Hücre yaşlanmasıyla ilintili DUSP10 and MTMR11 fosfataz genlerinin moleküler analizi

Liver cancer is the fifth most common cancer in the world. Until recently, tumor cellswere thought to proliferate indefinitely. In a previous study, our group showedspontaneous induction of replicative senescence in p53- and p16INK4a-deficientHCC (hepatocellular carcinoma) cell clones. The gene expression profiling was laterdone 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 andcarcinogenesis, their partners in signaling, protein phosphatases should also beimportant in these processes. Hence analysis and targeting of protein phosphatasesgenes with differential expression between immortal and senescent clones mightprove beneficial for HCC therapeutics. Among the phosphatase genes withdifferential expression patterns, we focused on two most upregulated genes insenescent clones with respect to immortal clones, DUSP10 and MTMR11. Aftergathering detailed information on these genes and their products by bioinformaticsanalysis, we confirmed the upregulation of the two genes in our senescent clonescompared to our immortal clones by semi-quantitative RT-PCR. We then checkedDUSP10 and MTMR11 expression in HCC and breast cancer cell lines to see if adifferential expression of these genes are observed in different subtypes of these celllines. Other experiments on MTMR11 focused on discovery of novel transcripts ofthis gene in HCC and breast cancer cell lines and checking the amounts of differenttranscripts in different subtypes of these cell lines, to form a bridge betweenMTMR11 transcript variants and carcinogenesis, however we did not observedifferential expression. Two microarray studies comparing non-tumor and HCCtissues have listed MTMR11 as upregulated in HCC. Hence, upregulation of this genein senescent clones may not be significant in hepatocarcinogenesis or replicativesenescence, and further experiments should be performed. Considering DUSP10, wechecked the subcellular localization of this protein in HCC cell lines byimmunostaining, to see if the two subtypes (well-differentiated and poorlydifferentiated)of HCC cell lines differed in DUSP10 localization. We observed somecell lines having only nuclear or only cytoplasmic DUSP10, whereas most had bothnuclear and cytoplasmic DUSP10. This lead the way for us to explore the factors thatmay be important in changing this protein?s localization, as this may be a type ofregulation on this protein, and may change during carcinogenesis or upon induction ofsenescence. For this purpose, we checked to see if DUSP10 changed its localization inaging MRC-5 cell passages compared to young, proliferating ones and in prematuresenescence-induced cells compared to normal ones. Interestingly, it was found thatupon replicative senescence induction, but not premature senescence, DUSP10localized more to the cell nucleus which indicated a connection between DUSP10localization and replicative senescence. We also checked to see if DUSP10 changedits localization upon disruption of the MAPK pathways it participates in, by kinaseinhibitor experiments. Interestingly, it was found that DUSP10 localized significantlymore to the cell nucleus upon inhibition of JNK pathway but not p38 pathway, inwell-differentiated subtype of HCC cell lines. DUSP10 localization did not changesignificantly in poorly-differentiated subtype of HCC cell lines. Although JNKs,which seem to regulate DUSP10 through its localization according to this study, act asoncogenes in HCC, the significance of the change in DUSP10 localization should becharacterized further before stating that DUSP10 can be a putative tumor suppressor.However, our other results indicate a relationship between DUSP10 localization andreplicative senescence, which is promising because DUSP10 has emerged from ourgroup?s microarray data as a replicative senescence-associated gene, and thisconnection should be analyzed further.