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Item Open Access Molecular analysis of senescence-associated protein phosphatases DUSP10 and MTMR11(2009) Gülay, Suna PelinLiver cancer is the fifth most common cancer in the world. Until recently, tumor cells were thought to proliferate indefinitely. In a previous study, our group showed spontaneous induction of replicative senescence in p53- and p16INK4a-deficient HCC (hepatocellular carcinoma) cell clones. The gene expression profiling was later done for these different clones, in an attempt to find novel therapeutic targets in HCC. Since protein kinases are known to be very important in disease formation and carcinogenesis, their partners in signaling, protein phosphatases should also be important in these processes. Hence analysis and targeting of protein phosphatases genes with differential expression between immortal and senescent clones might prove beneficial for HCC therapeutics. Among the phosphatase genes with differential expression patterns, we focused on two most upregulated genes in senescent clones with respect to immortal clones, DUSP10 and MTMR11. After gathering detailed information on these genes and their products by bioinformatics analysis, we confirmed the upregulation of the two genes in our senescent clones compared to our immortal clones by semi-quantitative RT-PCR. We then checked DUSP10 and MTMR11 expression in HCC and breast cancer cell lines to see if a differential expression of these genes are observed in different subtypes of these cell lines. Other experiments on MTMR11 focused on discovery of novel transcripts of this gene in HCC and breast cancer cell lines and checking the amounts of different transcripts in different subtypes of these cell lines, to form a bridge between MTMR11 transcript variants and carcinogenesis, however we did not observe differential expression. Two microarray studies comparing non-tumor and HCC tissues have listed MTMR11 as upregulated in HCC. Hence, upregulation of this gene in senescent clones may not be significant in hepatocarcinogenesis or replicative senescence, and further experiments should be performed. Considering DUSP10, we checked the subcellular localization of this protein in HCC cell lines by immunostaining, to see if the two subtypes (well-differentiated and poorlydifferentiated) of HCC cell lines differed in DUSP10 localization. We observed some cell lines having only nuclear or only cytoplasmic DUSP10, whereas most had both nuclear and cytoplasmic DUSP10. This lead the way for us to explore the factors that may be important in changing this protein’s localization, as this may be a type of regulation on this protein, and may change during carcinogenesis or upon induction of senescence. For this purpose, we checked to see if DUSP10 changed its localization in aging MRC-5 cell passages compared to young, proliferating ones and in premature senescence-induced cells compared to normal ones. Interestingly, it was found that upon replicative senescence induction, but not premature senescence, DUSP10 localized more to the cell nucleus which indicated a connection between DUSP10 localization and replicative senescence. We also checked to see if DUSP10 changed its localization upon disruption of the MAPK pathways it participates in, by kinase inhibitor experiments. Interestingly, it was found that DUSP10 localized significantly more to the cell nucleus upon inhibition of JNK pathway but not p38 pathway, in well-differentiated subtype of HCC cell lines. DUSP10 localization did not change significantly in poorly-differentiated subtype of HCC cell lines. Although JNKs, which seem to regulate DUSP10 through its localization according to this study, act as oncogenes in HCC, the significance of the change in DUSP10 localization should be characterized further before stating that DUSP10 can be a putative tumor suppressor. However, our other results indicate a relationship between DUSP10 localization and replicative senescence, which is promising because DUSP10 has emerged from our group’s microarray data as a replicative senescence-associated gene, and this connection should be analyzed further.