Interferon-resistant Human Melanoma Cells Are Deficient in ISGF3 Components, STAT1, STAT2, and p48-ISGF3γ

Abstract

The mechanism of IFN resistance was examined in three long-term cell lines, SK-MEL-28, SK-MEL-3, and MM96, exhibiting significant variation in responsiveness to the antiproliferative and antiviral effects of type I IFNs. The JAK-STAT components involved in IFN signal transduction were analyzed in detail. After exposure to IFN, activation of the IFN type I receptor-linked tyrosine kinases, JAK-1 and TYK-2, was detected at similar levels in both IFN-sensitive and IFN-resistant cell types, indicating that IFN resistance did not result from a deficiency in signaling at the level of receptor-associated kinase activation. However, analysis of ISGF3 transcription factor components, STAT1, STAT2, and p48-ISGF3␥, revealed that their expression and activation correlated with cellular IFN responsiveness. The analysis was extended to also include IFN-sensitive primary melanocytes, three additional IFN-resistant melanoma cell lines, and seven cell cultures recently established from melanoma patient biopsies. It was consistently observed that the most marked difference in ISGF3 was a lack of STAT1 in the resistant versus the sensitive cells. Transfection of the IFN-resistant MM96 cell line to express increased levels of STAT1 protein partially restored IFN responsiveness in an antiviral assay. We conclude that a defect in the level of STAT1 and possibly all three ISGF3 components in IFN-resistant human melanoma cells may be a general phenomenon responsible for reduced cellular responsiveness of melanomas to IFNs.The interferons (IFNs) comprise a family of multifunctional polypeptides, recognized for their antiviral, antiproliferative, and immunoregulatory functions (reviewed in Refs. 1 and 2). Type I IFNs, IFN-␣ and -␤, exert their biological actions by binding to high-affinity cell-surface receptors that stimulate phosphorylation of tyrosine residues on type I receptor components (3, 4) and on the receptor-associated tyrosine kinases, TYK-2 and JAK-1 (5-7). Following tyrosine phosphorylation, the activated tyrosine kinases then induce the formation and activation by tyrosine phosphorylation of the latent cytoplasmic transcription factor IFN-stimulated gene factor 3␣ (ISGF3␣) (8). The ISGF3␣ transcription factor is a complex of STAT molecules, including STAT1 and STAT2. STAT1 exists in two forms of 91 kDa (STAT1␣) and 84 kDa (STAT1␤), and STAT2 is a 113-kDa protein (reviewed in Refs. 9 and 10). During the classical type I IFN response, the tyrosine-phosphorylated STAT1 and STAT2 proteins form heterodimers (11), which complex with p48-ISGF3␥, a DNA-binding protein. (23), and in many other cancer cell types (24). In addition, melanoma cell lines with a wide variation in their responsiveness to the antiproliferative (25) and antiviral (26) activities of IFNs have been described. We have previously demonstrated that the difference in responsiveness among these melanoma cell lines is related neither to the receptor binding affinity nor to the numbers of IFN receptors on the cell surface (27). In addition, the cDNA sequence encodi...

High density oligonucleotide array analysis of interferon-α2a sensitivity and transcriptional response in melanoma cells

“…STAT genes included in the array (2, 4, 5a and 5b) were found to be expressed at relatively low levels (nAD ≤ 50) in all cell lines, irrespective of IFN-α responsiveness. These results are in agreement with data obtained by using different technologies, suggesting the presence of relatively functional signal transduction in IFN-α insensitive cells (Ralph et al, 1995;Wong et al, 1997). Interestingly, similar results were also obtained by testing some of the cell lines under investigation in the current work by conventional gene and protein expression techniques (Pansky et al, 2000).…”

Constitutive activation of STAT3 and STAT5 is present in the majority of nasopharyngeal carcinoma and correlates with better prognosis

“…In a recent study, activated STAT1 was also shown to negatively regulate angiogenesis, tumorigenicity and metastasis of tumour cells (Huang et al, 2002). STAT1 deficiency has also been found in a variety of tumour cell lines and this deficiency is responsible for the lack of INF-g-mediated tumour suppression effects (Wong et al, 1997;Abril et al, 1998;Sun et al, 1998;Pansky et al, 2000). Therefore, it was not unexpected that constitutively activated STAT1 was found in only eight (13.1%) of the 61 NPC specimens.…”

Interleukin-6 and oncostatin M-induced growth inhibition of human A375 melanoma cells is STAT-dependent and involves upregulation of the cyclin-dependent kinase inhibitor p27/Kip1

“…Loss of components of the signaling machinery may be regarded as an alternative strategy to attain a growth advantage: recently, melanoma cells resistant to the anti-proliferative e ect of IFNa were described to contain less STAT1 than IFNa-sensitive cells. Overexpression of STAT1 improved the responsiveness to IFNa (Wong et al, 1997). It will be of interest to ®nd out whether defects in Jak/ STAT signaling contribute to IL-6-resistance observed in several late-stage melanoma cell lines (Lu et al, 1992(Lu et al, , 1993.…”