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

De Angelis PM, Kravik KL, Tunheim SH, Haug T, Reichelt WH - Mol. Cancer (2004)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pathology, Rikshospitalet, 0027 Oslo, Norway. a.p.de@labmed.uio.no

ABSTRACT

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.

Conclusion: 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.

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Apoptotic response of HCT116 parental and treatment-derivatives to transient 5-FU challenge for 24 hours. 5-FU was continuously present in the media of exponentially-growing cells for 24 hours. Apoptotic fractions were measured using the TUNEL assay as outlined in Materials and Methods. % apoptosis refers to the number of TdT-positive cells, i.e., cells that have been end-labeled with a biotin-labeled nucleotide and counterstained with a streptavidin-conjugated FITC fluorochrome.
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Figure 1: Apoptotic response of HCT116 parental and treatment-derivatives to transient 5-FU challenge for 24 hours. 5-FU was continuously present in the media of exponentially-growing cells for 24 hours. Apoptotic fractions were measured using the TUNEL assay as outlined in Materials and Methods. % apoptosis refers to the number of TdT-positive cells, i.e., cells that have been end-labeled with a biotin-labeled nucleotide and counterstained with a streptavidin-conjugated FITC fluorochrome.

Mentions: Transient challenge with several different concentrations of 5-FU resulted in significantly lower apoptotic fractions for the ContinB and ContinD derivatives, respectively, compared to the sensitive parental cells at 24 hours (Figure 1). The Bolus3 derivative responded to the same 5-FU concentrations by inducing dose-dependent levels of apoptosis; the apoptotic fractions at 24 hours were very similar to those seen in the parental cell line (Figure 1).


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

De Angelis PM, Kravik KL, Tunheim SH, Haug T, Reichelt WH - Mol. Cancer (2004)

Apoptotic response of HCT116 parental and treatment-derivatives to transient 5-FU challenge for 24 hours. 5-FU was continuously present in the media of exponentially-growing cells for 24 hours. Apoptotic fractions were measured using the TUNEL assay as outlined in Materials and Methods. % apoptosis refers to the number of TdT-positive cells, i.e., cells that have been end-labeled with a biotin-labeled nucleotide and counterstained with a streptavidin-conjugated FITC fluorochrome.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC411052&req=5

Figure 1: Apoptotic response of HCT116 parental and treatment-derivatives to transient 5-FU challenge for 24 hours. 5-FU was continuously present in the media of exponentially-growing cells for 24 hours. Apoptotic fractions were measured using the TUNEL assay as outlined in Materials and Methods. % apoptosis refers to the number of TdT-positive cells, i.e., cells that have been end-labeled with a biotin-labeled nucleotide and counterstained with a streptavidin-conjugated FITC fluorochrome.
Mentions: Transient challenge with several different concentrations of 5-FU resulted in significantly lower apoptotic fractions for the ContinB and ContinD derivatives, respectively, compared to the sensitive parental cells at 24 hours (Figure 1). The Bolus3 derivative responded to the same 5-FU concentrations by inducing dose-dependent levels of apoptosis; the apoptotic fractions at 24 hours were very similar to those seen in the parental cell line (Figure 1).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pathology, Rikshospitalet, 0027 Oslo, Norway. a.p.de@labmed.uio.no

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus