Figure 7
Figure 7. Targeting of IGF-IR induces significant effects on downstream survival proteins in ALK+ ALCL cells. To explore possible explanations for the negative effects of down-regulation of IGF-IR signaling on ALK+ ALCL, the effect of selective blockade of IGF-IR by PPP on proteins downstream of IGF-IR was studied. These proteins are also known to significantly contribute to the pathogenesis of ALK+ ALCL. (A) WB (Karpas 299 cell line is shown as a representative example) confirmed that at 24 hours, PPP induces marked down-regulation of pIGF-IR, without a noticeable change in IGF-IR levels. The decrease in pIGF-IR is associated with decreases in pNPM-ALK (Tyr664), pAkt (Ser473), and pSTAT3 (Tyr705). In addition, PPP induces significant decreases in the antiapoptotic proteins Mcl-1 and Bcl-2. Furthermore, PPP induces a notable increase and decrease in the cell-cycle regulatory proteins cyclin B1 and pCdc, respectively. Changes are not seen in p16 and Cdc. The effects of PPP on the cell cycle regulatory proteins are consistent with the occurrence of G2/M-phase cell-cycle arrest. (B) We also used specific targeting of IGF-IR by siRNA to confirm some of the results and to rule out the possibility of nonspecific effects of the pharmacologic agent PPP. IGF-IR siRNA decreased pAkt, pSTAT3, Bcl-XL, and Bcl-2 levels. To further explore the mechanisms by which IGF-IR induces its oncogenic effects in ALK+ ALCL, the binding of STAT3 (C) and FKHR (D) to DNA was studied by using 2 different techniques, namely EMSA (top panels) and ELISA (bottom panels), after treatment with PPP (2 μM for 24 hours). (C) PPP significantly decreases STAT3 binding to DNA in the 5 ALK+ ALCL cell lines. The difference in the migration of the STAT3-DNA binding band between the control and the ALK+ ALCL cells could be explained by the difference in the target sequence. A vertical line has been inserted to indicate prepositioned gel lanes. (D) In contrast to its effect on STAT3, PPP enhances FKHR binding to DNA in SU-DHL-1, SUP-M2, SR786, and DEL cells. These results are in agreement with PPP-induced cell death of these cells. Vertical lines have been inserted to indicate repositioned gel lanes. The controls (CP, control probe; CNE, control nuclear extract; CCP, cold control probe; COMP, competition) confirm that the reagents performed properly.

Targeting of IGF-IR induces significant effects on downstream survival proteins in ALK+ ALCL cells. To explore possible explanations for the negative effects of down-regulation of IGF-IR signaling on ALK+ ALCL, the effect of selective blockade of IGF-IR by PPP on proteins downstream of IGF-IR was studied. These proteins are also known to significantly contribute to the pathogenesis of ALK+ ALCL. (A) WB (Karpas 299 cell line is shown as a representative example) confirmed that at 24 hours, PPP induces marked down-regulation of pIGF-IR, without a noticeable change in IGF-IR levels. The decrease in pIGF-IR is associated with decreases in pNPM-ALK (Tyr664), pAkt (Ser473), and pSTAT3 (Tyr705). In addition, PPP induces significant decreases in the antiapoptotic proteins Mcl-1 and Bcl-2. Furthermore, PPP induces a notable increase and decrease in the cell-cycle regulatory proteins cyclin B1 and pCdc, respectively. Changes are not seen in p16 and Cdc. The effects of PPP on the cell cycle regulatory proteins are consistent with the occurrence of G2/M-phase cell-cycle arrest. (B) We also used specific targeting of IGF-IR by siRNA to confirm some of the results and to rule out the possibility of nonspecific effects of the pharmacologic agent PPP. IGF-IR siRNA decreased pAkt, pSTAT3, Bcl-XL, and Bcl-2 levels. To further explore the mechanisms by which IGF-IR induces its oncogenic effects in ALK+ ALCL, the binding of STAT3 (C) and FKHR (D) to DNA was studied by using 2 different techniques, namely EMSA (top panels) and ELISA (bottom panels), after treatment with PPP (2 μM for 24 hours). (C) PPP significantly decreases STAT3 binding to DNA in the 5 ALK+ ALCL cell lines. The difference in the migration of the STAT3-DNA binding band between the control and the ALK+ ALCL cells could be explained by the difference in the target sequence. A vertical line has been inserted to indicate prepositioned gel lanes. (D) In contrast to its effect on STAT3, PPP enhances FKHR binding to DNA in SU-DHL-1, SUP-M2, SR786, and DEL cells. These results are in agreement with PPP-induced cell death of these cells. Vertical lines have been inserted to indicate repositioned gel lanes. The controls (CP, control probe; CNE, control nuclear extract; CCP, cold control probe; COMP, competition) confirm that the reagents performed properly.

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