In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of cyclin-dependent kinase (CDK) by WEE1 kinase, and, at mitosis, WEE1 protein is removed through the action of the 26S proteasome. Although in higher plants WEE1 function has been confirmed in the DNA replication checkpoint, Arabidopsis wee1 insertion mutants grow normally, and a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be confirmed. Here data are presented showing that the inhibitory effect of WEE1 on CDK activity in tobacco BY-2 cell cultures is cell cycle regulated independently of the DNA replication checkpoint: it is high during S-phase but drops as cells traverse G2 and enter mitosis. To investigate this mechanism further, a yeast two-hybrid screen was undertaken to identify proteins interacting with Arabidopsis WEE1. Three F-box proteins and a subunit of the proteasome complex were identified, and bimolecular fluorescence complementation confirmed an interaction between AtWEE1 and the F-box protein SKP1 INTERACTING PARTNER 1 (SKIP1). Furthermore, the AtWEE1–green fluorescent protein (GFP) signal in Arabidopsis primary roots treated with the proteasome inhibitor MG132 was significantly increased compared with mock-treated controls. Expression of AtWEE1–YFPC (C-terminal portion of yellow fluorescent protein) or AtWEE1 per se in tobacco BY-2 cells resulted in a premature increase in the mitotic index compared with controls, whereas co-expression of AtSKIP1–YFPN negated this effect. These data support a role for WEE1 in a normal plant cell cycle and its removal at mitosis via the 26S proteasome.
The NME1 gene represents the prototypical metastasis suppressor, whose expression inhibits cell motility and metastasis without impact on primary tumor growth in a number of different human cancers. This report outlines our recent efforts to define the molecular mechanisms through which NME1 both suppresses cell motility and promotes genomic integrity in the setting of human melanoma. Forced NME1 expression in a variety of melanoma-derived cell lines was shown to induce dynamic changes in cell morphology and reorganization of the actin cytoskeleton, with formation of a network of thick stress fibers and assembly of fibronectin fibrils at large focal adhesions. Moreover, NME1 expression results in adhesion reprogramming through an impact on integrin repertoire and focal adhesion dynamics. Having previously demonstrated that NME1 expression promotes repair of DNA damage induced by ultraviolet radiation (UVR) in both yeast and mammalian cells, probably via the nucleotide excision repair pathway, we have more recently demonstrated that NME1 is rapidly recruited to double-strand breaks. This preliminary result represents the first evidence of direct interactions between NME1 and DNA in the context of DNA repair, and has set the stage for current efforts to probe its functional interactions with double-strand break repair pathways. Discussed herein are molecular models to explain the interactions of NME1 with such diverse cellular functions as cell motility and DNA repair, potentially through its nucleoside diphosphate kinase and 3′-5′ exonuclease activities.
Metastasis suppressor activity of NM23-M1 (NM23-H1 in human) in melanoma has been established using melanoma cell lines in culture and, more recently, in a model of UV-induced melanoma using NM23 knockout mice. While NM23-M1/H1 is clearly a suppressor of melanoma cell motility, we have recently shown it also exhibits genome-stabilizing activity, promoting repair of both spontaneous and UV-induced DNA damage via the nucleotide excision repair pathway. These findings suggest NM23 proteins may also inhibit metastasis by suppressing acquisition of metastasis-driving mutations and other genomic aberrations. To dissect the molecular mechanisms through which NM23 contributes to genomic stability, this study focused on the double-strand break repair (DSBR) pathway. All members of the NM23 family of proteins possess nucleoside diphosphate kinase (NDPK) activity, which could enhance nucleotide availability during polymerase-mediated repair, and 3'5' exonuclease activity which has been suggested to be critical in the non-homologous end-joining (NHEJ) pathway of DSBR. To first determine whether NM23 is indeed recruited to DSBs, these lesions were produced in the human melanoma cell line WM793 using a tamoxifen-inducible form of the restriction endonuclease I-PpoI via a retroviral vector. Induction of I-PpoI-catalyzed DSBs resulted in recruitment of both endogenous NM23 and recombinant Flag-tagged H1 (Flag-H1) to a known I-PpoI cleavage site within 30 min, as demonstrated by chromatin immunoprecipitation. The rapid appearance of NM23-H1 at the DSB coincided with that of the damage sensor ATM, and disappearance of histone 2B. Forced expression of NM23-H1 resulted in significantly enhanced repair of the known I-PpoI-induced DSB site, shown using a quantitative PCR across the lesion, indicating a functional contribution of the protein to DSBR. Induction of DSBs resulted in colocalization of Flag-H1 with γ-histone 2AX within small intranuclear foci, also consistent with recruitment of NM23-H1 directly to DSB lesions. Induction of DSBs using the DNA topoisomerase II inhibitor etoposide resulted in rapid nuclear colocalization within 5 minutes of Flag-H1 and ribonucleotide reductase, suggesting cooperative actions of these two enzymatic activities during DSBR. These studies are the first to demonstrate recruitment of NM23-H1 directly to sites of DNA damage, and suggest the possibility that NM23 deficiency in melanoma and other human cancers accelerates the acquisition of metastasis-driving mutations during cancer progression. Citation Format: Stuart G. Jarrett, Marian Novak, Gemma S. Cook, David M. Kaetzel. Potential contributions of genome-stabilizing activity of NM23 to its metastasis suppressor function in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C27.
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