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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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Cluster analysis of signal log2 ratios (gene expression changes) for four derivative cell lines. Similar gene expression changes are demonstrated in the grouped pairs of treatment-derivatives, varying in degree of difference according the linkage distance. The smaller the linkage distance, the more similar the gene expression changes. ContinB and Bolus3 derivatives had similar gene expression changes, as did ContinD and Bolus1 derivatives.
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Figure 3: Cluster analysis of signal log2 ratios (gene expression changes) for four derivative cell lines. Similar gene expression changes are demonstrated in the grouped pairs of treatment-derivatives, varying in degree of difference according the linkage distance. The smaller the linkage distance, the more similar the gene expression changes. ContinB and Bolus3 derivatives had similar gene expression changes, as did ContinD and Bolus1 derivatives.

Mentions: Correlation analyses of raw signal intensity values (gene expression levels) demonstrated strong correlations between each pair of cell lines tested (correlation coefficients typically greater than 0.90), but the strongest correlations were demonstrated between the Bolus3 and ContinB derivatives, the Bolus3 and Bolus1 derivatives, and the ContinD and parental cell lines, indicating that these pairs of cell lines tended to have the same gene expression level patterns (Table 1). For example, if one cell line in a correlated pair of cell lines had an elevated expression level for a particular gene, the other cell line tended to have an elevated expression level for the same gene, and vice versa, if one cell line had a low gene expression level for a particular gene, the other cell line tended to have the same. However, the actual signal intensity values were not exactly the same for each gene in a correlated pair of cell lines, although in some cases they could be. Cluster analysis performed for all cell lines using signal intensity values demonstrated groupings between ContinB and Bolus3, and between ContinD and Bolus1 (Figure 2). The length of the vertical bars on the cluster plot indicates the degree of difference in gene expression level patterns between cell lines, showing that the ContinB/Bolus3 and ContinD/Bolus1 cell line pairs were more similar to each other than either of these pairs was to the parental line for this parameter. ContinB and Bolus3 derivatives and Bolus3 and Bolus1 derivatives were strongly correlated for the signal log2 ratio parameter, demonstrating that gene expression changes in the derivative cells (relative to the parental cells against which they were compared) were very similar in these pairs of cell lines. The ContinD derivative demonstrated moderate correlations with the Bolus3 derivative, with the ContinB derivative, and with the Bolus1 derivative for the same parameter (Table 1), indicating that gene expression changes were more dissimilar in these pairs of cell lines. The length of the vertical bars on the cluster plot in Figure 3 (cluster analysis for signal log2 ratios) indicates the degree of difference in gene expression changes between cell lines, showing that the ContinB/Bolus3 and ContinD/Bolus1 cell line pairs were again more similar to each other than either of these pairs was to the other pair for this parameter.


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)

Cluster analysis of signal log2 ratios (gene expression changes) for four derivative cell lines. Similar gene expression changes are demonstrated in the grouped pairs of treatment-derivatives, varying in degree of difference according the linkage distance. The smaller the linkage distance, the more similar the gene expression changes. ContinB and Bolus3 derivatives had similar gene expression changes, as did ContinD and Bolus1 derivatives.
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Related In: Results  -  Collection

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Figure 3: Cluster analysis of signal log2 ratios (gene expression changes) for four derivative cell lines. Similar gene expression changes are demonstrated in the grouped pairs of treatment-derivatives, varying in degree of difference according the linkage distance. The smaller the linkage distance, the more similar the gene expression changes. ContinB and Bolus3 derivatives had similar gene expression changes, as did ContinD and Bolus1 derivatives.
Mentions: Correlation analyses of raw signal intensity values (gene expression levels) demonstrated strong correlations between each pair of cell lines tested (correlation coefficients typically greater than 0.90), but the strongest correlations were demonstrated between the Bolus3 and ContinB derivatives, the Bolus3 and Bolus1 derivatives, and the ContinD and parental cell lines, indicating that these pairs of cell lines tended to have the same gene expression level patterns (Table 1). For example, if one cell line in a correlated pair of cell lines had an elevated expression level for a particular gene, the other cell line tended to have an elevated expression level for the same gene, and vice versa, if one cell line had a low gene expression level for a particular gene, the other cell line tended to have the same. However, the actual signal intensity values were not exactly the same for each gene in a correlated pair of cell lines, although in some cases they could be. Cluster analysis performed for all cell lines using signal intensity values demonstrated groupings between ContinB and Bolus3, and between ContinD and Bolus1 (Figure 2). The length of the vertical bars on the cluster plot indicates the degree of difference in gene expression level patterns between cell lines, showing that the ContinB/Bolus3 and ContinD/Bolus1 cell line pairs were more similar to each other than either of these pairs was to the parental line for this parameter. ContinB and Bolus3 derivatives and Bolus3 and Bolus1 derivatives were strongly correlated for the signal log2 ratio parameter, demonstrating that gene expression changes in the derivative cells (relative to the parental cells against which they were compared) were very similar in these pairs of cell lines. The ContinD derivative demonstrated moderate correlations with the Bolus3 derivative, with the ContinB derivative, and with the Bolus1 derivative for the same parameter (Table 1), indicating that gene expression changes were more dissimilar in these pairs of cell lines. The length of the vertical bars on the cluster plot in Figure 3 (cluster analysis for signal log2 ratios) indicates the degree of difference in gene expression changes between cell lines, showing that the ContinB/Bolus3 and ContinD/Bolus1 cell line pairs were again more similar to each other than either of these pairs was to the other pair for this parameter.

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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Related in: MedlinePlus