Real-time ECAR (as a measure of lactic acid production) and accumulated lactate levels of hepatocytes were measured as

previously established in our laboratory20, 21 with alterations detailed in the Supporting Materials and Methods. These assays were performed as described.11, 17 The details are described in the Supporting Materials and Methods. For statistical analyses, the two-tailed Selleck LY2109761 Student’s t-test was used. Significance was defined as P < 0.05. Two murine models of HCC were established and examined in this study. First, de novo HCC formation was induced in C57/BL6 WT and Cd39-null mice. Although all mice developed liver cancers, the tumor burden in Cd39-null mice was markedly increased when compared to WT controls (Fig. 1A-C). Incidence of tumors greater than 5 mm in diameter was 69% (9/13) in null livers and 6.7% (1/15) in WT livers (Supporting Table S1A, P < 0.001). Using a morphological classification of “mouse liver tumors” (MLT),22 30% of null tumors ABT-199 solubility dmso had high-grade malignancy (MLT type III and IV) (Fig. 1C, lower), whereas tumors arising in WT livers were of low-grade malignancy (chiefly MLT type I and II) (Fig. 1C, upper). We next studied HCC

arising spontaneously and in an autochthonous manner in mice. The incidence of liver neoplasia in aged (18-24 months old) Cd39-null mice (70%, 19/27) was significantly higher than that of WT mice (29%, 2/7) (Table S1B; P = 0.04; Fig. 1D,E). Accordingly, the mouse tumor biology database at Jackson Laboratories indicates that the frequency of spontaneous liver tumor varies from 8.8% to 30% in aged WT C57/BL6J mice (tumor.informatics.jax.org). Phospholipase D1 Hematoxylin-eosin (H&E) staining of liver tumor sections from Cd39-null mice confirmed that tumors were of the hepatocellular type with curiously marked hypervascularity

(Fig. 1E). Cd39-null mice developed spontaneous hepatic necrosis concomitantly accompanied by adjacent hepatocellular dysplasia at a young age (as early as 5 weeks old) (Fig. 2A). These mutant mice, in comparison to WT controls, exhibited significantly increased liver-to-body weight ratios, (Fig. 2B; P < 0.0001). Next we used RT-PCR to characterize the complement of P2 receptors expressed by WT hepatocytes. Mouse hepatocytes expressed messenger RNA (mRNA) transcripts for P2X4, P2X5, P2X7 (weaker), P2Y1, P2Y2, and P2Y12-14 (Supporting Fig. S1). We then examined the impact of Cd39 deletion on hepatocyte proliferation. First, Cd39-null cells exhibited a heightened baseline proliferation rate compared to WT cells (Fig. 2C). This occurred regardless of the culture conditions and appeared indicative of prior “set-points” in null cells (considered as possible “preprogramming”). Second, insofar as ATP has differential effects on cell proliferation at different concentrations, we evaluated the responses of WT cells to increasing concentrations of ATP.