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. 2008 Feb 27:5:3.
doi: 10.1186/1476-9255-5-3.

Inhibition of oncogene-induced inflammatory chemokines using a farnesyltransferase inhibitor

Katharine C Degeorge  1 , Brent R Degeorge Jr, James S Testa, Jay L Rothstein
Affiliations

Inhibition of oncogene-induced inflammatory chemokines using a farnesyltransferase inhibitor

Katharine C Degeorge et al. J Inflamm (Lond). .

Abstract

Background: Farnesyltransferase inhibitors (FTI) are small molecule agents originally formulated to inhibit the oncogenic functions of Ras. Although subsequent analysis of FTI activity revealed wider effects on other pathways, the drug has been demonstrated to reduce Ras signaling by direct measurements. The purpose of the current study was to determine if FTI could be used to inhibit the inflammatory activities of a known Ras-activating human oncoprotein, RET/PTC3. RET/PTC3 is a fusion oncoprotein expressed in the thyroid epithelium of patients afflicted with thyroid autoimmune disease and/or differentiated thyroid carcinoma. Previous studies have demonstrated that RET/PTC3 signals through Ras and can provoke nuclear translocation of NFkappaB and the downstream release of pro-inflammatory mediators from thyroid follicular cells in vitro and in vivo, making it an ideal target for studies using FTI.

Methods: For the studies described here, an in vitro assay was developed to measure FTI inhibition of RET/PTC3 pro-inflammatory effects. Rat thyrocytes transfected with RET/PTC3 or vector control cDNA were co-cultured with FTI and examined for inhibition of chemokine expression and secretion measured by RT-PCR and ELISA. Immunoblot analysis was used to confirm the level at which FTI acts on RET/PTC3-expressing cells, and Annexin V/PI staining of cells was used to assess cell death in RET/PTC3-expressing cells co-cultured with FTI.

Results: These analyses revealed significant mRNA and protein inhibition of chemokines Ccl2 and Cxcl1 with nanomolar doses of FTI. Neither RET/PTC3 protein expression nor apoptosis were affected at any dose of FTI investigated.

Conclusion: These data suggest that FTI may be applied as an effective inhibitor for RET/PTC3-oncogene induced pro-inflammatory mediators.

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Figures

Figure 1
Figure 1
Oncogene-induced expression of pro-inflammatory genes in RP3-expressing thyrocytes. Shown is the baseline gene expression of the housekeeping gene G3pdh, pro-inflammatory genes Ccl2 and Cxcl1, and the oncogene RP3 in PC Cl3RP3 and PC CL3pMV7 stably-transfected thyrocytes. Bars depict densitometric analysis of RT-PCR data. Cells expressing RP3 also express the pro-inflammatory genes Ccl2 and Cxcl1, whereas vector controls do not.
Figure 2
Figure 2
Reduced expression of pro-inflammatory chemokines in FTI-treated thyrocytes. (A) mRNA expression of the pro-inflammatory chemokine Ccl2 in PC Cl3RP3 thyrocytes cultured with indicated concentrations of FTI for 24 hours. (B) mRNA expression of the pro-inflammatory chemokine Cxcl1 in PC Cl3RP3 thyrocytes cultured with indicated concentrations of FTI for 24 hours. For both chemokines investigated, significant reduction in gene expression is seen, even at nanomolar concentrations of FTI. Data are normalized to G3pdh expression within each sample. Error bars represent s.e.m. and are representative of three independent experiments performed in triplicate (n = 3). *p < 0.05, **p < 0.01.
Figure 3
Figure 3
Reduced pro-inflammatory protein secretion in FTI-treated thyrocytes. (A) Ccl2 protein secretion measured by ELISA from PC Cl3RP3 thyrocytes cultured with indicated concentrations of FTI for 24 hours. (B) Cxcl1 protein secretion measured by ELISA from PC Cl3RP3 thyrocytes cultured with indicated concentrations of FTI for 24 hours. Significant reduction of protein secretion compared to control is seen for both chemokines. Error bars represent s.e.m. and are representative of three independent experiments performed in triplicate (n = 3). *p < 0.05, **p < 0.01.
Figure 4
Figure 4
No effect of FTI on oncogene expression and phosphorylation. Shown is an immunoblot analysis of RP3 and phospho-RP3 protein expression in PC CL3RP3 thyrocytes cultured with indicated concentrations of FTI for 24 hours. RP3 protein expression and phosphorylation was unchanged at any concentration of FTI. The data are representative of three independent experiments performed in triplicate.
Figure 5
Figure 5
Apoptosis is unaffected in FTI-treated thyrocytes. (A) Shown are data from FITC-conjugated Annexin V and PI stained PC Cl3RP3 thyrocytes cultured with indicated concentrations of FTI for 24 hours and analyzed by flow cytometry. Cells staining with Annexin V only represent early apoptotic cells, and the percentages of this population at indicated concentrations of FTI are depicted in the histogram (n = 3). There is no significant change in induction of apoptosis at any concentration of FTI. (B) Shown are representative dot plots from flow cytometry. Error bars represent s.e.m. and are representative of three independent experiments.
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