Development of IFN-gamma resistance is associated with attenuation of SOCS genes induction and constitutive expression of SOCS 3 in melanoma cells - PubMed (original) (raw)

Development of IFN-gamma resistance is associated with attenuation of SOCS genes induction and constitutive expression of SOCS 3 in melanoma cells

M Fojtova et al. Br J Cancer. 2007.

Abstract

The resistance to interferons (IFNs) limits their anticancer therapeutic efficacy. Here we studied the evolution of an IFN-resistant state in vitro using melanoma cell lines. We found that the cells became less sensitive to antiproliferative effect of IFN-gamma after prolonged cultivation enabling us to isolate sensitive and resistant subclones of the parental line. We investigated transcription of signal transducer and activator of transcription (STAT) 1-6 and suppressor of cytokine signalling (SOCS) 1-3 genes, and phosphorylation of STAT 1 protein. The resistant subline (termed WM 1158R) differed from the sensitive subline (WM 1158S) by a constitutive expression of SOCS 3, lack or weak SOCS 1-3 activation following IFN-gamma, and short duration of cytokine activatory signal. Similar correlations were observed in additional melanoma lines differing in IFN sensitivities. At the protein level, IFN-gamma induced strong and prolonged STAT 1 activation at serine 727 (S727) in WM 1158R while in WM 1158S cells phosphorylation of this amino acid was much less pronounced. On the other hand, phosphorylation of tyrosine 701 (Y701) was stimulated regardless of the sensitivity phenotype. In conclusion, constitutive expression of SOCS 3 is correlated with attenuation of its induction following IFN treatment. These results suggest that progression of melanoma cells from IFN sensitivity to IFN insensitivity associates with changes in SOCS expression.

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Figures

Figure 1

Figure 1

Antiproliferative effect of IFN-γ in WM 1158S and WM 1158R sublines measured by WST-1 colourimetric test. (A and B) The growth is expressed as the amount of viable cells in treated and untreated samples. Each value represents a mean from three independent experiments. Error bars indicate s.d. (C) The cell viability normalised to untreated controls. The differences between WM 1158S and WM 1158R sublines were statistically significant (P<0.01) for all time intervals (24, 48 and 72 h).

Figure 2

Figure 2

Analysis of STAT and SOCS transcripts in the sensitive WM 1158S subline. (A) Total RNAs were prepared from cells treated with IFN-γ for 30 min, 24 and 72 h and from non-treated controls. The DNA products of RT–PCRs were separated on 2% agarose gels. (B) Transcription is expressed as a SOCS/GAPDH ratio or (C) fold increase over basal level. Means of three independent experiments are shown.

Figure 3

Figure 3

Analysis of STAT and SOCS transcripts in the resistant WM 1158R subline. The conditions and experimental set-up were as in Figure 2.

Figure 4

Figure 4

Analysis of STAT and SOCS transcripts in normal human melanocytes. Description of lanes and panels is as in Figure 2. Results of a single experiment are shown.

Figure 5

Figure 5

STAT 1 phosphorylation levels in both IFN-resistant and IFN-sensitive WM 1158 sublines. The proteins were extracted from the same cellular pool as used for RNA analysis and analysed by Western blot. All experiments were performed in triplicates.

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