Background: Treatment of cells with the anti-cancer drug 5-fluorouracil (5-FU) causes DNA damage, which in turn affects cell proliferation and survival. Two stable wild-type TP53 5-FU-resistant cell lines, ContinB and ContinD, generated from the HCT116 colon cancer cell line, demonstrate moderate and strong resistance to 5-FU, respectively, markedly-reduced levels of 5-FU-induced apoptosis, and alterations in expression levels of a number of key cell cycle-and apoptosis-regulatory genes as a result of resistance development. The aim of the present study was to determine potential differential responses to 8 and 24-hour 5-FU treatment in these resistant cell lines. We assessed levels of 5-FU uptake into DNA, cell cycle effects and apoptosis induction throughout treatment and recovery periods for each cell line, and alterations in expression levels of DNA damage response-, cell cycle-and apoptosis-regulatory genes in response to short-term drug exposure.

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The spindle checkpoint, the primary mechanism to ensure that two daughter cells receive the same amount of DNA, is compromised in many malignant tumors and has been implicated as a contributor to aneuploidy and carcinogenesis. The extent of expression and subcellular localization of the spindle proteins Aurora A, Mad2, and BUBR1 varies considerably in different immunohistochemical (IHC) reports from archival tumor tissues. Given the conflicting reports in the literature about the localization of these proteins, we examined the subcellular localization of Aurora kinase A, Mad2, and BUBR1 in normal and cancerous human tissues by IHC. In normal tissues, Aurora A was mainly localized to the nucleus when monoclonal or purified polyclonal antibodies were used, and Mad2 was localized to the nucleus, whereas BUBR1 was localized to the cytoplasm. In malignant tissues, Aurora A showed additional staining in the cytoplasm in the majority of tumors analyzed. Furthermore, BUBR1 was also localized to both the nucleus and cytoplasm in a significant fraction of tumors. Subcellular localization of Mad2 was similar in normal and malignant tissues. Thus, the validity of some earlier IHC studies of Aurora A, Mad2, and BUBR1 should be reconsidered, indicating that high-quality antibodies and a high-alkaline antigen-retrieval technique are required to achieve optimal results. We conclude that the subcellular localizations of these spindle proteins are different, although they have overlapping biological functions, and that Aurora A and BUBR1 undergo a shift in the subcellular localization during malignant transformation.

Background: Established colorectal cancer cell lines subjected to different 5-fluorouracil (5-FU) treatment protocols are often used as in vitro model systems for investigations of downstream cellular responses to 5-FU and to generate 5-FU-resistant derivatives for the investigation of biological mechanisms involved in drug resistance. We subjected HCT116 colon cancer cells to two different 5-FU treatment protocols in an attempt to generate resistant derivatives: one that simulated the clinical bolus regimens using clinically-achievable 5-FU levels, the other that utilized serial passage in the presence of increasing 5-FU concentrations (continuous exposure). HCT116 Bolus3, ContinB, and ContinD, corresponding to independently-derived cell lines generated either by bolus exposure or continuous exposure, respectively, were characterized for growth-and apoptosis-associated phenotypes, and gene expression using 8.5 K oligonucleotide microarrays. Comparative gene expression analyses were done in order to determine if transcriptional profiles for the respective treatment derivatives were similar or substantially different, and to identify the signaling and regulatory pathways involved in mediating the downstream response to 5-FU exposure and possibly involved in development of resistance.

Loss of TP53 function may contribute to 5fluorouracil (5-FU) resistance in colorectal cancer since TP53-deficient cells may be unable to undergo apoptosis in response to 5-FU-induced DNA damage. 5-FU treatment of TP53-deficient cells would provide useful information on the apoptotic response to drug-induced DNA damage in the absence of TP53 and its transcriptional targets. We investigated apoptosis induction and cell cycle alterations in response to short-term treatment with two different 5-FU concentrations following siRNA-mediated knockdown of TP53 in the TP53-proficient HCT116 colon cancer cell line. We focused on high-dose 5-FU treatment to investigate the apoptotic phenotype in 5-FU-treated cultures since this dose resulted in apoptosis induction at 24 h of treatment, whereas clinically-relevant bolus 5-FU treatment of HCT116 cultures did not. Gene expression alterations were also assessed in 5-FU-treated HCT116 cultures using whole genome expression arrays. Compared to 5-FU-treated TP53-proficient HCT116 cultures, 5-FU-treated TP53depleted HCT116 cultures showed lack of CDKN1A induction, decreased apoptotic levels, decreased FAS and TNFRSF10B transcript levels and cleaved PARP protein levels, G 1 /S transition arrests, decreased CCND1 protein levels, and smaller intra-S phase arrests. Alterations in gene expression in 5-FU-treated TP53-depleted HCT116 cultures confirmed previously-reported TP53 target genes and suggested potentially novel TP53 target genes (e.g. APOBEC3C, BIRC3, JMJD2B, LAMP3, MYO1E, PRRG1, SULF2, TACSTD2, TncRNA, ZFYVE20) that may play a role in mediating the 5-FU-induced DNA damage response in TP53-proficient cells. Abrogation of TP53 function in 5-FUtreated HCT116 cultures results in reduced apoptosis, TP53and CDKN1A-independent G 1 /S phase arrests that may be protective against apoptosis, smaller intra-S phase arrests, and transcript level decreases of both reported TP53 target genes as well as potentially novel TP53 target genes.