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Few studies have investigated the relationship between blood type and trauma outcomes according to the type of injury. We conducted a retrospective multicenter observational study in twelve emergency hospitals in Japan. Patients with isolated severe abdominal injury (abbreviated injury scale for the abdomen ≥ 3 and that for other organs p p = 0.012). Furthermore, type O was associated with significantly higher cause-specific mortalities, fewer VFD, and larger transfusion volumes. Blood type O was associated with significantly higher mortality and larger transfusion volumes in patients with isolated severe abdominal trauma.

The incidence of rectal cancer with a pelvic abscess is rare; hence, treatment strategies are difficult because both malignant and infectious inflammation need to be addressed. Here, we report the case of a 53-year-old man diagnosed with rectal cancer accompanied by a pelvic abscess. We performed transrectal drainage of the abscess, and a transanal rectal drainage tube was inserted into the abscess cavity. His symptoms rapidly improved, and computed tomography showed that the pelvic abscess had disappeared. Six weeks after drainage, radical laparoscopic Hartmann’s procedure with resection of the rectal cancer and incision drainage scar was performed. After adjuvant chemotherapy, laparoscopic stoma closure was performed a year after the operation. The patient showed no evidence of cancer recurrence 1.5 years after radical surgery. Transrectal drainage followed by laparoscopic radical resection can be a less invasive and effective treatment for rectal cancer accompanied by a pelvic abscess.

BACKGROUND:We sought to investigate the efficacy of perioperative tight glycemic control (TGC) in reducing of postoperative infectious complications (POICs) and study its impact on early inflammatory mediators in patients who underwent pancreaticoduodenectomy., METHODS:In this non-randomized trial, the artificial pancreas (AP) group received TGC (target glucose range of 80-110mg/dL; n=14), while the control group received conventional glycemic control (range of 80-180mg/dL; n=15). The primary endpoint was POICs., RESULTS:The AP group had a markedly decreased POIC rate (28.6% vs. 73.3%; P= 0.027), mean glycemic variability (13.5±3.5% vs. 16.4±5.9%; P=0.038), and plasma interleukin-6 level (26.3±33.8 vs 98.3±89.1pg/ml; P=0.036) compared to the control group, but insulin dosage (27.0±13.4 vs. 10.2±16.2 U; P=0.002) and the adiponectin ratio (i.e., postoperative/preoperative adiponectin; 0.8±0.2 vs. 0.6±0.3; P=0.021) were markedly higher in the AP group., CONCLUSIONS:Among patients undergoing PD with impaired glucose tolerance, AP facilitated strict glycemic control and resulted in a reduction of anti-inflammatory mediators and POICs., SUMMARY:Perioperative hyperglycemia increases postoperative infectious complications; however, tight glycemic control using artificial pancreas can reduce them via a dual effect. Artificial pancreas facilitates strict and safe glycemic control while reducing anti-inflammatory mediators, including adiponectin, following pancreaticoduodenectomy.

A Bochdalek hernia (BH) is a congenital abnormality with incomplete closure of the diaphragm. It is usually manifested in infants but rarely in adults. Here, we report an adult patient with gastric volvulus and giant BH that were safely repaired by endoscopic reduction and elective laparoscopic surgery, respectively. A 79-year-old woman presented with left upper abdominal pain but no history of trauma. CT revealed a giant BH with gastric volvulus. After emergency endoscopic reduction of the volvulus, elective laparoscopic repair of the BH was performed. The 8 × 8-cm defect was repaired with interrupted nonabsorbable sutures and a mesh. The patient's postoperative course was uneventful, and no complications or recurrence were observed in the 6 months that followed.

Background: A rectovaginal fistula (RVF) after low anterior resection for rectal cancer is troublesome and refractory. Although various surgical procedures have been previously described, no definitive procedure has shown satisfactory outcomes. We present two consecutive Japanese patients who underwent successful surgery for a RVF after low anterior resection. Case presentation: The patients were a 61-year-old woman and a 64-year-old woman. They were admitted to our hospital with a chief complaint of fecal discharge from the vagina after low anterior resection using the double-stapling technique for rectal cancer. They were diagnosed with RVF. Local surgical procedures, including diverting ileostomy, were unsuccessful in previous hospitals. Therefore, we performed laparoscopy-assisted repair of the RVF. In both the patients, laparoscopically robust pelvic adhesions were dissected, and the sigmoid colon was transected just before the RVF. Thereafter, in combination with a perineal approach, the rectum along with a previous anastomosis and fistula were completely removed. Surgeries were completed after vaginal repair, redo coloanal anastomosis, and interposition of the dissected connective tissue. In both the patients, the postoperative courses were uneventful. They have complained of neither recurrence of any RVF nor fecal incontinence 1 year and 10 months respectively after diverting stoma closure.Conclusions: A laparoscopy-assisted procedure with reanastomosis and interposition of the perineal connective tissue can be an effective treatment for an RVF after low anterior resection for rectal cancer.

Adoptive cell transfer (ACT) is an emerging and promising cancer immunotherapy that has been improved through various approaches. Here, we described the distinctive characteristics and functions of tumor Ag-specific effector CD8+ T-cells, co-cultured with a tumor-specific peptide and a stimulatory anti-OX40 antibody, before being used for ACT therapy in tumor-bearing mouse recipients. Splenic T-cells were obtained from wild-type FVB/N mice that had been injected with a HER2/neu (neu)-expressing tumor and a neu-vaccine. The cells were then incubated for 7 days in vitro with a major histocompatibility complex (MHC) class I peptide derived from neu, in the presence or absence of an agonistic anti-OX40 monoclonal antibody, before CD8+ T cells were isolated for use in ACT therapy. The proliferative ability of OX40-driven tumor Ag-specific effector CD8+ T-cells in vitro was less than that of non-OX40-driven tumor Ag-specific effector CD8+ T-cells, but they expressed significantly more early T-cell differentiation markers, such as CD27, CD62L and CCR7, and significantly higher levels of Bcl-2, an anti-apoptotic protein. These OX40-driven tumor Ag-specific effector CD8+ T-cells, when transferred into tumor-bearing recipients, demonstrated potent proliferation capability and successfully eradicated the established tumor. In addition, these cells exhibited long-term antitumor function, and appeared to be established as memory T-cells. Our findings suggest a possible in vitro approach for improving the efficacy of ACT, which is simple, requires only a small amount of modulator, and can potentially avoid several toxicities associated with co-stimulation in vivo.

Recently, accelerometers measuring physical activity level have been available to the public. In the present study, it was examined whether the accelerometer could evaluate postoperative outcomes for 12 patients subjected to hepatic resection from August-November 2016. The association was evaluated between the changing pattern of activity level until the postoperative day (POD) 7 and the occurrence of postoperative complications. The median age of patients was 79 years (range, 58-85). Postoperative complications were identified in 6 patients. The activity level in patients with complications was low from POD 1 and was significantly lower than patients without complications following POD 6. The changing pattern of activity level with all included patients could be divided into the following 3 types: Increase type, bell curve type and flat type. Patients without complications exhibited an accelerated increase of postoperative activity level, categorized as increase type. Bell curve type and flat type demonstrated delay of recovery in postoperative activity levels, and were suggested to be associated with the occurrence of postoperative complications. These findings may provide rationale for larger sample studies to evaluate whether physical activity level measured via accelerometer may be a surrogate marker for postoperative complications.

Interferon (IFN) has been identified to suppress carcinogenesis when used for treating hepatitis C virus (HCV) infections. Treatment with IFN-free direct-acting antiviral agents (DAAs) is an acceptable alternative, even in elderly patients or patients who have been treated for hepatocellular carcinoma (HCC), because it has a lower incidence of side effects and higher sustained virological response (SVR) rate compared with IFN treatment. However, the suppression of carcinogenesis by DAAs is unclear. In the present study, 19 patients who underwent DAA treatment following treatment for HCC between January 2015 and March 2017 were retrospectively investigated. The clinical data were compared between 9 patients with HCC recurrence following DAA treatment (recurrence group) and 10 patients without HCC recurrence (no-recurrence group). The 1-year cumulative recurrence rate of HCC following SVR was as high as 50.2%. Age and sex did not significantly differ between the two groups, and the average number of HCC treatments prior to DAA treatment was also not significantly different between the recurrence and no-recurrence groups (3.2 and 2.2, respectively). The median interval between the final HCC treatment and the commencement of DAA treatment was 88 days in the recurrence group, which was significantly less compared with 790 days in the no-recurrence group (P=0.018). An interval of 120 days or more from final HCC treatment to the commencement of DAA treatment was a significant independent factor of no HCC recurrence following DAA treatment (P=0.028). A high HCC recurrence rate was identified following DAA treatment in patients with a history of HCC treatment. Therefore, there should be at least a 4-month interval from the final HCC treatment to the commencement of DAA treatment to ensure no HCC recurrence.

Hepatocyte growth factor (HGF) has an inhibitory effect on human HepG2 hepatoma cell proliferation. Previously, it was shown that HGF treatment downregulated Id1 and upregulated p16INK4a in an ERK-dependent manner, leading to the inhibition of cellular proliferation. Here, new insight suggests that Skp2, an SCF complex component and potential prognosticator in cancer, is downregulated by injection of HGF into established HepG2 xenograft tumors. The downregulation was evident at both the mRNA and protein level and in an ERK-dependent manner. Critically, high expression of Skp2 restored HGF-inhibited cell proliferation, indicating that the inhibitory effect of HGF required the downregulation of Skp2. However, downregulation was not involved in the HGF-induced upregulation of a CDK inhibitor, p27Kip1, a known SCF-Skp2 target. Instead, data revealed that Skp2 regulated Myc activity, which has oncogenic potential in the generation of hepatocellular carcinoma. Elevated expression of Skp2 or a mutant that is unable to associate with the SCF complex was capable of activating Myc, suggesting that Skp2 does not act on Myc as a component of the SCF complex, and thus functions as an activator of Myc independent of its role in ubiquitination. Furthermore, Skp2 regulated Id1 expression by regulating Myc activity, and the regulation of Skp2 is involved in the activity of p16 promoter through regulation of Id1 expression. Overall, these mechanistic findings provide the first evidence that ERK-dependent downregulation of Skp2 reduced Myc activity, leading to HGF-induced inhibition of cell proliferation through decreased Id1 expression. Implications: This study elucidates the molecular details of HGF-induced inhibition of cellular proliferation in liver cancer cells. Mol Cancer Res; 11(11); 1437–47. ©2013 AACR.

Hepatocyte growth factor (HGF) is a multifunctional molecule that acts as mitogen, motogen, and/or morphogen in a variety of cells. MET, a specific receptor tyrosine kinase for HGF, is upregulated in various tumors including squamous cell carcinoma of the human head and neck (HNSCC), but how HGF affects the expression of downstream functional genes has not yet been elucidated in detail. In the present study, we examined the expression of microRNA (miRNA), non-coding small RNA that regulate cell proliferation and functions by interfering with the translation of target mRNA, with or without HGF stimulation in HNSCC cell line HSC3. Among several miRNAs, in which the expression was altered after HGF stimulation, we focused on miR-200c and miR-27b, both of which were drastically downregulated after HGF stimulation. Expression of ZEB1, a target mRNA for miR-200c, was upregulated 3 and 6 h after HGF stimulation, and that of E-cadherin, a downstream molecule of ZEB1, was downregulated 12 h after HGF stimulation. Expression of ST14/matriptase, an enzyme for extracellular matrix (ECM) degradation and HGF activation and a target mRNA for miR-27b, was drastically upregulated in the protein level after HGF stimulation, although it was not statistically altered in the mRNA level. These results suggest that miR-200c and miR-27b downregulated by HGF might play an important role in epithelial–mesenchymal transition mediated by ZEB1/E-cadherin and ECM degradation and HGF autoactivation mediated by ST14/matriptase, respectively. Altered expression of miRNA directly regulated by HGF might contribute enhanced progressive and invasive characteristics of HNSCC by regulating the translation of HGF-induced functional molecules. (Cancer Sci 2011; 102: 2164–2171)

The complete mechanism of labor induction in eutherian mammals remains unclear. Although important roles for the fetus and placenta in triggering labor have been proposed, no gene has been shown to be required in the fetus/placenta for labor induction. Here we show that Nrk, an X-linked gene encoding a Ser/Thr kinase of the germinal center kinase family, is essential in the fetus/placenta for labor in mice. Nrk was specifically expressed in the spongiotrophoblast layer, a fetus-derived region of the placenta, and Nrk disruption caused dysregulated overgrowth of the layer. Due to preferential inactivation of the paternally derived X chromosome in placenta, Nrk heterozygous mutant placentas exhibited a similar defect to that in Nrk-null tissues when the wild-type allele was paternally derived. However, the phenotype was weaker than in Nrk-null placentas due to leaky Nrk expression from the inactivated X chromosome. Crossing of Nrk-null females to wild-type and Nrk-null males, as well as uterine transfer of Nrk-null fetuses to wild-type females, revealed that pregnant mice exhibit a severe defect in delivery when all fetuses/placentas are Nrk-null. In addition, Nrk was not expressed in female reproductive tissues such as the uterus and ovary, as well as the fetal amnion and yolk sac, in pregnant mice. Progesterone and estrogen levels in the maternal circulation and placenta, which control the timing of labor, were unaffected upon Nrk disruption. We thus provide evidence for a novel labor-inducing fetoplacental signal that depends on the X chromosome and possibly arises from the placenta.

鼠径ヘルニアを有する慢性腎不全患者は一般的に腹膜透析療法を選択されない．一方，腎不全患者の高齢化に伴い，鼠径ヘルニアを合併することが多くなっている．われわれは，Kugel法によるヘルニア修復を行った後，腹膜透析を行った経験を報告する．Kugel法は腹膜前腔にパッチを挿入して，1枚のパッチで内鼠径輪・外鼠径輪・大腿輪を同時にカバーし，鼠径部の全てのタイプのヘルニアに同時に対応する方法である．従来のMesh plug法に比べ理論上強度が強く，本法を行うことで鼠径ヘルニア修復と安全な腹膜透析が同時に可能と考えられる．

Type II transmembrane serine proteases (TTSPs) are structurally defined by the presence of a transmembrane domain located near the N-terminus and a C-terminal extracellular serine protease domain. The human TTSP family consists of 17 members. Some members of the family have pivotal functions in development and homeostasis, and are involved in tumorigenesis and viral infections. The activities of TTSPs are regulated by endogenous protease inhibitors. However, protease inhibitors of most TTSPs have not yet been identified. In this study, we investigated the inhibitory effect of hepatocyte growth factor activator inhibitor type 1 (HAI-1), a Kunitz-type serine protease inhibitor, on several members of the TTSP family. We found that the protease activity of a member, TMPRSS13, was inhibited by HAI-1. A detailed analysis revealed that a soluble form of HAI-1 with one Kunitz domain (NK1) more strongly inhibited TMPRSS13 than another soluble form of HAI-1 with two Kunitz domains (NK1LK2). In addition, an in vitro protein binding assay showed that NK1 formed complexes with TMPRSS13, but NK1LK2 did not. TMPRSS13 converted single-chain pro-hepatocyte growth factor (pro-HGF) to a two-chain form in vitro, and the pro-HGF converting activity of TMPRSS13 was inhibited by NK1. The two-chain form of HGF exhibited biological activity, assessed by phosphorylation of the HGF receptor (c-Met) and extracellular signal-regulated kinase, and scattered morphology in human hepatocellular carcinoma cell line HepG2. These results suggest that TMPRSS13 functions as an HGF-converting protease, the activity of which may be regulated by HAI-1.

Regulatory T cells (Tregs) play an important role in maintaining immunological tolerance that is one of the main obstacles to overcome for improving antitumor immunity. Recently, the Treg has been shown to constitutively express OX40 (CD134), which is a member of the TNF-receptor family that is transiently expressed on effector T cells after TCR triggering, and through which the signal enhances effector T-cell proliferation and memory T-cell development. However, little is known about the role of OX40 costimulation to Tregs in tumor immunology. Here we show that OX40 signaling modulates the function of naturally occurring Tregs in vitro and in vivo. Foxp3 expression on Tregs was reduced by OX40 costimulation, but not by IL-2 stimulation. Tregs suppressed the proliferation of naïve CD4(+) CD25(-) T cells after TCR triggering, in contrast, OX40 costimulated Tregs that reduced Foxp3 expression reversed the suppressive function. In addition, Tregs inhibited the proliferation of TCR-stimulated (primed) CD4(+) T cells and naïve CD8(+) T cells after TCR-mediated activation, however, Tregs with OX40 costimulation lost their suppressive function. Interestingly, Tregs minimally suppressed the proliferation or the cytokine secretion of Ag-specific CD8(+) T cells after Ag-restimulation. Furthermore, Tregs suppressive function to the antitumor effect was reversed by OX40 costimulation in vivo. Our data indicate that, in addition to controlling effector T-cell function, OX40 costimulation directly controls Treg-mediated suppression in tumor immunity.

Hepatocyte growth factor (HGF) inhibits the proliferation of several tumor cell lines and tumor growth in vivo. We showed previously that HGF induces cell cycle arrest at G1 in a human hepatoma cell line, HepG2, by up-regulating the expression of p16INK4a through strong activation of extracellular signal-regulated kinase (ERK). However, although essential, the activation was not sufficient for the up-regulation of p16. In this study, we examined regulatory mechanisms of p16 expression through a transcription factor, Ets, which has been shown previously to bind to the promoter. The treatment of HepG2 cells with HGF induced ERK-dependent phosphorylation of Ets, which leads to its activation, before the up-regulation of p16, suggesting that another factor suppresses Ets activity. We found that HGF reduces the amount of Id1, which is a dominant-negative inhibitor of Ets, leading to a decrease in Ets associated with Id1. Id1 was down-regulated via transcriptional regulation not via the ubiquitin-proteasome-mediated pathway. Inhibition of the HGF-induced high-intensity ERK activity had a modest effect on the Id1 down-regulation, and inhibition of the phosphatidylinositol 3-kinase pathway had no effect, showing that Id1 is regulated by ERK-dependent and -independent pathways other than the phosphatidylinositol 3-kinase pathway. Exogenously expressed Id1 suppressed the up-regulation of p16 by HGF and the antiproliferative effect of HGF. Knockdown of Id1 significantly enhanced the activity of the p16 promoter coordinately with the activation of ERK. Our results indicated that down-regulation of Id1 plays a key role in the inhibitory effect of HGF on cell proliferation and provides a molecular basis for cancer therapy with HGF. (Mol Cancer Res 2009;7(7):1179–88)

The nucleolus is a subnuclear compartment and the site of ribosome biogenesis. Previous studies have implicated protein ubiquitylation in nucleolar activity. Here we show that USP36, a deubiquitylating enzyme of unknown function, regulates nucleolar activity in mammalian cells. USP36 localized to nucleoli via the C-terminal region, which contains basic amino acid stretches. Dominant-negative inhibition of USP36 caused the accumulation of ubiquitin-protein conjugates in nucleoli, suggesting that nucleoli are the site of USP36 action. USP36 deubiquitylated the nucleolar proteins nucleophosmin/B23 and fibrillarin, and stabilized them by counteracting ubiquitylation-mediated proteasomal degradation. RNAi-mediated depletion of cellular USP36 resulted in reduced levels of rRNA transcription and processing, a less-developed nucleolar morphology and a slight reduction in the cytoplasmic ribosome level, which eventually led to a reduced rate of cell proliferation. We conclude that by deubiquitylating various nucleolar substrate proteins including nucleophosmin/B23 and fibrillarin, USP36 plays a crucial role in regulating the structure and function of nucleoli.

Activation of the extracellular signal-regulated kinase (ERK) pathway is a key factor in the regulation of cell proliferation by growth factors. Hepatocyte growth factor (HGF)-induced cell cycle arrest in the human hepatocellular carcinoma cell line HepG2 requires strong activation of the ERK pathway. In this study, we investigated the molecular mechanism of the activation. We constructed a chimeric receptor composed of the extracellular domain of the NGF receptor and the cytoplasmic domain of the HGF receptor (c-Met) and introduced a point mutation (N1358H) into the chimeric receptor, which specifically abrogates the direct binding of Grb2 to c-Met. The mutant chimeric receptor failed to mediate the strong activation of ERK, up-regulation of the expression of a Cdk inhibitor p16(INK4a) and inhibition of HepG2 cell proliferation by ligand stimulation. Moreover, the mutant receptor did not induce tyrosine phosphorylation of the docking protein Gab1. Knockdown of Gab1 using siRNA suppressed the HGF-induced strong activation of ERK and inhibition of HepG2 cell proliferation. These results suggest that coupling of Grb2 to Gab1 mediates the HGF-induced strong activation of the ERK pathway, which is required for the inhibition of HepG2 cell proliferation.

The association of hepatocyte growth factor (HGF) with its high-affinity receptor (c-Met) has been shown to induce mitogenesis, motogenesis and morphogenesis in a variety of cell types. Various point mutations in c-Met have been identified in hereditary and sporadic papillary renal carcinomas as well as in other carcinomas. In the present study, we examined the effects of c-Met point mutations on the morphology of a porcine aortic endothelial (PAE) cell line. When cultured in three-dimensional collagen gel, PAE cells formed branching tubule structures, and HGF treatment caused breakdown of the structures and induced a scattered morphology. The exogenous expression of c-Met point mutants inhibited the formation of tubules. HGF treatment induced the formation of tubules by PAE cells expressing some c-Met mutants, but it induced the scattering of PAE cells expressing other c-Met mutants. The presence of a low concentration of a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor cancelled the inhibitory effect of the c-Met point mutations on the formation of tubules. These results suggest that c-Met point mutations affect the extracellular signal-regulated kinase (ERK) signaling required for the formation of tubules by PAE cells, and HGF binding changes the conformation of c-Met mutants, leading to the different signals required for formation of tubules and cell scattering.

Monoubiquitination of endocytosed cell surface receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. The sorting of ubiquitinated proteins is executed by concerted actions of class E vacuolar protein sorting (Vps) proteins. Some proteins in the sorting machinery undergo monoubiquitination, suggesting that their functions are also regulated by ubiquitination. The Hrs-STAM complex, a class E Vps protein complex essential for the initial step of the sorting pathway, binds two deubiquitinating enzymes, UBPY and AMSH. Here we examined the effects of inactivating UBPY on protein ubiquitination at endosomes. Overexpression of a catalytically inactive UBPY mutant or depletion of UBPY by RNA interference resulted in the accumulation of ubiquitinated proteins on morphologically aberrant endosomes. Electron microscopy showed that they are aggregates of multivesicular endosomes. Among the sorting machinery proteins that undergo ubiquitination, Eps15 was monoubiquitinated at an elevated level in UBPY-inactivated cells. UBPY also deubiquitinated Eps15 in vitro, suggesting that Eps15 is a cellular substrate for UBPY. Furthermore, inactivation of UBPY caused the accumulation of Eps15 on the endosomal aggregates. These results suggest that UBPY regulates the level of protein ubiquitination on endosomes, which is required for maintaining the morphology of the organelle.

Background & Aims: Hepatocyte growth factor activator (HGFA) is a serum proteinase that specifically converts an inactive single-chain form of hepatocyte growth factor (HGF) into an active 2-chain form. HGFA is produced in its precursor form and then activated in injured tissues. To address the precise role of HGFA and to investigate the mechanisms of HGF activation in injured tissues, we generated mice deficient in HGFA. Methods: HGFA-deficient mice were generated using targeted gene disruption. The regenerating process of intestinal mucosa damaged by oral administration of dextran sodium sulfate (DSS) or by rectal administration of acetic acid was examined in both HGFA-deficient and control mice. HGF processing activity was analyzed using Western blotting and an HGF activation assay. Results: Homozygous mutant mice were viable and fertile without obvious abnormalities. When mice were treated with 3% DSS in drinking water for 6 days followed by distilled water without DSS, 72% of HGFA-deficient mice died through day 12 while 75% of control mice survived injury. Similar results were also observed in the acetic acid-induced intestinal injury; the survival rate was 36.6% in HGFA-deficient mice and 84.2% in control mice. In HGFA-deficient mice, the injured mucosa was not sufficiently covered by regenerated epithelium and the activation of HGF was impaired in the injured colon. Conclusions: These results indicate that HGFA is required for repair of injured intestinal mucosa but is not essential for normal development during embryogenesis or after birth.

c-Met is a high-affinity receptor for hepatocyte growth factor (HGF) and plays a crucial role in embryonic development, as well as in the process of tissue repair. Overexpression and amplification of c-Met are often observed in various cancer tissues, especially in gastric carcinoma. It has, however, been unclear whether the overexpression leads to activation of the c-Met receptor. To address this point, we prepared an antibody (anti-phospho-Met) which specifically recognizes c-Met that is phosphorylated at Y1235, a major phosphorylation site of c-Met. Normal as well as cancerous gastric tissue was positive for anti-total-Met staining, whereas only cancerous tissue was strongly positive for anti-phospho-Met staining; cells near the basal layer were moderately positive, and the proliferative zone in normal tissue was only weakly positive. Among cancerous tissues from seven patients examined in the present study, those from six patients were strongly positive for phospho-Met staining. These results indicate that c-Met is actually activated in gastric carcinoma tissue, and may trigger proliferation/anti-apoptotic signals.

Metastatic pancreas tumors from breast cancer are comparatively uncommon and patients with this tumor usually remain asymptomatic during their life. A 55-year-old woman presented with obstructive jaundice following mastectomy for invasive ductal carcinoma. We diagnosed obstructive jaundice due to a pancreatic tumor demonstrated on computed tomography and performed percutaneous transhepatic cholangio-drainage. Although the patient recovered from the jaundice, she had exacerbation of pneumonia from which she died. At autopsy, invasive ductal carcinoma was found in the pancreas tumor. Immunohistochemical staining was performed to confirm whether the pancreatic tumor was primary or secondary. Human milk fat globules 1 and 2 and gross cystic disease fluid protein-15, which characteristically exist in normal breast tissue or breast carcinoma, were expressed both in the primary breast tumor and the pancreatic tumor. In contrast, both the anti-estrogen receptor and anti-progesterone receptor antibodies stained positively in the primary breast cancer; however, neither of them was positive in the metastatic pancreatic tumor. We report a rare case of a patient who presented with obstructive jaundice from a pancreatic tumor metastasizing from breast cancer and in whom immunohistochemical staining using the antibodies unique to the mammary gland was effective for the diagnosis of this secondary tumor.

The cleavage cycle, which is initiated by fertilization, consists of only S and M phases, and the gap phases (G1 and G2) appear after the midblastula transition (MBT) in the African clawed frog, Xenopus laevis. During early development in Xenopus, we examined the E2F activity, which controls transition from the G1 to S phase in the somatic cell cycle. Gel retardation and transactivation assays revealed that, although the E2F protein was constantly present throughout early development, the E2F transactivation activity was induced in a stage-specific manner, that is, low before MBT and rapidly increased after MBT. Introduction of the recombinant dominant negative E2F (dnE2F), but not the control, protein into the 2-cell stage embryos specifically suppressed E2F activation after MBT. Cells in dnE2F-injected embryos appeared normal before MBT, but ceased to proliferate and eventually died at the gastrula. These cells contained decreased cdk activity with enhanced inhibitory phosphorylation of Cdc2 at Tyr15. Thus, E2F activity is required for cell cycle progression and cell viability after MBT, but not essential for MBT transition and developmental progression during the cleavage stage.

Background: Neoadjuvant chemotherapy (NAC) is widely performed for patients with locally advanced breast carcinomas. It is important to obtain precise information using imaging modalities about the distribution of residual carcinomas after NAC to predict the success of breast conserving surgery. However, the relationship between magnetic resonance imaging (MRI) shrinkage patterns and molecular subtypes of breast carcinomas has not yet been investigated. Methods: We analyzed the MRI shrinkage and pathological patterns of residual carcinomas after NAC in 54 consecutive breast carcinomas. The shrinkage patterns were classified into five categories: Type I and II (concentric shrinkage with or without any surrounding lesion), type III (shrinkage with residual multinodular lesions), type IV (diffuse contrast enhancement in the entire quadrant), and non-visualization. Results: The most common MRI shrinkage pattern was type I (39%) followed by non-visualization (26%). The most common pathological shrinkage pattern was type III (33%), followed by type II (28%) and non-visualization (22%). The concordance rates of the MRI patterns of non-visualization and type I were significantly lower than those of the MRI patterns of type II, III, and IV (p = 0.018). Moreover, in the luminal subtypes, the concordance rates of the MRI patterns of non-visualization and type I were significantly lower than those of the MRI patterns of type II, III, and IV (p = 0.026); however, in the non-luminal subtypes, this correlation was not significant. Conclusions: The results of the present study suggest that the cases with MRI shrinkage patterns of non-visualization and type I require caution regarding the surgical margins compared with the other types, especially the luminal subtypes. Citation Format: Tsuyoshi Mori, Kaori Tomida, Mina Kitamura, Sachiko Sakai, Yuki Kawai, Tomoharu Shimizu, Tomoko Umeda, Tomoyuki Ueki, Sachiko Kaida, Toru Miyake, Hiroya Iida, Hiroya Akabori, Naomi Kitamura, Tsuyoshi Yamaguchi, Hiromichi Sonoda, Shigeyuki Naka, Masaji Tani. Magnetic resonance imaging shrinkage patterns after neoadjuvant chemotherapy for breast carcinomas: Correlation with molecular subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3743. doi:10.1158/1538-7445.AM2017-3743

The mechanisms by which growth factors trigger signal transduction pathways leading to the regulation of c-Fos expression are of great interest. In this study we investigated the effect of hepatocyte growth factor (HGF/SF) and epidermal growth factor (EGF) on the expression of c-fos and its product, c-Fos, in human epithelial cell line MKN74. The expression level of c-Fos protein in HGF/SF-stimulated cells was 5--10-fold higher than that in EGF-stimulated cells, whereas the level of c-fos mRNA induced by HGF/SF was similar to that by EGF. The hyperphosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), indicative of an increased number of translation initiation complexes, was detected only in HGF/SF-induced MKN74 cells. Activation of phosphatidylinositol-3'-OH kinase and FKBP12-rapamycin associated mammalian target of rapamycin (FRAP/mTOR) was observed after the treatment with HGF/SF. Pretreatment with an inhibitor of either one, i.e. LY294002 for phosphatidylinositol-3'-OH kinase or rapamycin for FRAP/mTOR, completely inhibited 4E-BP1 phosphorylation and decreased the c-Fos synthesis induced by HGF/SF down to the level found in EGF-induced cells. These results suggest that the phosphorylation of 4E-BP1 is stimulated by HGF/SF in a manner requiring both phosphatidy-linositol-3'-OH kinase-dependent and FRAP/mTOR-dependent pathways, thereby stimulating c-fos mRNA translation. Regulation of the translation process of c-fos mRNA in addition to the immediate activation of c-fos transcription is necessary for the transient increase in the level of c-Fos protein to stimulate cell proliferation.

Hrs-binding protein (Hbp) is a Src homology 3 (SH3) domain-containing protein that tightly associates with Hrs. Hbp together with Hrs is thought to play a regulatory role in endocytic trafficking of growth factor-receptor complexes through early endosomes. Association of Hbp with a binding partner(s) via the SH3 domain seems to be essential for Hbp to exert its function. In this study, we searched for Hbp-binding proteins by a far Western screening and isolated a mouse cDNA clone encoding a deubiquitinating enzyme mUBPY as an Hbp SH3-binding protein. mUBPY has two Hbp-SH3 domain binding sites. Mutagenic analysis identified a consensus sequence PX(V/I)(D/N)RXXKP as the Hbp-SH3 domain binding motif. It is a novel SH3-binding motif and does not contain the canonical proline-rich consensus binding motif, PXXP. Ubiquitination of growth factor receptors is thought to regulate their intracellular degradation. Thus, UBPY may play a regulatory role in the degradation by interaction with the SH3 domain of Hbp via the novel SH3-binding motif.

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a prominent substrate for activated tyrosine kinase receptors that has been proposed to play a role in endosomal membrane trafficking. The protein contains a FYVE domain, which specifically binds to the lipid phosphatidylinositol (PI) 3-phosphate (PI 3-P). We show that this interaction is required both for correct localization of the protein to endosomes that only partially coincides with early endosomal autoantigen 1 and for efficient tyrosine phosphorylation of the protein in response to epidermal growth factor stimulation. Treatment with wortmannin reveals that Hrs phosphorylation also requires PI 3-kinase activity, which is necessary to generate the PI 3-P required for localization. We have used both hypertonic media and expression of a dominant-negative form of dynamin (K44A) to inhibit endocytosis; under which conditions, receptor stimulation fails to elicit phosphorylation of Hrs. Our results provide a clear example of the coupling of a signal transduction pathway to endocytosis, from which we propose that activated receptor (or associated factor) must be delivered to the appropriate endocytic compartment in order for Hrs phosphorylation to occur.

The c-Jun N-terminal kinase (JNK) signaling pathway plays a crucial role in cellular responses stimulated by stress-inducing agents and proinflammatory cytokines. The group I germinal center kinase family members selectively activate the JNK pathway. In this study, we have isolated a mouse cDNA encoding a protein kinase homologous to Nck-interacting kinase (NIK), a member of the group I germinal center kinase family. This protein kinase is expressed during the late stages of embryogenesis, but not in adult tissues, and thus named NESK (NIK-like embryo-specific kinase). NESK selectively activated the JNK pathway when overexpressed in HEK 293 cells but did not stimulate the p38 kinase or extracellular signal-regulated kinase (ERK) pathways. NESK-induced JNK activation was inhibited by the dominant negative mutants of MEKK1 and MKK4. Tumor necrosis factor (TNF)-alpha or TNF receptor-associated factor 2 (TRAF2) stimulated the NESK activity. Furthermore, the dominant negative NESK mutant inhibited the JNK activation induced by TNF-alpha or TRAF2. These results suggest that NESK, a novel activator of the JNK pathway, functions in coupling TRAF2 to the MEKK1 --MKK4 --JNK kinase cascade during the late stages of mammalian embryogenesis.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) are recently discovered Kunitz-type serine protease inhibitors which can be purified and cloned from human stomach cancer cell line MKN45 as specific inhibitors against hepatocyte growth factor activator (HGFA). HAI-2 was identical with the protein originally reported as placental bikunin. Both proteins contain two Kunitz inhibitor domains (KDs), of which the first domain (KD1) is mainly responsible for the inhibitory activity against HGFA, and are expressed ubiquitously in various tissues. In this study, we cloned the genes coding for these two structurally similar proteins by screening of human genomic bacterial artificial chromosome (BAC) library and their genomic structures were compared. HAI-1 and -2 genes consist of 11 and 8 exons spanning 12 kbp and 12.5 kbp, respectively. Three exons were inserted between KD1 and KD2 of each gene, of which the middle one was the low-density lipoprotein (LDL) receptor-like domain (HAI-1) and the testis specific exon (HAI-2). Apparently homologous regions between HAI-1 and -2 were not found in 5'-flanking region and neither TATA nor CAAT box was present. The genes were mapped to chromosome 15q15 (HAI-1) and 19q13.11 (HAI-2). These results suggested that although HAI-1 and -2 genes might be derived from same ancestor gene, they acquired distinctive in vivo roles during their evolution.

Hepatocyte growth factor (HGF) activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) are new Kunitz-type serine protease inhibitors that were recently purified and cloned from the human stomach cancer cell line MKN45 as specific inhibitors against HGF activator. Both proteins contain two Kunitz inhibitor domains and are expressed abundantly throughout the gastrointestinal tract, in addition to the placenta, pancreas, and kidney. In this study, to assess the possible roles of HAI-1 and HAI-2 in the intestinal mucosa, we examined the expression of HAI-1 and HAI-2 during regeneration of the intestinal mucosa. Immunohistochemical studies revealed that HAI-1 but not HAI-2 was detected more strongly in regenerative epithelium than in normal epithelium, although both proteins were detected throughout the human gastrointestinal tract. During the course of acetic acid-induced experimental colitis in an in vivo mouse model, HAI-1 but not HAI-2 was upregulated in the recovery phase, suggesting that HAI-1 but not HAI-2 is associated with the regeneration of damaged colonic mucosa. Upregulation of HAI-1 may serve to downregulate the proliferative response after initial activation of MET receptor by HGF/scatter factor after an injury.

Background Hrs (hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate) is an early endosomal protein that is rapidly tyrosine-phosphorylated in cells stimulated with growth factors. Hrs is thought to play a regulatory role in the endocytosis of growth factor/receptor complexes through early endosomes. In this study, we searched for Hrs-interacting molecules which may regulate the function of Hrs, using a yeast two-hybrid system. Results We isolated a cDNA clone encoding a novel Src homology 3 (SH3)-containing protein, and named it ‘Hrs binding protein’ (Hbp). Hbp was co-immunoprecipitated with Hrs, and its intracellular localization was similar to that of Hrs. The association between Hbp and Hrs was mediated through the coiled coil motifs in Hbp and Hrs. Deletion mutants of Hbp lacking either the SH3 domain or the Hrs binding domain showed dominantly negative effects on the intracellular degradation of a growth factor and its receptor, but not on the internalization of growth factor/receptor complexes. Conclusions Hbp is thought to be closely associated with Hrs on early endosomes. Hbp, together with Hrs may play a regulatory role in the vesicular transport of growth factor/receptor complexes through early endosomes, for their degradation.

We have reported that infusion of recombinant human hepatocyte growth factor (rhHGF) stimulates liver regeneration after hepatectomy in cirrhotic rats and increases the level of serum lipids and secretion of very-low density lipoprotein (VLDL). Studies were now performed to determine whether rhHGF directly influences lipid synthesis and its secretion in cultured rat hepatocytes. Isolated cells were cultured in the presence or absence of rhHGF (20 ng/mL) for 2 days. During the first 12 hours, rhHGF transiently inhibited the release of lipids (triacylglycerol, total cholesterol, and phospholipids), but stimulated their releases with maximal levels achieved at 36 hours. [3H]-glycerol experiment with the transcriptional and translational inhibitors revealed that rhHGF stimulated de novo synthesis of lipids by affecting activities of lipid metabolic gene. [35S]-Methionine experiment also revealed de novo synthesis of apolipoprotein B by rhHGF. Furthermore, lipid analysis of lipoprotein fractions in the conditioned medium showed that rhHGF enhanced levels of triacylglycerol, total cholesterol, and phospholipids by 50% to 200% in both VLDL and low-density lipoproteins (LDL)/high-density lipoprotein (HDL). Genistein, a tyrosine kinase inhibitor, blocked the secretion of VLDL, as well as synthesis of lipids and apolipoprotein B stimulated by rhHGF. These results indicate that HGF likely stimulates lipid biosynthesis and lipoprotein secretion in hepatocytes through its tyrosine kinase-associated receptor, c-met, and accelerates the progress of cell maturation in liver regeneration.

Hepatocyte growth factor (HGF) activator is a serine protease responsible for proteolytic activation of HGF in response to tissue injury and thus plays an important role in the regulation of biological functions of HGF in regenerating tissue. We previously purified an inhibitor of HGF activator (HGF activator inhibitor type 1, HAI-1) from the conditioned medium of a human stomach carcinoma cell line MKN45 and cloned its cDNA. HAI-1 is a novel member of the Kunitz family of serine protease inhibitors. In the present study, we purified a second type of HGF activator inhibitor (HAI-2) from the conditioned medium of MKN45 cells and molecularly cloned its cDNA. The cDNA sequence revealed that HAI-2 is derived from a precursor protein of 252 amino acids and contains two Kunitz domains, indicating that HAI-2 is also a member of the Kunitz family of serine protease inhibitors. The primary translation product of HAI-2 has a hydrophobic sequence in the COOH-terminal region, suggesting that, like HAI-1, HAI-2 is produced in a membrane-associated form and secreted in a proteolytically truncated form. Because HAI-2 and HAI-1 are potent inhibitors specific for HGF activator, they may be involved in regulation of proteolytic activation of HGF in injured tissues.

Hepatocyte growth factor (HGF) activator is a serine protease that is produced and secreted by the liver and circulates in the blood as an inactive zymogen. In response to tissue injury, the HGF activator zymogen is converted to the active form by limited proteolysis. The activated HGF activator converts an inactive single chain precursor of HGF to a biologically active heterodimer in injured tissue. The activated HGF may be involved in the regeneration of the injured tissue. In this study, we purified an inhibitor of HGF activator from the conditioned medium of a human MKN45 stomach carcinoma cell line and molecularly cloned its cDNA. The sequence of the cDNA revealed that the inhibitor has two well defined Kunitz domains, suggesting that the inhibitor is a member of the Kunitz family of serine protease inhibitors. The sequence also showed that the primary translation product of the inhibitor has a hydrophobic sequence at the COOH-terminal region. Inhibitory activity toward HGF activator was detected in the membrane fraction as well as in the conditioned medium of MKN45 cells. These results suggest that the inhibitor may be produced as a membrane-associated form and secreted by the producing cells as a proteolytically truncated form.

Hepatocyte growth factor activator (HGFA) is a serine protease that is responsible for localized activation of hepatocyte growth factor (HGF) in injured tissue. The activated HGF may be involved in regeneration of the injured tissue. HGFA is produced and secreted by the liver and circulates in the plasma as an inactive zymogen. In response to tissue injury, the HGFA zymogen is converted to the active form by limited proteolysis. In this study, we isolated a rat HGFA complementary DNA (cDNA) clone and analyzed the production of HGFA messenger RNA (mRNA) in response to tissue injury using this cDNA clone as a probe. The nucleotide sequence of the cDNA revealed that the amino acid sequences of rat and human HGFA showed a high degree of conservation in the regions of the characteristic domain structures, suggesting that rat and human HGFA are activated by a similar mechanism and have similar enzymatic activities in vivo. Tissue distribution analysis showed that the liver was the major site of rat HGFA mRNA synthesis. Moreover, the cells producing HGFA mRNA were identified as parenchymal liver cells. The level of HGFA mRNA increased in the liver after hepatotoxin or nephrotoxin treatment. This increase was also observed during acute inflammation induced by turpentine. These results suggest that the increase in production of HGFA mRNA in response to tissue injury is the result of an inflammatory response, and that HGFA is an acute phase protein.

Hrs is a 115-kDa double zinc finger protein that is rapidly tyrosine phosphorylated in growth factor-stimulated cells. However, its function remains unknown. Here we show that Hrs is localized to early endosomes. Intracellular localization of endogenous Hrs and exogenously expressed Hrs tagged with the hemagglutinin epitope was examined by immunofluorescence staining using anti-Hrs and anti-hemagglutinin epitope antibodies, respectively. Hrs was detected in vesicular structures and was colocalized with the transferrin receptor, a marker for early endosomes, but only partially with CD63, a marker for late endosomes. A zinc finger domain deletion mutant of Hrs was also colocalized with the transferrin receptor, suggesting that the zinc finger domain is not required for its correct localization. Immunoelectron microscopy showed that Hrs was localized to the cytoplasmic surface of these structures. By subcellular fractionation, Hrs was recovered both in the cytoplasmic and membrane fractions. The membrane-associated Hrs was extracted from the membrane by alkali treatment, suggesting that it is peripherally associated with early endosomes. These results, together with our finding that Hrs is homologous to Vps27p, a protein essential for protein traffic through a prevacuolar compartment in yeast, suggest that Hrs is involved in vesicular transport through early endosomes.

The function of hepatocyte growth factor/scatter factor (HGF/SF) is to increase proliferation as well as to stimulate motility and disperse cell colonies of epithelial cells. In this study, we examined the motogenic and mitogenic responses of two human gastric carcinoma cell types, MKN7 and MKN74. Cell motility of both cell lines was markedly stimulated by HGF/SF. In contrast, HGF/SF stimulated cell growth of MKN74 cells, but did not stimulate growth of MKN7 cells. To address the cause of the difference in response of these cells, which may reflect some differences in signaling pathways downstream from the HGF/SF receptor, c-Met, we investigated the induction of the proto-oncogene c-fos. The level of c-fos mRNA increased and reached a maximum approximately 40 min after HGF/SF stimulation in MKN74 cells, and thereafter its level rapidly decreased. In contrast, the level of c-fos expression was very low irrespective of the stimulation in MKN7 cells. c-Fos protein was transiently induced only in MKN74 cells l h after treatment with HGF/SF, and its levels subsequently decreased. We subsequently examined the activation of mitogen-activated-protein kinase, which is a major mediator in the signaling pathway leading to the stimulation of c-fos transcription, after HGF/SF treatment in both cell lines. Mitogen-activated-protein kinase was markedly activated by this treatment in MKN74 cells, but was only slightly activated in MKN7 cells. These results suggest that although mitogen-activated-protein kinase activation and c-fos induction play an essential role in the signaling pathway leading to cell growth, they are not required for the motility response induced by HGF/SF.

Hepatocyte growth factor (HGF) is secreted as an inactive single-chain precursor from the producing cells, and normally remains in this form associated with the extracellular matrix. In response to tissue injury, the single-chain precursor is converted to a biologically active heterodimer by a serine protease, the activity of which is induced in the injured tissue. We have previously identified HGF activator, a serum serine protease that activates single-chain HGF. The sequence of HGF activator cDNA revealed that the HGF activator is homologous to blood-coagulation factor XIIa. In this study, we found that coagulation factor XIIa has an ability to activate single-chain HGF. Factor XIIa exhibited a significant level of HGF-converting activity in the presence of dextran sulfate, although the specific activity of factor XIIa was slightly lower than that of the HGF activator. Since factor XIIa is activated during the initiation of contact activation induced by tissue injury, factor XIIa may function as an HGF-converting enzyme together with HGF activator in the injured tissue. C1-inhibitor, antithrombin III and alpha 2-antiplasmin, that regulate the blood-clotting activity of factor XIIa, were also effective against the HGF-converting activity of factor XIIa. Furthermore, factor XIIa was not active in the HGF-converting activity in serum. Thus, the HGF-converting activity of factor XIIa may be regulated by these serum inhibitors.

p120 was originally identified as a substrate of pp60src and several receptor tyrosine kinases, but its function is not known. Recent studies revealed that this protein shows homology to a group of proteins, beta-catenin/Armadillo and plakoglobin (gamma-catenin), which are associated with the cell adhesion molecules cadherins. In this study, we examined whether p120 is associated with E-cadherin using the human carcinoma cell line HT29, as well as other cell lines, which express both of these proteins. When proteins that copurified with E-cadherin were analyzed, not only alpha-catenin, beta-catenin, and plakoglobin but also p120 were detected. Conversely, immunoprecipitates of p120 contained E-cadherin and all the catenins, although a large subpopulation of p120 was not associated with E-cadherin. Analysis of these immunoprecipitates suggests that 20% or less of the extractable E-cadherin is associated with p120. When p120 immunoprecipitation was performed with cell lysates depleted of E-cadherin, beta-catenin was no longer coprecipitated, and the amount of plakoglobin copurified was greatly reduced. This finding suggests that there are various forms of p120 complexes, including p120/E-cadherin/beta-catenin and p120/E-cadherin/plakoglobin complexes; this association profile contrasts with the mutually exclusive association of beta-catenin and plakoglobin with cadherins. When the COOH-terminal catenin binding site was truncated from E-cadherin, not only beta-catenin but also p120 did not coprecipitate with this mutated E-cadherin. Immunocytological studies showed that p120 colocalized with E-cadherin at cell-cell contact sites, even after non-ionic detergent extraction. Treatment of cells with hepatocyte growth factor/scatter factor altered the level of tyrosine phosphorylation of p120 as well as of beta-catenin and plakoglobin. These results suggest that p120 associates with E-cadherin at its COOH-terminal region, but the mechanism for this association differs from that for the association of beta-catenin and plakoglobin with E-cadherin, and thus, that p120, whose function could be modulated by growth factors, may play a unique role in regulation of the cadherin-catenin adhesion system.

HEPATOCYTE growth factor/scatter factor (HGF/SF) functions as a mitogen, motogen and morphogen for a variety of cultured cells1–7. The genes for HGF/SF and its receptor (the c-met protooncogene product8) are expressed in many tissues during the embryonic periods and in the adult9–14. HGF/SF is thought to mediate a signal exchange between the mesenchyme and epithelia during mouse development15. To examine the physiological role of HGF/SF, we generated mutant mice with a targeted disruption of the HGF/SF gene. Here we report that homozygous mutant embryos have severely impaired placentas with markedly reduced numbers of labyrinthine trophoblast cells, and die before birth. The growth of trophoblast cells was stimulated by HGF/SF in vitro, and the HGF/SF activity was released by allantois in primary culture of normal but not mutant embryos. These findings suggest that HGF/SF is an essential mediator of allantoic mesenchyme-trophoblastic epithelia interaction required for placental organogenesis.

Ligand-induced tyrosine kinase activation of the scatter factor/hepatocyte growth factor receptor (c-Met) is thought to be essential for the biological responses of target cells. To assess the regulatory role of the major tyrosine autophosphorylation site (tyrosine 1233) of the mouse c-Met receptor in the tyrosine kinase activation of the receptor, we constructed a mutant receptor in which the tyrosine residue was replaced with phenylalanine. When the cells expressing the mutant receptor were incubated with the ligand, no biological responses were observed, and the level of tyrosine phosphorylation of the receptor was very low compared with that of the wild-type receptor. The in vitro kinase activity of the mutant receptor toward an exogenous substrate and the receptor itself was also low. Furthermore, tyrosine phosphorylation of the cellular proteins by ligand stimulation was not detected in intact cells expressing the mutant receptor. The low level of kinase activity and the lack of biological activity of the mutant receptor indicate that the major autophosphorylation site positively regulates the tyrosine kinase of the c-Met receptor and phosphorylation of cellular substrates in the scatter factor/hepatocyte growth factor signaling pathway.

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes and renal tubular epithelial cells and may play an important role in regeneration following hepatic and renal injury. HGF is first synthesized as a single chain precursor which is then converted to a heterodimeric form by proteolytic processing. This proteolytic conversion is required for HGF to function as a mitogen. In this study, we examined whether the proteolytic activation of HGF occurred in response to hepatic and renal injury. HGF remained as an inactive single chain form in the liver, kidney, lung, and spleen of normal rats. The production of HGF markedly increased in the liver after hepatotoxin treatment and in the kidney after nephrotoxin treatment. A significant portion of the increased HGF was converted to the active heterodimeric form, whereas in other tissues, conversion did not occur although the production of HGF increased in some of them. Furthermore, an enzymatic activity that converts the single chain form of HGF to the active heterodimeric form was detected in the injured liver but not in the normal liver. These results indicate that HGF is proteolytically activated in response to tissue injury, and this activation is mediated by an enzymatic activity which is induced exclusively in the injured tissues. Thus, the proteolytic activation system functions in vivo as a mechanism for localizing HGF activities to injured tissues.

Regulatory T cells (Tregs) are a major obstacle to the establishment of effective cancer immunotherapy. As mediators of immune tolerance, they are a critical target for pre-conditioning for adoptive immunotherapy. Here, we show that pre-treatment with cyclophosphamide or agonistic anti-OX40 mAb augments the anti-tumor immune effect of adoptive CD8+ T cell therapy in a clinically relevant wild-type model, as opposed to a TCR-transgenic mouse model. Tumor antigen-stimulated CD8+ T cells (7x106), including a small number (2.17x105) of tumor antigen-specific effector CD8+ T cells, were transferred into tumor-bearing mice. A response was detected in the adoptively transferred antigen-specific CD8+ T cells, but was insufficient for the eradication of the established tumor. However, pre-treatment with cyclophosphamide to reduce Tregs was shown to enhance the anti-tumor immune effect of the adoptively transferred CD8+ T cells. Moreover, we demonstrated for the first time that pre-treatment with OX40 costimulation, with the aim of nullifying Treg-mediated suppression, maintained the tumor-specific immune response of adoptively transferred CD8+ T cells, resulting in the eradication of the established tumor. These findings suggest that pre-conditioning with the aim of depleting Tregs is a useful strategy for adoptive cancer immunotherapy.