To use the equilibrium established by creatine kinase (CK) to determine hepatic free ADP levels, the transcriptional control elements of the transthyretin gene were used to direct expression of the CK B isozyme to the livers of transgenic mice. Activities of CK ranging from 80-250 pmol per min per g (wet weight) were detected in liver extracts from five founder mice. The CK activity was stably transmitted to subsequent generations. Isozyme gels and immunoblots confirmed that the activity detected in extracts was due to the B isozyme of CK. Immunohistology indicated that the protein was expressed uniformly throughout the liver and was localized primarily to the cytoplasm. 31P NMR spectroscopy was used to detect the metabolic product of the CK reaction, phosphocreatine, demonstrating that the enzyme was active in vivo. The phosphocreatine level fell rapidly during anoxia (tQ12 = 1 min), indicating that the CK reaction was integrated into hepatic energy metabolism. The equilibrium established by CK was used to calculate a hepatic free ADP level of 0.059 ± 0.004 ,umol/g (wet weight). In vivo NMR studies of these mice will be valuable for studying the role of free ADP in regulating liver metabolism.Adenosine diphosphate (ADP) is an important regulator of many metabolic pathways in vitro, including oxidative phosphorylation (1), glycolysis (2), gluconeogenesis (2), and ion transport (3). Due to difficulties in measuring free ADP levels in intact liver it has been impossible to assess the role of this key metabolite in regulating hepatic metabolism in vivo (4). To study changes that may occur during perturbations of liver metabolism, ADP is usually assayed from whole cell extracts or from liver cells after subcellular fractionation into cytoplasmic and mitochondrial compartments (4-6). However, it is generally acknowledged that for metabolites such as ADP, the concentrations of which are low and comparable to the number of intracellular binding sites, measurements from extracts lead to an overestimation of free metabolite levels (4).Hans Krebs introduced the idea of using cellular reactions near equilibrium to determine the levels of free metabolites at low concentrations (7). This technique relies on measuring compounds present in relatively high concentrations that are in chemical equilibrium with the metabolite of interest. Ideally, a single equilibrium using metabolites that can be monitored in vivo should be used. The reaction catalyzed by creatine kinase (CK), phosphocreatine (PCr) + MgADP + H+ = MgATP + creatine, is the method of choice for the determination of ADP (4). The value of the equilibrium constant is known for a variety of conditions, and free ADP levels can be calculated from in vivo nuclear magnetic resonance (NMR) measurements of PCr, ATP, creatine, Mg2+, and pH. This approach has been successfully used to study the role of ADP in metabolic regulation in heart (8), muscle (9), and brain metabolism (10). However, it has not been useful for liver due to the lack of significant levels of CK.In...

The simian virus 40 (SV40) large tumor antigen (T antigen) under its natural regulatory elements induces choroid plexus papillomas in transgenic mice. Because these tumors develop focally after several months, it has been suggested that secondary cellular alterations are required to induce a tumor in this tissue. In contrast to SV40, the related lymphotropic papovavirus early region induces rapid nonfocal choroid plexus neoplasia in transgenic mice. Here, using hybrid gene constructs, we showed that T antigen from either virus is in fact sufficient to induce these tumors. Their abilities to induce proliferative abnormalities in other tissues, such as kidney and thymus, were also indistinguishable. Differences in the rate of choroid plexus tumorigenesis reflected differences in the control regions of the two viruses, rather than differences in T antigen per se. Under SV40 regulation, expression was limited to a fraction of the choroid plexus cells prior to the formation of focal tumors. When SV40 T antigen was placed under lymphotropic papovavirus control, in contrast, expression was generally uniform in the choroid plexus and rapid expansion of the tissue ensued. We found a direct relationship between T-antigen expression, morphological transformation, and proliferation of the choroid plexus epithelial cells. Analysis of mosaic transgenic mice indicated further that T antigen exerts its mitogenic effect cell autonomously. These studies form the foundation for elucidating the role of various T-antigen subactivities in tumorigenesis.The natural induction of neoplasia appears to involve multiple genetic events (28), including alteration of cellular proto-oncogenes (dominant events) and of tumor suppressor genes (recessive events) (2,8,15,28,34,40). In transgenic mice, simian virus 40 (SV40) large T antigen induces a variety of neoplasms depending on the transcriptional signals used for its expression (for reviews, see references 25 and 26); included are eye lens (37), acinar (47, 49) and beta (24) cell pancreatic tumors, B-cell and histiocytic lymphomas (58), rhabdosarcoma (1, 19), stomach carcinoma (9, 49), osteosarcoma (1, 31), hepatoma (41,52,54), hibernoma (21), melanoma (3), retinoblastoma (64), and choroid plexus papilloma (7, 55). The wide spectrum of susceptible cell types could mean that T antigen acts through targeting proteins common to all these cell types. Alternatively, the subset of T-antigen functions, and the corresponding target proteins, could differ depending on the cell type. T antigen encodes multiple transformation-related activities (43), including complex formation with at least two cellular tumor suppressor proteins, pRB (16) and p53 (33, 35). Our ultimate goal using transgenic mice is to determine the role of each T-antigen subactivity in the tumorigenesis of a variety of cell types. A necessary first step, however, is to establish the effects of the complete protein on a particular tissue. Because previous studies raised questions about whether wildtype T antigen is sufficient to induce ...