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Gene trapping identifies chloride channel 4 as a novel inducer of colon cancer cell migration, invasion and metastases.

Ishiguro T, Avila H, Lin SY, Nakamura T, Yamamoto M, Boyd DD - Br. J. Cancer (2010)

Bottom Line: Rapid amplification of cDNA ends identified the trapped gene as the chloride channel 4 (CLCN4) exchanger.Stable expression of a CLCN4 cDNA enhanced motility, whereas cells knocked down or for this transcript showed reduced migration/invasion.Intra-splenic delivery of RKO-CLCN4 transfectants, but not controls, yielded liver metastases, and transcript levels were higher in colon cancer metastases to the liver when compared with primary tumours.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology Department, MD Anderson Cancer Center, Houston, TX 77030, USA.

ABSTRACT

Background: To date, there are few reports on gene products contributing to colon cancer progression.

Methods: We used a gene trap comprised of an enhanced retroviral mutagen (ERM) cassette that includes a tetracycline-responsive promoter upstream of a haemagglutinin (HA) tag and a splice donor site. Integration of the ERM within an endogenous gene yields a tetracycline-regulated HA-tagged transcript. We transduced RKO colon cancer cells expressing a tetracycline trans-activator-off with the ERM-encoding retrovirus and screened for enhanced migration.

Results: One clone showed fivefold enhanced migration with tetracycline withdrawal. Rapid amplification of cDNA ends identified the trapped gene as the chloride channel 4 (CLCN4) exchanger. Stable expression of a CLCN4 cDNA enhanced motility, whereas cells knocked down or for this transcript showed reduced migration/invasion. CLCN4-overexpressing RKO colon cancer cells were more resistant than controls to proton load-induced cytotoxicity, consistent with the H(+)-extruding function of this antiporter. Intra-splenic delivery of RKO-CLCN4 transfectants, but not controls, yielded liver metastases, and transcript levels were higher in colon cancer metastases to the liver when compared with primary tumours.

Conclusions: CLCN4 is a novel driver of colon cancer progression.

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Related in: MedlinePlus

CLCN4-overexpressing colon cancer cells are more resistant to acid-induced cytotoxicity. Pooled RKO clones overexpressing full-length CLCN4 cDNA (RKO-CLCN4) or the empty vector (RKO-control) were cultured for 72 h at the specified pH (using a HEPES-buffered medium with hydrochloric acid/sodium hydroxide) and assayed for viability using MTT. Data are expressed relative to the value at pH 7.4. The experiment was undertaken three times. Statistical analysis was performed using an unpaired t-test.
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fig9: CLCN4-overexpressing colon cancer cells are more resistant to acid-induced cytotoxicity. Pooled RKO clones overexpressing full-length CLCN4 cDNA (RKO-CLCN4) or the empty vector (RKO-control) were cultured for 72 h at the specified pH (using a HEPES-buffered medium with hydrochloric acid/sodium hydroxide) and assayed for viability using MTT. Data are expressed relative to the value at pH 7.4. The experiment was undertaken three times. Statistical analysis was performed using an unpaired t-test.

Mentions: Considering the fact that CLCN4, as a proton/Cl exchanger, extrudes protons from the cell against its electrochemical gradient (Picollo and Pusch, 2005), we argued that cells overexpressing the exchanger would be more resistant to the cytotoxic effect of an extracellular proton load. To answer this question, we cultured RKO cells overexpressing CLCN4, or the vector only, in an acidic medium akin to the pH evident in the tumour microenvironment (Gatenby and Gillies, 2004). Indeed, whereas over 35% of CLCN4-overexpressing RKO cells survived the reduced pH of 6.6, only 10% of vector controls tolerated the increased acidity (Figure 9), and this difference was statistically significantly (P=0.01).


Gene trapping identifies chloride channel 4 as a novel inducer of colon cancer cell migration, invasion and metastases.

Ishiguro T, Avila H, Lin SY, Nakamura T, Yamamoto M, Boyd DD - Br. J. Cancer (2010)

CLCN4-overexpressing colon cancer cells are more resistant to acid-induced cytotoxicity. Pooled RKO clones overexpressing full-length CLCN4 cDNA (RKO-CLCN4) or the empty vector (RKO-control) were cultured for 72 h at the specified pH (using a HEPES-buffered medium with hydrochloric acid/sodium hydroxide) and assayed for viability using MTT. Data are expressed relative to the value at pH 7.4. The experiment was undertaken three times. Statistical analysis was performed using an unpaired t-test.
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Related In: Results  -  Collection

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fig9: CLCN4-overexpressing colon cancer cells are more resistant to acid-induced cytotoxicity. Pooled RKO clones overexpressing full-length CLCN4 cDNA (RKO-CLCN4) or the empty vector (RKO-control) were cultured for 72 h at the specified pH (using a HEPES-buffered medium with hydrochloric acid/sodium hydroxide) and assayed for viability using MTT. Data are expressed relative to the value at pH 7.4. The experiment was undertaken three times. Statistical analysis was performed using an unpaired t-test.
Mentions: Considering the fact that CLCN4, as a proton/Cl exchanger, extrudes protons from the cell against its electrochemical gradient (Picollo and Pusch, 2005), we argued that cells overexpressing the exchanger would be more resistant to the cytotoxic effect of an extracellular proton load. To answer this question, we cultured RKO cells overexpressing CLCN4, or the vector only, in an acidic medium akin to the pH evident in the tumour microenvironment (Gatenby and Gillies, 2004). Indeed, whereas over 35% of CLCN4-overexpressing RKO cells survived the reduced pH of 6.6, only 10% of vector controls tolerated the increased acidity (Figure 9), and this difference was statistically significantly (P=0.01).

Bottom Line: Rapid amplification of cDNA ends identified the trapped gene as the chloride channel 4 (CLCN4) exchanger.Stable expression of a CLCN4 cDNA enhanced motility, whereas cells knocked down or for this transcript showed reduced migration/invasion.Intra-splenic delivery of RKO-CLCN4 transfectants, but not controls, yielded liver metastases, and transcript levels were higher in colon cancer metastases to the liver when compared with primary tumours.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology Department, MD Anderson Cancer Center, Houston, TX 77030, USA.

ABSTRACT

Background: To date, there are few reports on gene products contributing to colon cancer progression.

Methods: We used a gene trap comprised of an enhanced retroviral mutagen (ERM) cassette that includes a tetracycline-responsive promoter upstream of a haemagglutinin (HA) tag and a splice donor site. Integration of the ERM within an endogenous gene yields a tetracycline-regulated HA-tagged transcript. We transduced RKO colon cancer cells expressing a tetracycline trans-activator-off with the ERM-encoding retrovirus and screened for enhanced migration.

Results: One clone showed fivefold enhanced migration with tetracycline withdrawal. Rapid amplification of cDNA ends identified the trapped gene as the chloride channel 4 (CLCN4) exchanger. Stable expression of a CLCN4 cDNA enhanced motility, whereas cells knocked down or for this transcript showed reduced migration/invasion. CLCN4-overexpressing RKO colon cancer cells were more resistant than controls to proton load-induced cytotoxicity, consistent with the H(+)-extruding function of this antiporter. Intra-splenic delivery of RKO-CLCN4 transfectants, but not controls, yielded liver metastases, and transcript levels were higher in colon cancer metastases to the liver when compared with primary tumours.

Conclusions: CLCN4 is a novel driver of colon cancer progression.

Show MeSH
Related in: MedlinePlus