Comparison of gene expression in HCT116 treatment derivatives generated by two different 5-fluorouracil exposure protocols.
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.


RESULTS
HCT116 ContinB and ContinD cells were respectively 27-fold and >100-fold more resistant to 5-FU and had reduced apoptotic fractions in response to transient 5-FU challenge compared to the parental cell line, whereas HCT116 Bolus3 cells were not resistant to 5-FU after 3 cycles of bolus 5-FU treatment and had the same apoptotic response to transient 5-FU challenge as the parental cell line. However, gene expression levels and expression level changes for all detected genes in Bolus3 cells were similar to those seen in both the ContinB (strongest correlation) and ContinD derivatives, as demonstrated by correlation and cluster analyses. Regulatory pathways having to do with 5-FU metabolism, apoptosis, and DNA repair were among those that were affected by 5-FU treatment.


CONCLUSIONS
All HCT116 derivative cell lines demonstrated similar transcriptional profiles, despite the facts that they were generated by two different 5-FU exposure protocols and that the bolus exposure derivative had not become resistant to 5-FU. Selection pressures on HCT116 cells as a result of 5-FU challenge are thus similar for both treatment protocols.
De Angelis
Paula M
PM
Institute of Pathology, Rikshospitalet, 0027 Oslo, Norway. a.p.de@labmed.uio.no
Kravik
Katherine L
KL
Tunheim
Siv H
SH
Haug
Terje
T
Reichelt
Wenche H
WH
D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D016000:Cluster Analysis; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D005472:Fluorouracil / Q000008:administration & dosage* / Q000494:pharmacology* / Q000627:therapeutic use; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics
D012334:RNA, Neoplasm; D005472:Fluorouracil