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Recombinant interleukin-24 lacks apoptosis-inducing properties in melanoma cells.

Kreis S, Philippidou D, Margue C, Rolvering C, Haan C, Dumoutier L, Renauld JC, Behrmann I - PLoS ONE (2007)

Bottom Line: To date, the physiological properties of IL-24 are still not well understood but available data suggest that IL-24 affects epidermal functions by increasing proliferation of dermal cells.Furthermore, evaluating four different sources and modes of IL-24 administration (commercial recombinant IL-24, bacterially expressed GST-IL-24 fusion protein, IL-24 produced from transfected Hek cells, transiently over-expressed IL-24) no induction or increase in cell death was detected when compared to appropriate control treatments.Thus, we conclude that the cytokine IL-24 itself has no cancer-specific apoptosis-inducing properties in melanoma cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biologie et Physiologie Intégrée, Life Science Research Unit, University of Luxembourg, Luxembourg. stephanie.kreis@uni.lu <stephanie.kreis@uni.lu>

ABSTRACT
IL-24, also known as melanoma differentiation antigen 7 (mda-7), is a member of the IL-10 family of cytokines and is mainly produced by Th(2) cells as well as by activated monocytes. Binding of IL-24 to either of its two possible heterodimeric receptors IL-20R1/IL-20R2 and IL-22R/IL-20R2 activates STAT3 and/or STAT1 in target tissues such as lung, testis, ovary, keratinocytes and skin. To date, the physiological properties of IL-24 are still not well understood but available data suggest that IL-24 affects epidermal functions by increasing proliferation of dermal cells. In stark contrast to its "normal" and physiological behaviour, IL-24 has been reported to selectively and efficiently kill a vast variety of cancer cells, especially melanoma cells, independent of receptor expression and Jak-STAT signalling. These intriguing properties have led to the development of adenovirally-expressed IL-24, which is currently being evaluated in clinical trials. Using three different methods, we have analysed a large panel of melanoma cell lines with respect to IL-24 and IL-24 receptor expression and found that none of the investigated cell lines expressed sufficient amounts of functional receptor pairs and therefore did not react to IL-24 stimulation with Jak/STAT activation. Results for three cell lines contrasted with previous studies, which reported presence of IL-24 receptors and activation of STAT3 following IL-24 stimulation. Furthermore, evaluating four different sources and modes of IL-24 administration (commercial recombinant IL-24, bacterially expressed GST-IL-24 fusion protein, IL-24 produced from transfected Hek cells, transiently over-expressed IL-24) no induction or increase in cell death was detected when compared to appropriate control treatments. Thus, we conclude that the cytokine IL-24 itself has no cancer-specific apoptosis-inducing properties in melanoma cells.

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RT-PCR of IL-24 and its receptor subunits.Eight different melanoma cell lines, primary NHEM and HaCaT cells were grown on Petri dishes to a confluence of 80–90% before cells were harvested. Total RNA was extracted, reverse transcribed and cDNA amounts equivalent to 100 ng RNA were used as input material for amplifications of IL-24, and the receptor subunits IL-20R1, -20R2, and -22R. To control for equal starting amounts and loading, actin was amplified. Size markers (bp) are indicated on the left.
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pone-0001300-g003: RT-PCR of IL-24 and its receptor subunits.Eight different melanoma cell lines, primary NHEM and HaCaT cells were grown on Petri dishes to a confluence of 80–90% before cells were harvested. Total RNA was extracted, reverse transcribed and cDNA amounts equivalent to 100 ng RNA were used as input material for amplifications of IL-24, and the receptor subunits IL-20R1, -20R2, and -22R. To control for equal starting amounts and loading, actin was amplified. Size markers (bp) are indicated on the left.

Mentions: IL-24 signals through two heterodimeric receptor complexes, which share the short IL-20R2 chain but have distinct longer alpha chains: IL-20R1 and IL-22R [7]. Since no Jak/STAT signalling was detected in any of the melanoma cell lines following IL-24 (or IL-19 and IL-20) treatment, the expression of the 3 possible receptor chains was analysed by standard RT-PCR, quantitative PCR (qPCR), Western Blot and FACS analyses. Primers used for PCR reactions are listed in Table 1. First, standard RT-PCR was performed using the equivalent of 100 ng RNA as starting material (Fig. 3). Some but not all cell lines showed positive results for IL-22R mRNA while hardly any mRNA for IL-20R1 and very low or absent mRNAs for IL-20R2 chains, the common subunit present in both heterodimeric receptor complexes, were detected. As expected and as previously published, HaCaT cells were positive for all 3 receptor chains but negative for IL-24. IL-24 mRNA was detectable to various levels in 6 out of 10 melanoma cell lines. The lower band present in IL-24 RT-PCR samples corresponds to a splice variant, which has been described before [41]. Previous reports have suggested that melanoma cells express receptors for IL-24 although it is unclear how this was analysed [28]. Performing RT-PCR with very high cDNA input quantities (equivalent to 1 µg of RNA), we also found all analysed cell lines to be positive for the 3 possible IL-24 receptor chains (data not shown). However, lower starting amounts of cDNA (10-100 ng/ml) reproducibly gave results as depicted (Fig. 3) and these results matched well with those from qPCR shown in Fig. 4. Again, in depth qPCR analysis was performed on different amounts of input material to achieve a standard curve and a PCR amplification efficiency of 100% for all samples. Fig. 4 shows a summary of triplicate qPCR data from 3 independent experiments using the equivalent of 12.5 ng RNA input material. mRNA amounts of IL-24 were normalised to TBP and are shown in correlation to NHEM (Fig. 4A). A375 and IPC298 showed moderate amounts of IL-24 mRNA with Wm9 reproducibly having IL-24 mRNA quantities ∼50 fold higher than primary melanocytes.


Recombinant interleukin-24 lacks apoptosis-inducing properties in melanoma cells.

Kreis S, Philippidou D, Margue C, Rolvering C, Haan C, Dumoutier L, Renauld JC, Behrmann I - PLoS ONE (2007)

RT-PCR of IL-24 and its receptor subunits.Eight different melanoma cell lines, primary NHEM and HaCaT cells were grown on Petri dishes to a confluence of 80–90% before cells were harvested. Total RNA was extracted, reverse transcribed and cDNA amounts equivalent to 100 ng RNA were used as input material for amplifications of IL-24, and the receptor subunits IL-20R1, -20R2, and -22R. To control for equal starting amounts and loading, actin was amplified. Size markers (bp) are indicated on the left.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2110900&req=5

pone-0001300-g003: RT-PCR of IL-24 and its receptor subunits.Eight different melanoma cell lines, primary NHEM and HaCaT cells were grown on Petri dishes to a confluence of 80–90% before cells were harvested. Total RNA was extracted, reverse transcribed and cDNA amounts equivalent to 100 ng RNA were used as input material for amplifications of IL-24, and the receptor subunits IL-20R1, -20R2, and -22R. To control for equal starting amounts and loading, actin was amplified. Size markers (bp) are indicated on the left.
Mentions: IL-24 signals through two heterodimeric receptor complexes, which share the short IL-20R2 chain but have distinct longer alpha chains: IL-20R1 and IL-22R [7]. Since no Jak/STAT signalling was detected in any of the melanoma cell lines following IL-24 (or IL-19 and IL-20) treatment, the expression of the 3 possible receptor chains was analysed by standard RT-PCR, quantitative PCR (qPCR), Western Blot and FACS analyses. Primers used for PCR reactions are listed in Table 1. First, standard RT-PCR was performed using the equivalent of 100 ng RNA as starting material (Fig. 3). Some but not all cell lines showed positive results for IL-22R mRNA while hardly any mRNA for IL-20R1 and very low or absent mRNAs for IL-20R2 chains, the common subunit present in both heterodimeric receptor complexes, were detected. As expected and as previously published, HaCaT cells were positive for all 3 receptor chains but negative for IL-24. IL-24 mRNA was detectable to various levels in 6 out of 10 melanoma cell lines. The lower band present in IL-24 RT-PCR samples corresponds to a splice variant, which has been described before [41]. Previous reports have suggested that melanoma cells express receptors for IL-24 although it is unclear how this was analysed [28]. Performing RT-PCR with very high cDNA input quantities (equivalent to 1 µg of RNA), we also found all analysed cell lines to be positive for the 3 possible IL-24 receptor chains (data not shown). However, lower starting amounts of cDNA (10-100 ng/ml) reproducibly gave results as depicted (Fig. 3) and these results matched well with those from qPCR shown in Fig. 4. Again, in depth qPCR analysis was performed on different amounts of input material to achieve a standard curve and a PCR amplification efficiency of 100% for all samples. Fig. 4 shows a summary of triplicate qPCR data from 3 independent experiments using the equivalent of 12.5 ng RNA input material. mRNA amounts of IL-24 were normalised to TBP and are shown in correlation to NHEM (Fig. 4A). A375 and IPC298 showed moderate amounts of IL-24 mRNA with Wm9 reproducibly having IL-24 mRNA quantities ∼50 fold higher than primary melanocytes.

Bottom Line: To date, the physiological properties of IL-24 are still not well understood but available data suggest that IL-24 affects epidermal functions by increasing proliferation of dermal cells.Furthermore, evaluating four different sources and modes of IL-24 administration (commercial recombinant IL-24, bacterially expressed GST-IL-24 fusion protein, IL-24 produced from transfected Hek cells, transiently over-expressed IL-24) no induction or increase in cell death was detected when compared to appropriate control treatments.Thus, we conclude that the cytokine IL-24 itself has no cancer-specific apoptosis-inducing properties in melanoma cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biologie et Physiologie Intégrée, Life Science Research Unit, University of Luxembourg, Luxembourg. stephanie.kreis@uni.lu <stephanie.kreis@uni.lu>

ABSTRACT
IL-24, also known as melanoma differentiation antigen 7 (mda-7), is a member of the IL-10 family of cytokines and is mainly produced by Th(2) cells as well as by activated monocytes. Binding of IL-24 to either of its two possible heterodimeric receptors IL-20R1/IL-20R2 and IL-22R/IL-20R2 activates STAT3 and/or STAT1 in target tissues such as lung, testis, ovary, keratinocytes and skin. To date, the physiological properties of IL-24 are still not well understood but available data suggest that IL-24 affects epidermal functions by increasing proliferation of dermal cells. In stark contrast to its "normal" and physiological behaviour, IL-24 has been reported to selectively and efficiently kill a vast variety of cancer cells, especially melanoma cells, independent of receptor expression and Jak-STAT signalling. These intriguing properties have led to the development of adenovirally-expressed IL-24, which is currently being evaluated in clinical trials. Using three different methods, we have analysed a large panel of melanoma cell lines with respect to IL-24 and IL-24 receptor expression and found that none of the investigated cell lines expressed sufficient amounts of functional receptor pairs and therefore did not react to IL-24 stimulation with Jak/STAT activation. Results for three cell lines contrasted with previous studies, which reported presence of IL-24 receptors and activation of STAT3 following IL-24 stimulation. Furthermore, evaluating four different sources and modes of IL-24 administration (commercial recombinant IL-24, bacterially expressed GST-IL-24 fusion protein, IL-24 produced from transfected Hek cells, transiently over-expressed IL-24) no induction or increase in cell death was detected when compared to appropriate control treatments. Thus, we conclude that the cytokine IL-24 itself has no cancer-specific apoptosis-inducing properties in melanoma cells.

Show MeSH
Related in: MedlinePlus