The SYK signal appears to be localized mainly in the cytoplasm, with an increased intensity in patch-like structures. and the use of small molecule SYK inhibitors significantly reduced the cell viability of neuroblastoma cell lines expressing SYK protein. Moreover, SYK inhibition decreased ERK1/2 and Akt phosphorylation. The SYK inhibitor BAY 61-3606 enhanced the effect of different chemotherapeutic medicines. Transient expression of a constitutive active SYK variant improved the viability of neuroblastoma cells self-employed of endogenous SYK levels. Collectively, our findings suggest that focusing on SYK in combination with standard chemotherapy should be further evaluated as a treatment option in neuroblastoma. gene manifestation using the publicly available R2: Genomics analysis and visualization platform (http://r2.amc.nl) and observed that manifestation was higher in four different neuroblastoma cohorts compared to neural crest cells and benign neurofibroma (Number 1A). Open in a separate window Number 1 SYK is definitely indicated in neuroblastoma cells. Gene manifestation data were analyzed using the R2 database http://r2.amc.nl. (A) The manifestation of was compared between neural crest (Etchevers n = 5), benign neurofibroma (Miller n = 86) and 4 neuroblastoma cohorts (cohort 1: Versteeg n = 88, cohort 2: Delattre n = 64, cohort 3: Hiyama n = 51, Rabbit Polyclonal to PSEN1 (phospho-Ser357) cohort 4: Lastowska n = 30). The presence of APY29 SYK protein (B,C) and phosphorylation at Tyr525 (D,E) were identified in neuroblastoma main cells using immunoperoxidase staining. (B,D) display a staining of a non-amplified9 (10)9 (9)* Treated cells11 (13)10 (11)* Untreated cells26 (26)25 (26)Ganglioneuroma3 (3)3 (3) Open in a separate windowpane * For three tumor cells samples the information concerning prior treatment was unavailable. Using Fishers precise test we identified that there was no significant difference in the presence of SYK protein between = 0.4239). However, analyzing different neuroblastoma datasets in the R2: Genomics analysis and visualization platform, we observed a significant negative correlation between and manifestation (Supplementary Number S1A showing a representative dataset). In contrast, we found a significant positive correlation between and manifestation (Supplementary Number S1B). Furthermore, we evaluated whether there was a difference in the presence of SYK in tumors that were treated with chemotherapy prior to surgery compared to untreated tumors. All 26 untreated tumor samples and 11 out of 13 treated tumor samples were SYK-positive. APY29 This difference was however not significant (Fishers precise test = 0.1053). Of notice, surgery treatment was performed after at least 10C14 days of washout. Hence, no acute chemotherapy-induced rules of genes should be expected. APY29 Additionally, the presence of SYK phosphorylated at Tyr525, located within the activation loop of the kinase website, was examined as an indication for active SYK [8,42]. Number 1D,E APY29 display a representative staining of p-SYK in non-mRNA and protein in neuroblastoma cell lines. The majority of the neuroblastoma cell lines express mRNA at varying levels (Number 2A). However, SYK protein was recognized by western blotting in only two of 10 neuroblastoma cell lines, actually after long exposure times (Number 2B). Interestingly, we noticed that the cell lines with absent or APY29 very low mRNA levels are mRNA and to a lesser lengthen SYK protein are indicated in neuroblastoma cell lines. (A) RT-PCR analysis demonstrating the manifestation of both mRNA variants in different neuroblastoma cell lines. U937 cells were used like a positive control (Personal computer). NTC, no template control. (B) Manifestation of SYK protein was determined by western blot. THP-1 cells were used like a positive control. Immunofluorescence labeling of SYK (green) in SH-SY5Y (C), LAN-6 (D) and SK-N-BE(2) cells (E). The nuclei (blue) were stained with Hoechst 33342. Panels (FCH) display isotype settings for SH-SY5Y (F), LAN-6 (G) and SK-N-BE(2) cells (H). The shorter SYK splice variant SYK B offers previously been recognized in different cell types [5,6,7,37]. We observed that SH-SY5Y, LAN-6 and SK-N-FI cells concomitantly communicate both splice variants of mRNA at related levels whereas SH-EP1, SK-N-SH, and IMR-32 show mainly the short SYK B variant. The monocytic cell lines U937 and THP-1 with known SYK manifestation were used as positive settings for RT-PCR and western blot, respectively . ICC was used to confirm the presence of SYK protein in SH-SY5Y and LAN-6 cells. A definite SYK labeling was observed in the cytoplasm of SH-SY5Y (Number 2C) and LAN-6 cells (Number 2D). The SYK transmission appears to be localized primarily in the cytoplasm, with an increased intensity in patch-like constructions. However, a faint staining was also observed.