Using a proteomic analysis of human hepatocellular carcinoma (HCC), we identified the overexpression in 4 tumors of RuvB-like 2 (RUVBL2), an ATPase and putative DNA helicase known to interact with ␤-catenin and cellular v-myc myelocytomatosis viral oncogene homolog (c-myc). RUVBL2 expression was further analyzed in tumors with quantitative reverse-transcription polymerase chain reaction analysis and immunohistochemistry; in addition, RUVBL2 expression in a HuH7 cell line was silenced by small interfering RNA or increased with a lentiviral vector. RUVBL2 messenger RNA overexpression was confirmed in 72 of 96 HCC cases, and it was associated with poorly differentiated tumors (P ‫؍‬ 0.02) and a poor prognosis (P ‫؍‬ 0.02) but not with ␤-catenin mutations or c-myc levels. Although RUVBL2 was strictly nuclear in normal hepatocytes, tumoral hepatocytes exhibited additional cytoplasmic staining. There was no mutation in the coding sequence of RUVBL2 in 10 sequenced cases. Silencing RUVBL2 in HuH7 HCC cells reduced cell growth (P < 0.001) and increased apoptosis, as shown by DNA fragmentation (P < 0.001) and caspase 3 activity (P < 0.005). This was associated with an increased expression of several proapoptotic genes and with an increased conformational activation of Bak-1 and Bax. On the other hand, HuH7 cells with an overexpression of RUVBL2 grew better in soft agar (P < 0.03), had increased resistance to C2 ceramide-induced apoptosis (P < 0.001), and gave rise to significantly larger tumors when injected into immunodeficient Rag2/␥c mice (P ‫؍‬ 0.016). Conclusion: RUVBL2 is overexpressed in a large majority of HCCs. RUVBL2 overexpression enhances tumorigenicity, and RUVBL2 is required for tumor cell viability. These results argue for a major role of RUVBL2 in liver carcinogenesis.

This paper describes the interaction of apamin, a bee venom neurotoxin, with the mouse neuroblastoma cell membrane. Voltage-clamp analyses have shown that apamin at low concentrations specifically blocks the Ca2"-dependent K+ channel in differentiated neuroblastoma cells. Binding experiments with highly radiolabeled toxin indicate that the dissociation constant of the apamin-receptor complex in differentiated neuroblastoma cells is 15-22 pM and the maximal binding capacity is 12 fmol/ mg of protein. The receptor is destroyed by proteases, suggesting that it is a protein.The binding capacity ofneuroblastoma cells for radiolabeled apamin dramatically increases during the transition from the nondifferentiated to the differentiated state.

Eight different polypeptide toxins from sea anemones of four different origins (Anemonia sulcata, Anthopleura xanthogrammica, Stoichactis giganteus, and Actinodendron plumosum) have been studied. Three of these toxins are new; the purification procedure for the five other ones has been improved. Sea anemone toxins were assayed (i) for their toxicity to crabs and mice, (ii) for their affinity for the specific sea anemone toxin receptor situated on the Na+ channels of rat brain synaptosomes, and (iii) for their capacity to increase, in synergy with veratridine, the rate of 22Na+ entry into neuroblastoma cells via the Na+ channel. Some of the toxins are more active on crustaceans, whereas others are more toxic to mammals. A very good correlation exists between the toxic activity to mice, the affinity of the toxin for the Na+ channel in rat brain synaptosomes, and the stimulating effect on 22 Na+ uptake by neuroblastoma cells. The observation has also been made that the most cationic toxins are also the most active on mammals and the least active on crustaceans. Toxicities (LD50) to mice of the most active sea anemone toxins and of the most active scorpion toxins are similar, and sea anemone toxins at high enough concentrations prevent binding of scorpion toxins to their receptor. However, scorpion toxins have affinities for the Na+ channel which are approximately 60 times higher than those found for the most active sea anemone toxins. Three sea anemone toxins appear to be more interesting than toxin II from A. sulcata (the "classical" sea anemone toxin) for studies of the Na+ channel structure and mechanism when the source of the channel is of a mammalian origin. Two of these three toxins can be radiolabeled with iodine while retaining their toxic activity; they appear to be useful tools for future biochemical studies of the Na+ channel.

Novel high-affinity, low-capacity binding sites in intestinal membranes for the heat-stable toxin produced by Escherichia coli have been defined. The appearance of these sites is observed in the presence of physiological concentrations of NaCl in binding reactions. Scatchard analyses of equilibrium binding in the absence of NaCl demonstrated a single class of binding sites with KD = 1.9 x 10(-9) M and Bmax = 0.75 pmol/mg of protein. In contrast, similar experiments in the presence of NaCl demonstrated, in addition to the previously described low-affinity site, a high-affinity site with a KD of 2.1 x 10(-11) M and a Bmax of 73 fmol/mg of protein. Confirmation of the presence of high- and low-affinity sites was obtained in studies of the kinetics of ST binding. These sites exhibited similar dissociation but markedly different association kinetics. Determination of the association and dissociation constants permitted calculation of the KD's for the high- and low-affinity sites, which were 1.15 x 10(-11) M and 1.89 x 10(-9) M, respectively. These data agree closely with those obtained in studies of equilibrium binding. Furthermore, similar values for the KD's of these sites were obtained in experiments of competitive displacement of labeled ST, confirming the presence of two receptors for this toxin. Binding of ST to high-affinity sites is completely reversible and does not appear to be coupled to activation of particulate guanylate cyclase. In contrast, binding of ST to low-affinity sites appears to be partially reversible and may be coupled to activation of guanylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)