Transcriptional profiling of MCF7 breast cancer cells in response to 5‐Fluorouracil: Relationship with cell cycle changes and apoptosis, and identification of novel targets of p53

Abstract

The availability of oral precursors of 5-Fluorouracil (5-FU) and its favorable results in treating advanced breast cancer have renewed the interest in the molecular mechanisms underlying its cytotoxicity. We have compared the changes in cell cycle and cell death parameters induced by 2 different concentrations of 5-FU (IC50 and IC80) in the breast adenocarcinoma cell line MCF7. G1/S cell cycle arrest was associated with both concentrations, whereas cell death was mainly induced after IC80 5-FU. These changes were correlated with gene expression assessed by cDNA microarray analysis. Main findings included an overexpression of p53 target genes involved in cell cycle and apoptosis (CDKN1A/ p21, TP53INP, TNFRSF6/FAS and BBC3/PUMA), and significant repression of Myc. High dose 5-FU also induced a higher regulation of the mitochondrial death genes APAF1, BAK1 and BCL2, and induction of genes of the ID family. Furthermore, we establish a direct causal relationship between p21, ID1 and ID2 overexpression, increased acetylation of histones H3 and H4 and binding of p53 to their promoters as a result of 5-FU treatment. The relevance of these findings was further studied after interfering p53 expression in MCF7 cells (shp53 cells), showing a lower induction of both, ID1 and ID2 transcripts, after 5-FU when compared with MCF7 shGFP control cells. This molecular characterization of dose-and time-dependent modifications of gene expression after 5-FU treatment should provide a resource for future basic studies addressing the molecular mechanisms of chemotherapy in breast cancer. ' 2006 Wiley-Liss, Inc.Key words: 5-Fluorouracil; cDNA microarrays; p53; E2F1; ID proteins Carcinogenesis is a multistep process involving genetic and epigenetic alterations that drive the progressive transformation of normal cells into malignant types. Deregulated processes involved in tumorigenesis, such as regulation of cell cycle progression, angiogenesis and apoptosis, provide rational targets for novel therapies. For this reason, there is now more emphasis on understanding the mechanisms of carcinogenesis and how these can be exploited when designing new therapeutic agents. 1 Capecitabine is an example of a rationally designed cytotoxic treatment. It is designed to generate 5-Fluorouracil (5-FU) preferentially in tumor cells, by exploiting the higher activity of the activating enzyme thymidine phosphorylase in tumors compared with healthy tissues. Tumor-specific activation has the potential to increase intratumor drug levels to enhance efficacy and minimize toxicity. Proof of this principle is provided by clinical trial results, showing that capecitabine is effective and has a favorable safety profile in the treatment of metastatic breast cancer. 1 Capecitabine active metabolite, 5-FU, is one of the most widely used chemotherapy drugs for the treatment of solid tumors. However, despite its extensive use in clinical practice, only in recent years we have began to understand the molecular basis for 5-FU toxicity on cancer cells. As a pyrimidine ana...

In Vitro and In Vivo Evaluation of a Folate-Targeted Copolymeric Submicrohydrogel Based on N-Isopropylacrylamide as 5-Fluorouracil Delivery System

“…The concentration range of free 5-FU was selected based on preliminary experiments, in which increasing concentrations of 5-drug from 0.01 µM to 1 mM were tested in culture. Those drug concentrations were in accordance with data reported in the literature [33][34][35]. The administration of 5-FU 10 μM in solution produced a decrease of the percentage of viability in MCF7 cells, which was between 91 ± 8% and 43 ± 3% after 3 days of incubation.…”

Molecular profiling of docetaxel cytotoxicity in breast cancer cells: uncoupling of aberrant mitosis and apoptosis

“…After culture at different time points, samples were harvested (including detached cells), suspended in phosphate-buffered saline (PBS), fixed in 70% ethanol, and their DNA content was evaluated after PI staining, as described previously (Herna´ndez-Vargas et al, 2006). Fluorescence-activated cell sorting analysis was carried out using a FACScan flow cytometer (Becton Dickinson, San Diego, CA, USA) and CellQuest software.…”

“…Three micrograms of total RNA from samples and Universal Human Reference RNA (Stratagene, La Jolla, CA, USA) were used for amplification using the Superscript Choice System (Life Technologies Inc., Gaithersburg, MD, USA) and in vitro transcription with Megascript T7 (Ambion, Austin, TX, USA), as described previously (MorenoBueno et al, 2003;Herna´ndez-Vargas et al, 2006). The cDNA array chip is the CNIO Oncochip manufactured by the CNIO Genomic Unit (http://bioinfo.cnio.es/data/oncochip).…”

Inhibitor of Differentiation/DNA Binding 1 (ID1) Inhibits Etoposide-induced Apoptosis in a c-Jun/c-Fos-dependent Manner

“…Cancer Cells-Previous studies indicated that ID1 was commonly up-regulated by chemotherapeutic drug treatment (18,19). To evaluate the possible role of ID1 in ESCC, we first analyzed ID1 expression in ESCC tumor tissues and ESCC cell lines Fig.…”

“…Notably, ID1 was involved in chemotherapy and radiotherapy resistance in human cancers including pancreatic adenocarcinoma, breast cancer, lung cancer, colorectal cancer, and esophageal cancer and becomes a new potential therapeutic target (13)(14)(15)(16)(17). ID1 is transactivated in the context of 5-fluorouracil therapy, which provides a resource for future study addressing the molecular mechanisms of chemotherapy in breast cancer (18). In the study of p53 protecting cells from arsenic caused cell cycle arrest, ID1 is more extensive induced by arsenite in p53 ϩ/ϩ cells rather than p53-deficient cells, which display greater resistance to arsenite-induced mitotic arrest and apoptosis (19).…”