engleski

Overexpression of NM23-H1 enhences proliferation of CAL 27 cells

Nm23-H1 and Nm23-H2 are two highly homologous subunits of nucleoside - diphosphate kinase (NDPK) whose role is to transfer the terminal phosphate from ATP to all other NDPs and dNDPs. Eukaryotic NDP kinases have been reported to be catalytically active only as homo- or hexamers. Up until today, six other members of the nm23 gene family have been discovered. Although the NDPK has been known for decades the nm23-H1 gene has been discovered in 1988 on the basis of its reduced expression in melanoma cell lines with low vs. high metastatic potential. Therefore, it has been proposed as a metastasis suppressor gene. There has been quite a body of evidence proving the involvement of Nm23 in several cellular processes and has been assigned diverse biological roles. It has been proved that, although forming a functional enzyme together, Nm23-H1 and Nm23-H2 also have other, distinct functions in the cell. Although nm23 family has been intensively studied for more than a decade the exact mechanisms of its actions in different cellular events, its cellular partners and downstream targets have not yet been discovered. Many authors predict a multifunctional and tissue specific nature of at least some of the Nm23 proteins. Previous results of our laboratory based on nm23-H1 expression correlated with SCCHN (squamous cell carcinoma of the head and neck) tumor stages and cell proliferation strongly indicate that nm23-H1 is involved in progression of SCCHN in a manner which is not consistent with its proposed role as a metastasis suppressor gene. Our present work has been concentrated exclusively on the role of nm23-H1 and nm23-H2 in oral squamous cell carcinoma i.e. the consequences of their overexpression in CAL 27 cells in vitro. Herein we present our preliminary results on the effect of nm23-H1 overexpression on their proliferation. nm23-H1 and nm23-H1H118N (with a point mutation in the kinase domain) cDNA fragments were subcloned into pcDNA3 with carrying a flag-tag and the gene responsible of G418 resistance. CAL 27 clones stably overexpressing Nm23-H1 and Nm23-H1H118N were isolated, synchronized and tested for proliferation rate (DNA cell-cycle analysis) using flow cytometry. The nm23-H1 overexpressing CAL27 cells exhibited enhanced proliferation rate compared to nm23-H1H118N overexpressing and control cells. The mechanism of this phenomenon remains to be elucidated.

nm23; proliferation; cell-cycle analysis; fow cytometry

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