Year of Publication

2008

Degree Name

Doctor of Philosophy (PhD)

Document Type

Dissertation

College

Medicine

Department

Pharmacology

First Advisor

Dr. David Kaetzel

Abstract

NM23-H1 represents the first identified metastasis suppressor, exhibiting reduced expression in breast carcinoma and melanoma, and an ability to inhibit metastatic growth without significant impact on the transformed phenotype. Although its molecular mechanism of action is not fully understood, NM23-H1 possesses at least three enzymatic activities that may mediate metastasis suppressor function. It catalyzes nucleoside diphosphate kinase (NDPK) activity, as well as protein histidine kinase and 3’-5’ exonuclease activities. As 3’-5’ exonucleases are generally required for maintenance of genomic integrity, this activity represents a plausible mediator to underlie the metastasis suppressor function of NM23-H1 protein. To investigate the relevant activity of NM23-H1 in metastasis suppression, we constructed a panel of NM23-H1 mutant variants with selective enzymatic lesions. Previous studies have identified some key amino acid residues important for the enzymatic characteristics of NM23-H1. However, none of them are selective for disrupting the 3’-5’ exonuclease activity. In this study, we show that a substitution of Glu5 to alanine results in a dramatic, selective loss in 3’-5’ exonuclease property without significant affecting other enzymatic activities. To measure the extent to which the exonuclease function opposes mutation and metastasis, NM23-deficient and metastatic cell lines with forced expression of NM23-H1 variants are analyzed in nude mice. In spontaneous metastasis models, NM23-H1 mutants deficient in 3‘-5’ exonuclease activity significantly disrupt the capacity of metastasis suppression of wild-type protein, indicating that the 3’-5’ exonuclease activity of NM23-H1 is necessary for the spontaneous metastasis-suppressing effects. As 3'-5' exonucleases are generally associated with DNA repair process, we have also studied the contributions of yeast NM23 homologue YNK1 to genomic integrity in Saccharomyces cerevisiae. Consistent with an antimutator function, ablation of YNK1 significantly results in increased mutation rates following exposure to UV irradiation and the alkylating agent methyl methanesulfonate (MMS). The impaired DNA-damage response of ynk1Δ cells suggests a role of human homologue NM23 in DNA repair. More evidence is being collected in our laboratory to demonstrate a role for NM23-H1 in maintaining genomic integrity. Collectively, our findings of DNA repair activity of NM23-H1 will contribute to the understandings of the mechanisms in metastasis suppression and new drug discoveries.

Included in

Pharmacology Commons

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