This report describes a new unroofing technique to secure an effective microwave coagulation therapy (MCT) for a case of hepatocellular carcinoma, which was deeply located in segment 5 adjacent to the confluence of right anterior and posterior portal pedicles. A 62 year-old man developed HCC in the context of liver cirrhosis caused by hepatitis C virus. Transcatheter arterial embolization was attempted initially because of the tumor location and poor hepatic functional reserve, but failed to achieve satisfactory remission. On laparotomy, hepatic parenchyma was superficially resected to uncover the ventral surface of deeply located HCC adjacent to major hepatic vessels, enabling an accurate as well as effective MCT. The patient has been alive and well without recurrence for five months following the surgery.
Glycoprotein nonmetastatic melanoma protein B (GPNMB) has a neuroprotective effect against neuronal cell death caused by the accumulation of abnormal mutated proteins. It is known that the accumulation of pathological proteins induces endoplasmic-reticulum (ER) stress leading to cell damage. The aim of this study was to determine the role of GPNMB in the ER stress response. GPNMB was greatly up-regulated by thapsigargin-induced ER stress. Under the ER stress conditions, GPNMB relocated to the nucleus and specifically up-regulated expression of BiP at the mRNA level by promoting the BiP pre-mRNA splicing, not through the pathways initiated by the three major transducers of the unfolded protein response: IRE1, PERK, and ATF6. Furthermore, we found that the protein level of BiP and the infarction were increased and attenuated, respectively, in Gpnmb-transgenic mice after occlusion of the middle cerebral artery, in comparison with wild-type mice. Thus, our findings indicate that GPNMB enhances the BiP expression by promoting the splicing (thereby preventing cell death caused by ER stress) and could be a therapeutic target in ER stress-related disorders.
Although reports of typical acute promyelocytic leukemia (APL) cases rarely mention dysplastic changes, this report concerns a rare case of APL with tri-lineage dysplastic changes resembling the characteristic features of myelodysplastic syndrome (MDS). The patient, a 77-year-old Japanese male, was diagnosed as having pancytopenia with hematologic morphological abnormalities comprising micro - megakaryocytes, neutrophils with hypo-granulation and negative peroxidase activity, and erythroblasts containing nuclei with abnormalities such as karyorrhexis. Although there is one report of a case of transformation of de novo MDS into APL and several reports of cases of therapy-related MDS transformed into APL, our patient had no history of cytopenia or of either chemo or radiation therapy. Our case can thus be considered to constitute a rare case of APL with dysplastic morphology.
A wide variety of biological activities including the major metabolic actions of insulin is regulated by phosphatidylinositol (PI) 3-kinase. However, the downstream effectors of the various signaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downstream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr308 and Ser473) targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt. Akt-AA thus acts in a dominant negative manner in intact cells. Insulin-stimulated protein synthesis, which is sensitive to wortmannin, a pharmacological inhibitor of PI 3-kinase, was abolished by overexpression of Akt-AA without an effect on amino acid transport into the cells, suggesting that Akt is required for insulin-stimulated protein synthesis. Insulin activation of p70 S6 kinase was inhibited by ∼75% in CHO cells and ∼30% in 3T3-L1 adipocytes, whereas insulin-induced activation of endogenous Akt was inhibited by 80 to 95%, by expression of Akt-AA. Thus, Akt activity appears to be required, at least in part, for insulin stimulation of p70 S6 kinase. However, insulin-stimulated glucose uptake in both CHO cells and 3T3-L1 adipocytes was not affected by overexpression of Akt-AA, suggesting that Akt is not required for this effect of insulin. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of PI 3-kinase.
A 70's man was admitted to our hospital because of lumbago and paresthesia in the right lower extremity. He underwent surgical resection of gastric gastrointestinal stromal tumor (GIST), which was classified to the high-risk group according to the modified-Fletcher's classification, one and half years ago. CT, MRI, and PET-CT showed metastases to a part of the liver (S3-4), the 12th thoracic vertebra, and the sacral bone. Subsequently, radiotherapy for the bone metastasis and administration of imatinib mesylate were started. Four months after the initial admission, the liver and the bone metastatic lesions achieved PET-complete response (CR). This report shows that multimodality therapy with radiotherapy and imatinib mesylate was effective for liver and bone metastases after complete resection of gastric GIST.
A 61-year-old woman, who was diagnosed in 1982 as having polycythemia vera, was admitted to our hospital in July 1998 because of a splenic tumor in an enlarged spleen due to myelofibrosis. As it was difficult to identify the etiology of the splenic tumor, partial splenectomy was carried out. The resected tumor proved to be an extremely proliferative lesion as the result of extramedullary hematopoiesis. Since it is difficult to diagnose the etiology of splenic tumor, the collection and analysis of reports of relevant cases may well facilitate diagnosis.(Internal Medicine 39: 416-118, 2000)
The roles of Akt (protein kinase B) and the atypical λ isoform of protein kinase C (PKCλ), both of which act downstream of phosphoinositide 3-kinase, in the activation of glycogen synthase and phosphorylation of 4E-BP1 (PHAS-1) in response to insulin were investigated. A mutant Akt (Akt-AA) in which the phosphorylation sites targeted by growth factors are replaced by alanine was shown to inhibit insulin-induced activation of both Akt and glycogen synthase in L6 myotubes. Expression of a mutant Akt in which Lys179 in the kinase domain was replaced by aspartate also inhibited insulin-induced activation of glycogen synthase but had no effect on insulin activation of endogenous Akt. A kinase-defective mutant of PKCλ (λΔNKD), which prevents insulin-induced activation of PKCλ, did not affect the activation of glycogen synthase by insulin. Insulin-induced phosphorylation of 4E-BP1 was inhibited by Akt-AA in Chinese hamster ovary cells. However, λΔNKD had no effect on 4E-BP1 phosphorylation induced by insulin. These data suggest that Akt, but not PKCλ, is required for insulin activation of glycogen synthase and for insulin-induced phosphorylation of 4E-BP1. The roles of Akt (protein kinase B) and the atypical λ isoform of protein kinase C (PKCλ), both of which act downstream of phosphoinositide 3-kinase, in the activation of glycogen synthase and phosphorylation of 4E-BP1 (PHAS-1) in response to insulin were investigated. A mutant Akt (Akt-AA) in which the phosphorylation sites targeted by growth factors are replaced by alanine was shown to inhibit insulin-induced activation of both Akt and glycogen synthase in L6 myotubes. Expression of a mutant Akt in which Lys179 in the kinase domain was replaced by aspartate also inhibited insulin-induced activation of glycogen synthase but had no effect on insulin activation of endogenous Akt. A kinase-defective mutant of PKCλ (λΔNKD), which prevents insulin-induced activation of PKCλ, did not affect the activation of glycogen synthase by insulin. Insulin-induced phosphorylation of 4E-BP1 was inhibited by Akt-AA in Chinese hamster ovary cells. However, λΔNKD had no effect on 4E-BP1 phosphorylation induced by insulin. These data suggest that Akt, but not PKCλ, is required for insulin activation of glycogen synthase and for insulin-induced phosphorylation of 4E-BP1. Insulin exerts a variety of effects on carbohydrate, lipid, and protein metabolism (1Ogawa W. Matozaki T. Kasuga M. Mol. Cell. Biochem. 1998; 182: 13-22Crossref PubMed Scopus (102) Google Scholar). Although the intracellular signaling cascades that mediate these divergent actions remain to be fully characterized, phosphoinositide (PI) 3-kinase, composed of a 110-kDa catalytic subunit and a Src homology 2 domain-containing regulatory subunit, is thought to play a central role in several metabolic effects of insulin (1Ogawa W. Matozaki T. Kasuga M. Mol. Cell. Biochem. 1998; 182: 13-22Crossref PubMed Scopus (102) Google Scholar, 2Holman G.D. Kasuga M. Diabetologia. 1997; 40: 991-1003Crossref PubMed Scopus (189) Google Scholar, 3Shepherd P.R Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (841) Google Scholar). Inhibition of PI 3-kinase with the use of pharmacological or molecular biological strategies has revealed a variety of molecules that act downstream of this enzyme (3Shepherd P.R Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (841) Google Scholar). One such molecule that appears to function immediately downstream of PI 3-kinase is the serine-threonine kinase Akt (also known as protein kinase B). Akt interacts through its pleckstrin homology domain with the phospholipids produced by PI 3-kinase and is thereby activated in vitro (4Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-667Crossref PubMed Scopus (1310) Google Scholar, 5Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (448) Google Scholar). Phosphorylation of Thr308 and Ser473 of Akt is also important for its activation (6Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2530) Google Scholar, 7Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. 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EMBO J. 1994; 13: 2313-2321Crossref PubMed Scopus (328) Google Scholar), Rac, a member of the Rho family of small GTPases, as well as one of its direct targets, p21-activated kinase, also may play a role in insulin action downstream of PI 3-kinase (16Tsakiridis T. Taha C. Grinstein S. Klip A. J. Biol. Chem. 1996; 271: 19664-19667Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Furthermore, the activities of protein kinases belonging to the mitogen-activated protein (MAP) kinase superfamily, including those of extracellular signal-regulated kinase (17Cross D.A. Alessi D.R. Vandenheede J.R. McDowell H.E. Hundal H.S. Cohen P. Biochem. J. 1994; 303: 21-26Crossref PubMed Scopus (421) Google Scholar, 18Welsh G.I. Stokes C.M. Wang X. Sakaue H. Ogawa W. Kasuga M. Proud C.G. FEBS Lett. 1997; 410: 418-422Crossref PubMed Scopus (89) Google Scholar, 19Sharma P.M. Egawa K. Huang Y. Martin J.L. Huvar I. Boss G.R. Olefsky J.M. J. Biol. Chem. 1998; 273: 18528-18537Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) and c-Jun NH2-terminal kinase (or stress-activated protein kinase) (20Logan S.K. Falasca M. Hu P. Schlessinger J. Mol. Cell. Biol. 1997; 17: 5784-5790Crossref PubMed Scopus (124) Google Scholar) isozymes, are sensitive to blockers of PI 3-kinase in intact cells, suggesting that these enzymes participate in signaling downstream of PI 3-kinase. However, despite the identification of these various molecules that act downstream of PI 3-kinase, relatively little is known of how signals diverge at this key step of insulin signaling and of which molecules are responsible for each of the specific biological activities of this hormone. Synthesis and breakdown of glycogen are important aspects of carbohydrate homeostasis. Glycogen synthase, a key enzyme that catalyzes glycogen synthesis, is activated by insulin in a PI 3-kinase-dependent manner (21Yamamoto-Honda R. Tobe K. Kaburagi Y. Ueki K. Asai S. Yachi M. Shirouzu M. Yodoi J. Akanuma Y. Yokoyama S. Yazaki Y. Kadowaki T. J. Biol. Chem. 1995; 270: 2729-2734Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 22Sakaue H. Hara K. Noguchi T. Matozaki T. Kotani K. Ogawa W. Yonezawa K. Waterfield M.D. Kasuga M. J. Biol. Chem. 1995; 270: 11304-11309Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 23Shepherd P.R. Nave B.T. Siddle K. Biochem. J. 1995; 305: 25-28Crossref PubMed Scopus (232) Google Scholar). Moreover, a constitutively active mutant of Akt stimulates glycogen synthase in quiescent cells (24Ueki K. Yamamoto-Honda R. Kaburagi Y. Yamauchi T. Tobe K. Burgering B.M. Coffer P.J. Komuro I. Akanuma Y. Yazaki Y. Kadowaki T. J. Biol. Chem. 1998; 273: 5315-5322Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar), suggesting that the PI 3-kinase-Akt pathway mediates activation of this enzyme in response to insulin. However, demonstration of such a role for Akt would require investigation of the effects of specific inhibition of Akt on insulin-induced activation of glycogen synthase. Furthermore, it is important to study the regulation of glycogen synthase in physiologically relevant cells, such as muscle cells or hepatocytes, because such regulation may differ among cell or tissue types (24Ueki K. Yamamoto-Honda R. Kaburagi Y. Yamauchi T. Tobe K. Burgering B.M. Coffer P.J. Komuro I. Akanuma Y. Yazaki Y. Kadowaki T. J. Biol. Chem. 1998; 273: 5315-5322Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 25Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar, 26Azpiazu I. Saltiel A.R. DePaoli-Roach A.A. Lawrence Jr., J.C. J. Biol. Chem. 1996; 271: 5033-5039Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Insulin rapidly promotes general protein synthesis in a variety of cells and tissues. Although insulin affects multiple steps of protein synthesis, initiation of mRNA translation is thought to be one of its most important targets (27Sonenberg N. Gingras A.C. Curr. Opin. Cell Biol. 1998; 10: 268-275Crossref PubMed Scopus (510) Google Scholar). The small, heat- and acid-stable protein 4E-BP1 (also known as PHAS-1) is rapidly phosphorylated in response to insulin (28Lin T.-A. Kong X. Haystead T.A.J. Pause A. Belsham G. Sonenberg N. Lawrence Jr., J.C. Science. 1994; 266: 653-656Crossref PubMed Scopus (602) Google Scholar, 29Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.-A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1063) Google Scholar). Insulin-induced phosphorylation of 4E-BP1 results in a decrease in its binding affinity for eIF-4E, an essential translation initiation factor for certain mRNAs that possess an m7(5′)Gppp(5′)N (where N is any nucleotide) cap structure (27Sonenberg N. Gingras A.C. Curr. Opin. Cell Biol. 1998; 10: 268-275Crossref PubMed Scopus (510) Google Scholar, 29Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.-A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1063) Google Scholar). Subsequent dissociation of eIF-4E from 4E-BP1 promotes cap structure-dependent translation initiation (29Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.-A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1063) Google Scholar). A constitutively active mutant of Akt was shown to stimulate phosphorylation of 4E-BP1 (24Ueki K. Yamamoto-Honda R. Kaburagi Y. Yamauchi T. Tobe K. Burgering B.M. Coffer P.J. Komuro I. Akanuma Y. Yazaki Y. Kadowaki T. J. Biol. Chem. 1998; 273: 5315-5322Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 30Gingras A.C. Kennedy S.G. O'Leary M.A. Sonenberg N. Hay N. Genes Dev. 1998; 12: 502-513Crossref PubMed Scopus (730) Google Scholar, 31Kohn A.D. Barthel A. Kovacina K.S. Boge A. Wallach B. Summers S.A. Birnbaum M.J. Scott P.H. Lawrence Jr., J.C. Roth R.A. J. Biol. Chem. 1998; 273: 11937-11943Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). In addition, expression of a kinase-defective Akt, in which Lys179 in the kinase domain was mutated, inhibited insulin-induced phosphorylation of 4E-BP1 (30Gingras A.C. Kennedy S.G. O'Leary M.A. Sonenberg N. Hay N. Genes Dev. 1998; 12: 502-513Crossref PubMed Scopus (730) Google Scholar). The relevance of this latter observation is unclear, however, because this and similar kinase-defective mutants of Akt do not exert a dominant inhibitory effect on insulin-induced activation of Akt (7Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar,32van Weeren P.C. de Bruyn K.M.T. de Vries-Smits A.M.M. van Lint J. Burgering B.M.T. J. Biol. Chem. 1998; 273: 13150-13156Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). We have recently shown that a mutant Akt in which the sites of ligand-induced phosphorylation are replaced with alanine (Akt-AA) inhibits the growth factor-induced activation of endogenous Akt when overexpressed in CHO cells or 3T3-L1 adipocytes (7Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar, 33Kuroda S. Ogawa W. Kitamura T. Konishi H. Kikkawa U. Kasuga M. Biochem. Biophys. Res. Commun. 1998; 249: 781-785Crossref PubMed Scopus (2) Google Scholar). With the use of this mutant, we have also shown that Akt is essential for stimulation of bulk protein synthesis by insulin (7Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar). In addition, we have shown that PKCλ is activated by insulin in a PI 3-kinase-dependent manner, and that a PKCλ mutant that prevents insulin-induced activation of endogenous PKCλ markedly, but not completely, inhibits insulin stimulation of glucose transport (14Kotani K. Ogawa W. Matsumoto M. Kitamura T. Sakaue H. Hino Y. Miyake K. Sano W. Akimoto K. Ohno S. Kasuga M. Mol. Cell. Biol. 1998; 18: 6971-6982Crossref PubMed Google Scholar). These observations indicate that PKCλ acts downstream of PI 3-kinase to mediate certain biological effects of insulin. In the present study, we have investigated the roles of Akt and PKCλ in the insulin-induced activation of glycogen synthase and phosphorylation of 4E-BP1 in L6 myotubes and CHO cells, respectively. In particular, the requirement for these two protein kinases in the effects of insulin have been evaluated by specifically inhibiting their activities with the use of dominant negative mutants. L6 myoblasts were maintained and induced to differentiate into myotubes as described previously (34Mitsumoto Y. Liu Z. Klip A. Endocr. J. 1993; 1: 307-315Google Scholar). Polyclonal antibodies to Akt (αpanAkt) were generated against a glutathione S-transferase fusion protein containing amino acids 428–480 of rat Akt1, as described (7Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar). Polyclonal antibodies to PKCλ were generated in response to a peptide corresponding to amino acids 197–213 of mouse PKCλ (1Ogawa W. Matozaki T. Kasuga M. Mol. Cell. Biochem. 1998; 182: 13-22Crossref PubMed Scopus (102) Google Scholar). Polyclonal antibodies to MAP kinase (αC92) (35Sakaue H. Ogawa W. Takata M. Kuroda S. Kotani K. Matsumoto M. Sakaue M. Nishio S. Ueno H. Kasuga M. Mol. Endocrinol. 1997; 11: 1552-1562Crossref PubMed Scopus (116) Google Scholar) or to 4E-BP1 (28Lin T.-A. Kong X. Haystead T.A.J. Pause A. Belsham G. Sonenberg N. Lawrence Jr., J.C. Science. 1994; 266: 653-656Crossref PubMed Scopus (602) Google Scholar, 29Pause A. Belsham G.J. Gingras A.-C. Donzé O. Lin T.-A. Lawrence Jr., J.C. Sonenberg N. Nature. 1994; 371: 762-767Crossref PubMed Scopus (1063) Google Scholar) were as described. Polyclonal antibodies specific to Akt2 (PKBβ) were obtained from Upstate Biotechnology, and those specific to Akt3 (PKBγ) (36Walker K.S. Deak M. Paterson A. Hudson K. Cohen P. Alessi D.R. Biochem. J. 1998; 331: 299-308Crossref PubMed Scopus (241) Google Scholar) were kindly provided by P. Cohen (University of Dundee, Dundee, Scotland, United Kingdom). Monoclonal antibodies to the hemagglutinin (HA) epitope tag (12CA5) or to the FLAG epitope tag were obtained from Roche Molecular Biochemicals and Kodak Scientific Imaging Systems, respectively. Adenovirus vectors encoding HA-tagged wild-type Akt (AxCAAkt-WT), HA-tagged mutant Akt in which Lys179 in the kinase domain was replaced by aspartate (AxCAAkt-K179D), HA-tagged mutant Akt in which Thr308 and Ser473 were replaced by alanine (AxCAAkt-AA), a constitutively active mutant of PKCλ that lacks the pseudosubstrate domain (AxCAλΔPD), or a dominant negative mutant of PKCλ that lacks the NH2-terminal region (amino acids 1–235) of the wild-type protein and in which Lys273 in the kinase domain was replaced by glutamate (AxCAλΔNKD) were as described previously (7Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar,14Kotani K. Ogawa W. Matsumoto M. Kitamura T. Sakaue H. Hino Y. Miyake K. Sano W. Akimoto K. Ohno S. Kasuga M. Mol. Cell. Biol. 1998; 18: 6971-6982Crossref PubMed Google Scholar). An adenovirus vector encoding FLAG epitope-tagged Akt-AA (AxCAAkt-AAFL) was constructed with the use of an adenovirus expression kit (Takara, Tokyo, Japan) and a cDNA (produced by the polymerase chain reaction) encoding Akt-AA (7Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar) fused at its NH2terminus with the FLAG epitope (DYKDDDDK). CHO cells or fully differentiated L6 myotubes were infected with adenovirus vectors at the indicated multiplicity of infection (m.o.i.) as described previously (7Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar, 35Sakaue H. Ogawa W. Takata M. Kuroda S. Kotani K. Matsumoto M. Sakaue M. Nishio S. Ueno H. Kasuga M. Mol. Endocrinol. 1997; 11: 1552-1562Crossref PubMed Scopus (116) Google Scholar). The cells were subjected to experiments 24–48 h after infection. L6 myotubes were deprived of serum for 16–20 h, incubated in the absence or presence of 100 nminsulin for 10 min, and then immediately frozen with liquid nitrogen. For assay of Akt activity, the frozen cells were lysed and subjected to immunoprecipitation with antibodies to either Akt or HA, as described previously (7Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar). The kinase activity in the resulting immunoprecipitates was then assayed as described (7Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar) with histone 2B as substrate. For assay of PKCλ activity, the frozen cells were lysed and subjected to immunoprecipitation with antibodies to PKCλ. The kinase activity in the resulting immunoprecipitates was then assayed with myelin basic protein as substrate, as described (14Kotani K. Ogawa W. Matsumoto M. Kitamura T. Sakaue H. Hino Y. Miyake K. Sano W. Akimoto K. Ohno S. Kasuga M. Mol. Cell. Biol. 1998; 18: 6971-6982Crossref PubMed Google Scholar). MAP kinase activity and PI 3-kinase activity were assayed in immunoprecipitates prepared with antibodies to MAP kinase or to phosphotyrosine, respectively, as described previously (35Sakaue H. Ogawa W. Takata M. Kuroda S. Kotani K. Matsumoto M. Sakaue M. Nishio S. Ueno H. Kasuga M. Mol. Endocrinol. 1997; 11: 1552-1562Crossref PubMed Scopus (116) Google Scholar). For analysis of 4E-BP1 phosphorylation, serum-deprived CHO cells were incubated in the absence or presence of 100 nm insulin for 10 min, scraped into a solution containing 50 mm Tris-HCl (pH 7.5), 100 mm potassium fluoride, 10 mmEDTA, 2 mm EGTA, leupeptin (10 μg/ml), aprotinin (10 μg/ml), and 0.1 mm phenylmethylsulfonyl fluoride (PMSF), and lysed by three cycles of freezing and thawing. After removal of debris by centrifugation in a microcentrifuge, the resulting supernatant was heated at 100 °C for 7 min. The resulting precipitated material was removed by centrifugation, and the new supernatant was subjected to immunoblot analysis with antibodies to 4E-BP1. Alternatively, serum-deprived CHO cells were washed twice with KRH buffer (25 mm Hepes-NaOH (pH 7.4), 119 mmNaCl, 4.95 mm KCl, 2.54 mm CaCl2, 0.3 mm potassium phosphate, 1.19 mmMgSO4) containing 1% bovine serum albumin (BSA), and then incubated for 2 h at 37 °C with KRH buffer containing 3% BSA and [32P]orthophosphate (0.5 mCi/ml). After subsequent incubation in the absence or presence of 100 nm insulin for 10 min, the cells were washed twice with KRH buffer containing 1% BSA and lysed in a solution containing 10% glycerol, 50 mmTris-HCl (pH 7.5), 60 mm KCl, 2 mm EDTA, 1% Triton X-100, 2 mm dithiothreitol, 20 mmβ-glycerophosphate, 10 mm sodium pyrophosphate, 50 mm NaF, 1 mm PMSF, 0.1 mm sodium orthovanadate, 50 mm p-nitrophenyl phosphate. The lysate was centrifuged (15,000 × g for 20 min), and the resulting supernatant was subjected to immunoprecipitation with polyclonal antibodies to 4E-BP1. The immunoprecipitates were washed three times with a lysis buffer, then two times with a solution containing 20 mm Tris-HCl (pH 7.5), 150 mmNaCl, 0.1% sodium deoxycholate, 5 mm EDTA, 10 mm NaF, 2 mm sodium orthovanadate, 1 mm PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1% Nonidet P-40, then washed two times with a solution containing 200 mm LiCl, 50 mm Tris-HCl (pH 7.5), 1 mm dithiothreitol. The precipitates were then boiled in SDS sample buffer, and subjected to SDS-PAGE on a 15% gel; the incorporation of radioactivity into 4E-BP1 was visualized or quantitated with a Fuji BAS2000 image analyzer. Glycogen synthase activity was assayed essentially as described (21Yamamoto-Honda R. Tobe K. Kaburagi Y. Ueki K. Asai S. Yachi M. Shirouzu M. Yodoi J. Akanuma Y. Yokoyama S. Yazaki Y. Kadowaki T. J. Biol. Chem. 1995; 270: 2729-2734Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In brief, serum-deprived differentiated L6 myotubes in 60-mm culture dishes were incubated in the absence or presence of 100 nminsulin for 30 min at 37 °C, and then lysed in 120 μl of a solution containing 50 mm Tris-HCl (pH 7.6), 100 mm potassium fluoride, 10 mm PMSF and 30% (v/v) glycerol. After removal of insoluble material by centrifugation, 30 μl of the supernatant were mixed with 60 μl of a solution containing 50 mm Tris-HCl (pH 7.6), 20 mm EDTA, 25 mm potassium fluoride, glycogen (10 mg/ml), and 6.7 mm uridine diphospho-d-[U-14C]glucose (0.05 μCi), in the absence or presence of 10 mm glucose 6-phosphate. After incubation for 30 min at 30 °C, the reaction was terminated by spotting the reaction mixture onto filter paper, and glycogen was precipitated on the paper by soaking in ice-cold 70% (v/v) ethanol. Each filter was washed four times with ice-cold 70% ethanol, and the radioactivity remaining on the paper was then measured with a liquid scintillation counter. The activity ratio (the ratio of the activity in the absence of glucose 6-phosphate to that in its presence) was calculated. L6 myotubes were incubated for 10 min in the absence or presence of 100 nm insulin, lysed, and subjected to immunoprecipitation with polyclonal antibodies that recognize all three known isoforms of Akt (αpanAkt) (7Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar). The resulting immunoprecipitates were then assayed for kinase activity with histone 2B as substrate. The amount of Akt activity in the precipitate from insulin-treated cells was about 5 times that in the precipitate from untreated cells (Fig.1 A). We have previously shown that a mutant Akt in which the sites of ligand-induced phosphorylation are replaced by alanine (Akt-AA) inhibits the activation of endogenous or transfected Akt by insulin, platelet-derived growth factor, or heat treatment in 3T3-L1 adipocytes or CHO cells (7Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar, 33Kuroda S. Ogawa W. Kitamura T. Konishi H. Kikkawa U. Kasuga M. Biochem. Biophys. Res. Commun. 1998; 249: 781-785Crossref PubMed Scopus (2) Google Scholar). We thus examined whether Akt-AA exerts similar effects in L6 myotubes. Myotubes were infected with an adenovirus vector encoding HA-tagged wild-type Akt (AxCAAkt-WT) at an m.o.i. of 0.5 plaque-forming units (pfu)/cell and, after 12 h, infected again with an adenovirus encoding FLAG-tagged Akt-AA (AxCAAkt-AAFL) at various MOIs. After an additional 20 h, the myotubes were incubated in the absence or presence of 100 nm insulin for 10 min, lysed, and subjected to immunoprecipitation with antibodies to HA. Assay of the resulting immunoprecipitates for Akt activity revealed that insulin increased the activity of HA-Akt to an extent similar to that observed for endogenous Akt (Fig. 1 C). Infection of the cells with AxCAAkt-AAFL inhibited insulin-induced activation of HA-Akt in an m.o.i.-dependent manner (Fig. 1 C), without affecting the amount of HA-Akt protein present in the immunoprecipitates (Fig. 1 B). At an AxCAAkt-AAFL m.o.i. of 10 pfu/cell, insulin-induced activation of HA-Akt was almost completely abolished. Infection with AxCAAkt-AAFL did not affect insulin activation of MAP kinase (Fig. 1 D) or of PI 3-kinase (as assayed in precipitates prepared with antibodies to phosphotyrosine) (data not shown), suggesting that the inhibitory effect of Akt-AA on Akt activation is specific. Infection of the myotubes with a control virus encoding β-galactosidase (AxCALacZ) at an m.o.i. of 10 pfu/cell did not affect insulin activation of Akt (data not shown). These results suggest that Akt-AA exerts a dominant negative effect on insulin-induced Akt activation. We next investigated the effect of Akt-AA on endogenous Akt activity in L6 myotubes by precipitating the endogenous protein with αpanAkt. Because these antibodies recognize both endogenous and recombinant Akt proteins, Akt-AA was immunodepleted from cell lysates with antibodies to FLAG before endogenous Akt was immunoprecipitated with the antibodies to Akt and assayed for kinase activity. Infection of L6 myotubes with AxCAAkt-AAFL resulted in a dose-dependent increase in total Akt protein (Fig.2 A); at an m.o.i. of 10 pfu/cell, the abundance of Akt-AA was ∼20 times that of endogenous Akt. After three sequential rounds of immunoprecipitation of cell lysates with antibodies to FLAG, the amounts of Akt protein remaining in the supernatant were similar for infected and noninfected cells (Fig. 2 A), indicating that Akt-AA was completely removed by this procedure. The insulin-induced activation of endogenous Akt was inhibited by AxCAAkt-AAFL in an m.o.i.-dependent manner, and at an m.o.i. of 20 pfu/cell, insulin-induced activation of endogenous Akt was almost completely abolished (Fig.2 B). We have previously shown that Akt-K179D, a mutant Akt in which Lys179 in the kinase domain is replaced by aspartate, did not inhibit insulin-induced activation of Akt when overexpressed in CHO cells (7Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar). We therefore examined the effect of Akt-K179D on endogenous Akt activity in L6 myotubes. Myotubes were infected with a virus encoding HA-tagged Akt-K179D (AxCAAkt-K179D), incubated in the absence or presence of insulin, and lysed. Akt-K179D was then immunodepleted from the cell lysates with antibodies to HA, after which endogenous Akt in the remaining supernatant was immunoprecipitated with αpanAkt and assayed for kinase activity. Although the amount of Akt protein in the cells infected with AxCAAkt-K179D at an m.o.i. of 10 pfu/cell was similar to that in cells infected with AxCAAkt-AAFL at the same m.o.i. (Fig. 2 C), activation of endogenous Akt by insulin was not affected by Akt-K179D (Fig. 2 D). Although we have previously shown that αpanAkt recognize all three known isoforms of rat Akt transiently expressed in COS cells (7Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar), a recent report has suggested that the previously cloned rat Akt3 (PKBγ) may be a minor splice variant of this isoform (37Brodbeck D. Cron P. Hemmings B.A. J. Biol. Chem. 1999; 274: 9133-9136Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). We therefore examined whether αpanAkt recognizes endogenous Akt3 in L6 myotubes. CHO cells, L6 myotubes, or 293 cells were lysed and immun
Abstract Background and Aims: Prognosis after hepatectomy for hepatocellular carcinoma (HCC) has been improved by progress in the evaluation of hepatic functional reserve, surgical techniques and perioperative management. However, even when curative resection is performed at a relatively early stage, a considerable number of patients develop early intrahepatic and/or extrahepatic recurrence postoperatively. This study analyzed the clinicopathologic features of HCC with early recurrence. Methods: We reviewed records of 513 consecutive patients who had undergone liver resection for HCC. There were 48 deaths within a year after surgery from recurrence, including 21 patients with stage II or III HCC (group I). Clinicopathologic parameters of group I patients were compared with those of 188 patients (group II) who developed recurrence following resection of stage II or III HCC and died more than 1 year after surgery. Results: On univariate analysis, age, tumor diameter (Φ), α‐fetoprotein (AFP):Φ and protein induced by vitamin K absence or antagonist II (PIVKA‐II):Φ were significantly greater in group I than in group II. Macroscopic portal vein invasion, microscopic vascular invasion, intrahepatic metastasis, poor differentiation, pleomorphism, sarcomatous change, vascular lake, and angiographic condensed pooling were more frequently observed in group I than group II. Five independent determinants were selected by multivariate analysis: AFP:Φ, histologic pleomorphism, sarcomatous change, vascular lake and angiographic condensed pooling. Conclusions: Highly malignant HCC with extremely poor prognosis exhibits peculiar clinicopathologic characteristics, particularly histologic immaturity, and can be predicted by preoperative indicators such as markedly elevated tumor marker concentrations and condensed pooling on angiography.
