Nucleoside diphosphate kinase (NDPK) 是一類廣泛存在的酵素，它通過乒乓機制將nucleoside diphosphates磷酸化為nucleoside triphosphates。該基因家族已知有8個成員，其中NDPK-A/NM23-H1和NDPK-B/NM23-H2研究較多。目前研究指出，雖然NDPK-A和NDPK-B的氨基酸序列同源性高達88%，NDPK-A與癌細胞轉移關係更密切。NDPK-B被證實為human c-myc transcription factor PuF，可結合至c-myc promoter上nuclease-hypersensitive III1 片段。c-myc 原致癌基因在正常細胞生理扮演關鍵性調控角色，其與細胞生長，繁殖，分化，凋亡有關。在大多數的腫瘤，常可發現c-myc表達失調。本實驗室先前發現NDPK-A也可結合至c-myc promoter in vitro並調節其活性in vivo。經由 EMSA 和 UV-crosslinking實驗分析，NDPK-A相較NDPK-B對c-myc的結合能力明顯較弱。於是我更進一步探討NDPK-A及NDPK-B如何分別及共同調控c-myc轉錄。藉由CAT assay分析，我發現NDPK-A對於c-myc調控不需要phosphotransferase活性。除了NDPK-AS120G/N82S，NDPKAWT和NDPKAS120G外生性的表現增加能抑制c-myc轉錄。相同地，NDPK-B也能抑制c-myc的轉錄。NDPK-A和NDPK-B的外生性表現同時增量則導致更強烈c-myc轉錄之抑制現象。相反地，利用shRNA降低內生性的NDPK-A或NDPK-B的表現則活化c-myc轉錄。在過氧壓力下，NDPK-A增進了細胞之存活，降低內生性的NDPK-A或NDPK-B的表現則增加細胞死亡。共同降低內生性NDPK-A與NDPK-B的表現使細胞存活率更低。雖然NDPK-A與NDPK-B對於細胞生長都不具影響，但兩者對於細胞的Wound healing 有相反的能力，本研究結果將癌症轉移所見到的NDPKs異常表現與c-myc 失調連結在一起。

Nucleoside diphosphate kinase (NDPK) is an ubiquitous enzyme, which catalyzes the transfer of the terminal phosphate from a nucleoside triphosphate to a nucleoside diphosphate through a ping-pong mechanism. At present, eight members of the human NDPK family have been identified. The most widely studied members are NDPK-A and NDPK-B which are encoded by NM23-H1 and NM23-H2 genes. Accumulating evidence indicates that NDPK-A, but not NDPK-B, is strongly correlated with tumor metastasis even though the two proteins share 88% identical amino acid residues. NDPK-B was identified as PuF, which is a transcriptional regulator of c-myc that binds to the nuclease-hypersensitive element III1 (NHE) III1 in the c-myc promoter. Our laboratory previously found that NDPK-A also specifically binds c-myc NHEIII1 in vitro and regulates the c-myc promoter activity in vivo. Compared to NDPK-B, however, NDPK-A displayed a significantly lower binding affinity toward c-myc DNA based on EMSA and UV-crosslinking. In my study, I have examined the individual and collaborative effects of NDPK-A and NDPK-B on c-myc transcriptional regulation in NB69 neuroblastoma cells and cervical cancer HeLa cells. Using the CAT assay, I found that the phosphotransferase activity or intra-molecular disulfide bond of NDPK-A was not essential for its regulation of c-myc transcription. Ectopic expression of wide-type NDPK-A (NDPKAWT) and metastasis-associated S120G mutation (NDPKAS120G), negatively regulated c-myc mediated transcription in a dose-dependent manner. Similarity, NDPK-B also displayed an inhibitory effect on c-myc transcription in both cell lines. When combined with NDPK-B, NDPK-A suppressed c-myc transcription more strongly than either protein alone. On the other hand, knockdown of endogenous NDPK-A and/or NDPK-B enhanced c-myc transcription. Under oxidative stress, knockdown of NDPK-A decreased cell viability by ~15% relative to the control cells. Knockdown of both NDPK-A and NDPK-B further decreased cell viability by ~50% after H2O2 treatment. Additionally, knockdown of NDPK-A increased the migration potential, whereas knockdown of NDPK-B reduced its migration capacity compared with the control. However, knockdown NDPK-A and/or NDPK-B did not significantly affect the proliferation of HeLa cells. These findings provide a link between metastasis-associated alterations of NDPK-A and c-myc deregulation.

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