BMC Genomics 2004, 5:46 http://www.biomedcentral.com/1471-2164/5/46 Table 1: Differential Gene Expression in LAPE Cells Relative to LAPN Cells (Continued) Miscellaneous parotid secretory protein amyloid-like protein 1 precursor liver receptor homologous protein oncomodulin tctex-1 beta-hydroxysteroid dehydrogenase type 2 C57BL/6J ob/ob haptoglobin angiotensin-converting enzyme Swiss Webster demilune cell-specific salivary gland protein neurexophilin 1 Accession fold-change X01697 4.0 Q03157 4.0 M81385 1.7 Z48238 -1.6 M25825 -1.6 X90647 -2.8 M96827 J04947 W15826 U56651 -2.9 -3.2 -7.1 -7.8 Cell cycle regulation NHE1 activity has a permissive effect in promoting cell proliferation [6,7] and cDNA microarray analysis indicated a number of genes with roles in DNA synthesis and cell cycle control had altered expression in LAPE cells compared with LAPN cells. We recently reported [5] that the proliferative rate of LAPE cells is ~3 to 4-fold less than that of LAPN cells and that LAPE cells lack a pH-depend- ent timing of cell cycle progression that is specifically associated with delayed G2/M entry and transition. Con- sistent with these findings there was an upregulation of genes associated with G2/M arrest and DNA repair responses (Fig. 3A). Decreased protein expression of GADD45 and FEN1 was confirmed by immunoblotting (Fig. 3B,3C). The decrease in FEN1 in LAPE cells was most marked when cells were synchronized by a double thymi- dine block, and released from the block for 3 to 9 hours (Fig. 3C). GADD45, which acts in DNA repair, is generally upregulated with cell cycle arrest [37,38], and FEN1 is thought to play an essential role in DNA replication and in base excision repair [39,40]. Although decreased expression of GADD45 and FEN1 in LAPE cells appears paradoxical, recent findings indicate that a decrease in GADD45 would contribute to p53 instability [38] and FEN1 is stimulated by proliferating nuclear antigen [41], which is decreased in LAPE cell (Fig. 3A). Additional growth arrest and DNA damage-inducible proteins, including Rad54 and GADD153, were upregulated. Rad54 functions in homologous recombination repair pathways to maintain telomere length [42] and it facilities chromatin remodeling [43], which correlates with increased histone H3 in LAPE cells (Fig. 2). As described above, increased GADD153 expression, which was con- firmed TaqMan analysis (Fig. 5B), is induced by growth arrest, DNA damage, and environmental stress. Despite an established role for GADD153 in inducing apoptosis [44,45], and its increased expression in LAPE cells (~45- fold), there was no indication that LAPE cells have increased necrosis or apoptosis compared with LAPN cells. Consistent with LAPE cells having delayed G2/M entry and progression [5], the array analysis indicated upregula- tion of genes negatively regulating G2/M, including 14-3- 3 θ and Wee1 kinase. Increased expression of Wee1 kinase, which induces inhibitory phosphorylation of Cdc2 on tyrosine 15 [46], was confirmed by TaqMan anal- ysis (Fig. 3D). Moreover, there was a downregulation of genes associated with promoting cell cycle progression, including CDC25 (Fig 3A) and CKS-2 (Fig. 3A), and involved in chromatin assembly, including histone H3 (Fig. 2A). Although cDNA array analysis of asynchronous LAPE cells did not indicate a change in cyclin B1 expres- sion, we found that with a time-dependent release of cells from a double thymidine block, cyclin B1, as indicated by immunoblotting, was significantly downregulated in LAPE cells compared with LAPN cells (Fig. 3C). Hence, loss of NHE1 activity likely decreases the stability of cyclin B1 protein rather than decreasing cyclin B1 gene expres- sion. Decreased cyclin B1 expression and increased Wee1 kinase expression is consistent with our previous finding that Cdc2 kinase activity is inhibited in LAPE cells com- pared with LAPN cells [5]. Carbohydrate metabolism, electron transport and oxidative phosphorylation A global pattern of metabolic genes differentially regu- lated in LAPE cells would favor glycolysis and oxidative phosphorylation, possibly in response to reduced ATP (Fig. 4A). Paradoxically, expression of genes encoding enzymes that regulate substrate entry for glycolysis was decreased. Decreased expression of the glucose trans- porter Glut-4 was confirmed by TaqMan analysis (Fig 4B). Additionally, decreased phosphorylase kinase would favor decreased conformational change of phosphorylase b to phosphorylase a, and reduced glycogen breakdown to glucose 6-phosphate and decreased galactokinase would favor decreased utilization of galactose for glycolysis. Key regulators of glycolytic flux, however, including hexoki- nase, phosphofusctose kinase, and pyruvate kinase were upregulated in LAPE cells compared with LAPN cells. Increases in hexokinase and phosphofusctose kinase, which catalyze the first and second ATP ultilization steps of glycolysis, respectively, and in pyruvate kinase, which catalyzes the final reaction of glycolysis, would favor increased production of pyruvate. Phosphorylation of fructose 6-phosphate by phosphofusctose kinase is a rate- determining reaction and the activity of phosphofusctose kinase is stimulated by low AMP and inhibited by high ATP and by citrate. In LAPE cells, a decrease in citrate transport protein would likely decrease cytosolic citrate, a negative regulator of phosphofusctose kinase, and indi- rectly increase phosphofusctose kinase activity. Glycolytic Page 6 of 13 (page number not for citation purposes)