cell development by controlling the number of cells being produced; it is also involved in the degradation of unhealthy cells (Ghobrial et al., 2005). Defects in apoptosis are linked with the formation of cancer since it can evade the pro-apoptosis pathways and this has been linked to chemotherapy resistant cancer (Johnstone et al., 2002). Apoptosis requires nuclear transport to get signals into and out of the nucleus and this is mediated by RanGTP.

the molecule coming into or out of the cell. RanGTP comes into play in both of these processes. For import, the karyopherins go through the nuclear pore complex then bind with a higher efficiency to RanGTP and release the cargo into the nucleus. For export in order for the designated molecule to move out of the nucleus the karyopherin has to bind to RanGTP before it can bind to the cargo (Gasiorowski and Dean, 2003; Went and Rout, 2010). If RanGTP is not inside of the nucleus then nuclear transport is disrupted.

Camptothecin is a cytotoxic alkaloid and anticancer agent that targets topoisomerase I by stopping it right after it cuts the DNA or RNA involved in replication, transcription, or repair (Cunha et al., 2002; Hsiang et al., 1985). This then creates DNA damage which is sensed by different stress signals and leads to the cascade pathway to apoptosis (Cunha et al., 2002). In previous studies it was shown that nuclear transport coincides with nuclear envelope breakdown (personal communication with Dr. Resendes).

5-Fluorouracil was originally created over 50 years ago as a chemotherapeutic agent and is still used to treat a variety of tumor types (Showalter et al., 2008). Once 5-FU enters the cell it is converted into florodeoxyuridine monophosphate (FdUMP) and forms a complex with another molecule that inhibits deoxythymidine monophosphate (dTMP) function (Saleh et al., 2013). dTMP is crucial for DNA replication and repair, without it this causes cytotoxicity and leads to the extrinsic pathway of apoptosis (Saleh et al., 2013). In a previous capstone study it was found that 5-FU disrupts nuclear transport before nuclear envelope breakdown. This study will investigate the disruption of nuclear transport in apoptosis activated by the combination of 5-fluorouracil and camptothecin on HeLa cells.

Introduction Results

Discussion

MethodsApoptosis:

Twenty-four hours prior to experimentation HeLa cells were seeded onto 12-20 coverslips each in separate wells. Cells were treated with 60μM camptothecin in combination with 100μM 5-fluorouracil, with controls treated with the vehicle methanol. Two to three treatment coverslips as well as a control coverslip were taken every hour and stained with propidium iodide (PI), which is an indicator of apoptosis, and DAPI mounting media, which stains the chromosomes, then adhered to a microscope slide. Using a fluorescent microscope 500 cells were counted and categorized as apoptotic or non-apoptotic based on the nuclear stain PI. Four trials were done to determine an apoptotic timeline. A student t-test was done to compare the control to the later time point to determine if there is a difference.RanGTP:

Cells were seeded into 12-16 coverslips each in separate wells and treated with 60μM camptothecin in combination with 100μM 5-fluorouracil. From the apoptotic timeline found by PI, four time points were tested, hour 4, 5, 6, 7, 8, and 9. Three coverslips were pulled and were fixed with 3% formaldehyde, permiablized with 10X-Triton, and blocked with 5% FBS. Each coverslip was treated with Ran-BD, a monoclonal mouse Ran antibody that would indicate proper nuclear transport, followed by a secondary Santi Cruz anti-mouse antibody that contains fluorphrore, which enables the staining to fluoresce under the microscope. Cells were then stained with DAPI mounting media, and adhered to a microscope slide. Using a fluorescent microscope the cells were categorized into normal RanGTP localization or abnormal RanGTP localization. A student t-test was used to compare the control to the last time point to determine if the difference is significant.Nuclear Integrity:

Cells were seeded into 12-16 coverslips each in separate wells and treated with 60 μM camptothecin in combination with 100μM 5-fluorouracil. From the apoptotic timeline found by PI staining, four hours were used, hour 4, 5, 6, 7, and 8 and three coverslips were pulled and were fixed with 3% formaldehyde, blocked with 10X-Triton, and permiablized with 5% FBS. Each coverslip was treated with mAb414, a monoclonal mouse anti-nuclear pore complex proteins antibody that indicates nuclear membrane integrity, followed by a secondary Santi Cruz anti-mouse antibody. Cells were then stained with DAPI mounting media, and adhered to a microscope slide. Using fluorescent microscope the cells were categorized into having an intact nuclear envelope or having a degraded nuclear envelope.

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Figure 3: Comparison of rates of apoptosis, nuclear degradation and mislocalization of RanGTP.

Figure 3: Comparison of apoptosis, nuclear degradation and mislocalization of RanGTP. An ANCOVA was done to compare the slopes of the line. Df=2 F=1.64 P=0.222. Since the p-value is more then 0.05, this shows that the slopes are not significantly different.

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Transport into the nucleus is mediated by a GTPase called Ran, which is found mainly in the nucleus, and during apoptosis this is interrupted and can be an indicator of apoptosis (Wilde and Zheng, 2009). In regular cell function, molecules larger than 4 kDa in mass and more than 5 nm in diameter have to be facilitated through the nuclear pore complex, which is done by proteins called karyopherins. These proteins bind to a specific sequence on

Overview of nuclearr import and export (Wente and Rout, 2010)

Figure 1. 5-Fluorouracil and camptothecin effectively induces apoptosis in HeLa cells.