Abstract

Spred proteins are inducible inhibitors of signalling induced by receptor tyrosine kinases. They are implicated in negative feedback interactions that regulate intracellular pathways. The repressive function of Spred proteins targets several TK receptors so resulting in a variety of biological effects. Spred proteins, after growth factor stimulation, translocate to the plasma membrane, become tyrosine phosphorilated and interact with components of the Ras/MAPK and Ras/Raf/Erk pathways. Abnormal activation of Ras/MAPK pathway has been demonstrated in many haematological disorders including AML. In order to assess whether the activation of this pathway, leading to the disruption of many biological process, could be ascribed not only to the constitutive activation of TKs but also to defective signalling inhibition, we studied the function of Spred1 in AML. Using a Real Time PCR we studied the expression level of Spred1 in 80 AML samples (15 PB and 65 BM), 26 normal controls (16 PB and 10 BM) and 12 CD34+ samples sorted from AML patients and 12 from healthy subjects. We found that Spred1 transcript amount is significant reduced in AML samples when compare to normal controls (2−DeltaDeltaCt =0,084 vs 2,4 p=0,0000001) (range in AML 0,3–0,003). This difference is even more sound in AML CD34+ cells where the Spred1 transcript is 3 logs lower compared to normal CD34+ (2−DeltaDeltaCt =0,001 vs 2,27 p=0,0000001). Western blot using an antibody against Spred1 demonstrated the reduction or the absence of Spred1 protein in AML cells. Sequence analysis allowed to exclude the presence of mutations in the coding and promoter regions. In order to better understand the mechanism leading to the abrogation of Spred1 we analyzed the factors responsible for Spred1 transcription. We demonstrated that the transcription factor WT1 binds to and activates the promoter region of Spred1. WT1 KTS- is the isoform with transcriptional activity, while the isoform KTS+ is mainly localized at the cytoplasmatic level as confirmed by immunofluorescence analysis. Immunofluorescence assay and western blot carried out in NIH3T3 cells transfected with plasmids containing KTS+ and KTS- isoforms demonstrated that Spred1 protein is present at high levels in WT1 KTS- cells but not in KTS+ transfected cells. Finally we demonstrated, using capillary electrophoresis, that in AML patients the WT1 KTS- isoform was reduced or completely absent (ratio KTS+/KTS- ranging from 2 to 9,6 compared to a normal ratio ranging from 1, to 1,4 in cells from healthy subjects. Finally, in patients who achieved a complete remission after chemotherapy, WT1 KTS+/KTS- ratio returned within the normal range and Spred1 transcript and protein were significantly upregulated. This study clearly demonstrates that WT1 transcription activity is altered in AML cells in which WT1 may probably play a role of oncogene rather than of tumor suppressor gene. This leads to the absence of Spred1 transcript and protein which is a physiological inhibitor of RTK mediated signalling.

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