Search for genes differently expressed when we inhibit NatB N-terminal acetyltransferase. We compare gene expression after inhibiting hNAT5 expression in Hela cells with specific siRNAs expressed with adenoviruses. Keywords: gene expression comparison Overall design: We analyzed duplicate samples and we used as controls Hela cells and Hela cells that express a siRNA that doesnt inhibit any gene expression. The N-terminal acetyltransferase NatB, composed in Saccharomyces cerevisiae by the Nat3p and Mdm20p subunits, is an important factor for yeast growth and resistance to several stress agents. However, the expression and functional role of the mammalian counterpart has not yet been analysed. Here, we report the identification of Nat3p human homologue (hNAT5/hNAT3) and the characterization of its biological function. We found that hNAT5/hNAT3 silencing in HeLa cells results in inhibition of cell proliferation and increased sensitivity to the proapoptotic agent MG132. Moreover, inhibition of hNAT5/hNAT3 expression induces p53 activation and upregulation of the antiproliferative protein p21(WAF1/CIP1). The changes of the cellular transcriptome after hNAT5/hNAT3 knockdown confirmed the involvement of this protein in cell growth and survival processes. Among the genes differentially expressed we observed upregulation of several p53-dependent antiproliferative and proapoptotic genes. In the c-myc transgenic mice which is a model of inducible hepatocarcinoma we found that hNAT5/hNAT3 was upregulated when the tumor was induced. In accordance with this observation we noticed increased hNAT5/hNAT3 protein level in neoplastic versus non-neoplastic tissue in a high proportion of patients with hepatocellular carcinoma. Consequently, our results suggest that the expression of the protein hNAT5/hNAT3 is required for cellular proliferation and tumor growth.