Hepatic arterial infusion chemotherapy with cisplatin before radical local treatment of early hepatocellular carcinoma (JIS score 0/1) improves survival.

BACKGROUND: It has not yet been determined whether hepatic arterial infusion (HAI) chemotherapy improves survival in patients with early hepatocellular carcinoma (HCC). We evaluated the effectiveness of HAI with high-concentration cisplatin (DDP-H) for the treatment of HCC by comparing outcomes between patients who received HAI with DDP-H before radical local treatment of early-stage HCC [Japan Integrated Staging (JIS) score 0/1] and patients who did not receive HAI chemotherapy. PATIENTS AND METHODS: Survival was analyzed in 114 patients with early-stage HCC who underwent radical local treatment. The patients were divided into two groups: a HAI group (n = 79) who received DDP-H infusion into the whole liver via the proper hepatic artery, and a non-HAI group (n = 35) who did not receive HAI chemotherapy. RESULTS: The cumulative survival rates at 1, 3, and 5 years were 77.4%, 69.2%, and 55.3% in the non-HAI group and 97.4%, 87.0%, and 84.4% in the HAI group, respectively. Survival time prolonged significantly in the HAI group compared with the non-HAI group (log-rank test: P = 0.023; generalized Wilcoxon test: P = 0.012) Multivariate analysis using the Cox proportional hazards model identified HAI with DDP-H as the most important factor affecting survival. CONCLUSIONS: Whole-liver HAI with DDP-H before radical local treatment can improve the prognosis of patients with early-stage HCC.

A novel gene, Translin, encodes a recombination hotspot binding protein associated with chromosomal translocations.

We have identified a novel gene, Translin, encoding a protein which specifically binds to consensus sequences at breakpoint junctions of chromosomal translocations in many cases of lymphoid malignancies. The encoded protein, Translin, is a previously undescribed type with no significant similarity to known proteins. In the native form, Translin polypeptides form a multimeric structure which is responsible for its DNA binding activity. Nuclear localization of Translin is limited to lymphoid cell lines, raising the intriguing possibility that nuclear transport of Translin is regulated in a physiologically significant way such that active nuclear transport is associated with the lymphoid specific process known as Ig/TCR gene rearrangement.

U.v.-induced nuclear accumulation of p53 is evoked through DNA damage of actively transcribed genes independent of the cell cycle.

Induction of p53 in u.v.-irradiated primary human fibroblasts was monitored by immunostaining and Western blotting. Minimum u.v. doses required for induction of nuclear accumulation of p53 (minimum response dose: MRD) were estimated in various cells with different DNA repair capacities. The MRD in repair deficient xeroderma pigmentosum (XP) group A cells is eightfold lower than in normal cells, indicating that nuclear accumulation of p53 is related to DNA repair capacity. Cells from patients with another u.v.-sensitive disorder, Cockayne syndrome (CS), which have normal repair capacity for the overall genome but have a specific defect in preferential repair of lesions in active genes, have the same low MRD as of XP-A cells. Furthermore, the MRD in XP-C cells, which have normal preferential repair but have defects in overall genome repair, is as high as that of normal cells. DNA damage induced by X-ray is repaired at similar rates in normal, XP and CS cells. In contrast to u.v.-irradiation, the minimum dose of X-rays that induces nuclear accumulation of p53 is the same in these cells. Inhibition of transcription with alpha-amanitin evokes nuclear accumulation of p53 both in normal cells and in XP cells. These results strongly suggest that u.v.-induced nuclear accumulation of p53 is evoked through DNA damage of actively transcribed genes. Nuclear accumulation of p53 is observed in any phase of the cell cycle at both low and high u.v. doses.

Adrenomedullin inhibits angiotensin II-induced expression of tissue factor and plasminogen activator inhibitor-1 in cultured rat aortic endothelial cells.

Adrenomedullin (AM) is a potent vasodilating peptide having a variety of pharmacological properties mainly in respect to vascular pathophysiology. We have previously demonstrated that angiotensin II (Ang II) or natriuretic peptides have influence on the expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) in vascular endothelial cells. The aim of this study was to elucidate the effects of AM on TF and PAI-1 mRNA and protein expression in endothelial cells. As a result, AM inhibited Ang II-induced TF and PAI-1 mRNA expression in a dose- and time-dependent manner. Because the expression of TF and PAI-1 mRNA induced by Ang II was attenuated by the increase of intracellular concentrations of cAMP by forskolin and 8-bromo-cAMP and because AM increased the intracellular level of cAMP in rat aortic endothelial cells, it was indicated that the inhibitory effect of AM on the expressions of TF and PAI-1 was mainly mediated by the cAMP-dependent signal transduction. Furthermore, the inhibitory effect of AM on TF and PAI-1 expression was partly attenuated by an NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester. In conclusion, AM is shown to contribute to the regulation of blood coagulation and fibrinolysis by vascular endothelial cells mainly via the cAMP pathway.

Primary culture of chicken hepatocytes in serum-free medium (pH 7.8) secreted albumin and transferrin for a long period in free gas exchange with atmosphere.

To study liver functions of chicken, we examined the primary culture of chicken hepatocytes, and found an easy method of long-term culture with free atmosphere exchange. Chicken hepatocytes were obtained by collagenase perfusion and cultured at 37 degrees C as a monolayer without substratum in serum-free L-15 medium (pH 7.8) with free atmosphere exchange. The amounts of albumin and transferrin in medium were assayed by ELISA. The culture of chicken hepatocytes was maintained in the serum-free L15-medium )pH 7.) and 37 degrees C with free atmosphere exchange for 20 days. The amount of albumin secreted in the medium decreased to low levels early in culture; however, this was followed by marked increase from day 9 to day 17 of culture. The amount of transferrin was constant until day 6, then it too increased with further culture. We reported an easy method for the simple monolayer culture of chicken hepatocytes in serum-free L12 medium (pH 7.8) with free atmosphere exchange over an extended period. Expression of liver-specific functions, viz. albumin and transferrin synthesis, was observed after 1 week of culture.

Preparation of human monoclonal antibodies against a cytomegalovirus glycoprotein complex of 130 and 55 kDa.

Nine human monoclonal antibodies (MAbs) with neutralization activity against cytomegalovirus (CMV) were obtained by screening human MAbs using a CMV glycoprotein complex of 130 and 55 kDa (gp130/55). The gp130/55 antigen was purified by immunoaffinity chromatography and the purified antigen used to detect anti-gp130/55 MAbs in an enzyme-linked immunosorbent assay. Relatively few of the human anti-CMV MAbs were directed against gp130/55 but all showed high neutralization activities to a variety of clinical isolates with titers (ED50 values) ranging from 0.15 to 7.9 micrograms/ml. Six of the nine anti-gp130/55 MAbs required complement for virus neutralization. Such human MAbs may prove to be useful for passive immunotherapy against CMV infection.

A microneutralization enzyme immunoassay for antibody to human cytomegalovirus.

We have developed a relatively rapid, sensitive and quantitative microneutralization assay for antibody to human cytomegalovirus (HCMV). Cell monolayers in 96-well microtiter plates inoculated with pre-incubated virus-antibody mixtures were fixed after 3 days. Infectious foci were stained with peroxidase-labeled human monoclonal antibody to a 64 kDa immediate early antigen of HCMV, and the plates were read at OD450. The 50% neutralization titer of the antibody was calculated. A study with 20 human sera and a human monoclonal antibody which neutralizes virus showed that this microneutralization enzyme immunoassay is more sensitive than, and as quantitative as, the conventional plaque reduction assay for antibody to HCMV. The neutralizing antibody titers of each sample measured by these two methods showed good correlation (n = 19, r = 0.884). Thus, this new assay is a useful and valid alternative to the conventional method for mass screening of sera and hybridoma fluids, and considerably more rapid.

Expression of adrenomedullin and proadrenomedullin N-terminal 20 peptide in PC12 cells after exposure to nerve growth factor.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are multi-functional peptides derived from the same precursor, proadrenomedullin. We have studied the regulatory mechanism of expression of these peptides during neuronal differentiation of rat pheochromocytoma PC12 cells by nerve growth factor (NGF). The cellular levels of the peptides increased slightly, and then progressively decreased below the control by NGF. Immunoreactive (ir)-AM in the medium was transiently increased by NGF. Cytochemical staining showed that ir-AM and ir-PAMP were abundantly present in cytoplasm in the undifferentiated cells, and were decreased during culture with NGF. There was no preferential localization of ir-AM or ir-PAMP in neurites in comparison with in cytoplasm in the differentiated cells. Northern blot analysis showed that mRNA encoding these peptides, as detected as a band of 1.6 kb, increased more than three-fold at 1 h after the addition of NGF and then progressively decreased to one fifth of the control during 72 h. Degradation rate of the mRNA was slowed by NGF even when mRNA level is decreased after 72 h of NGF treatment. The transcription rate of their gene increased transiently and then decreased by the long-term treatment with NGF. These results demonstrate that expression of AM and PAMP is regulated by NGF along with time-dependent differentiation: AM gene transcription is transiently activated by NGF, whereas it was suppressed during neuronal differentiation of the cells.

Genetic analysis in Japanese kindreds of congenital type I antithrombin deficiency causing thrombosis.

We have identified two novel minor deletions (case 1; -TA or -AT at nucleotide 9831-3 in exon 5 and case 2; -A at nucleotide 7640-1 in exon 4), one novel nonsense mutation (case 3; TAT to TAA at nucleotide 7491 in exon 4), and one recurrent nonsense mutation (case 4; CGA to TGA at nucleotide 5381 in exon 3A) in Japanese kindreds with congenital type I antithrombin deficiency. The deletion detected in case 1 represented a symmetric element (CTCTGTCTC) and possessed a direct repeat (CTCTATGTCTC). The deletion in case 2 was recognized in a consensus sequence (TGAAT) and possessed a direct repeat (GATGAA). The nonsense mutation in case 3 formed a palindrome (CCGTTAACGG) and that in case 4 was caused by a CpG dinucleotide mutation. These results confirm that the mutations of congenital type I antithrombin deficiency are not random events but are influenced strongly by DNA sequences.

Natriuretic peptides regulate the expression of tissue factor and PAI-1 in endothelial cells.

In the present study, we demonstrate that brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) interact with angiotensin II (Ang II) in regulative blood coagulation and fibrinolysis by suppressing the expressions of both tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) induced by Ang II. The expressions of TF and PAI-1 mRNA were analyzed by northern blotting methods, and the activities of TF on the surface of rat aortic endothelial cells (RAECs) and PAI-1 in the culture media were respectively measured by chromogenic assay. Both BNP and CNP suppressed the expressions of TF and PAI-1 mRNA induced by Ang II in a time- and concentration-dependent manner via cGMP cascade, which suppressions were accompanied by respective decrease in activities of TF and PAI-1. However, neither the expression of tissue factor pathway inhibitor (TFPI) nor tissue-type plasminogen activator (TPA) mRNA was affected by the treatment of BNP and CNP.

Angiotensin II increases plasminogen activator inhibitor-1 and tissue factor mRNA expression without changing that of tissue type plasminogen activator or tissue factor pathway inhibitor in cultured rat aortic endothelial cells.

Angiotensin converting enzyme inhibitors (ACE-I) have been reported to prevent the recurrence of cardiovascular events. The mechanism of this decrease, however, can not be completely explained by anti-hypertensive and anti-hypertrophic effects of ACE-I. To investigate the mechanism of this decrease, we studied the regulation of plasminogen activator inhibitor-1 (PAI-1), tissue type plasminogen activator (TPA), tissue factor (TF), and tissue factor pathway inhibitor (TFPI) by angiotensin II (Ang II) in cultured rat aortic endothelial cells. Ang II increased PAI-1 and TF mRNA expression without affecting that of TPA or TFPI. These inductions were accompanied by increases in PAI-1 and TF activities and were inhibited by a type I Ang II receptor antagonist. The results suggest that Ang II decreases the antithrombotic properties of endothelial cells which increases the chance of thrombosis. Thus, inhibition of the renin-angiotensin system may be beneficial to prevent thrombus formation in treatment of ischemic heart disease.

Atrial natriuretic peptide inhibits the expression of tissue factor and plasminogen activator inhibitor 1 induced by angiotensin II in cultured rat aortic endothelial cells.

The pharmacological characteristics of atrial natriuretic peptide (ANP), such as natriuresis, vasodilation, or suppression of smooth muscle cell proliferation, are well investigated. However, this is the first study to report its role on blood coagulation and fibrinolysis mediated by vascular endothelial cells. In this study, the effects of ANP on the enhanced expression of tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) by angiotensin II (Ang II) in cultured rat aortic endothelial cells (RAECs) were examined. The expressions of TF and PAI-1 mRNA were detected by northern blotting methods. The activities of TF on the surface of RAECs and PAI-1 in the culture media were measured by chromogenic assay. ANP suppressed mRNA expressions of TF and PAI-1 induced by Ang II in a concentration-dependent manner. This suppression was accompanied by the decreased activities of TF and PAI-1.

The effects of angiotensin metabolites on the regulation of coagulation and fibrinolysis in cultured rat aortic endothelial cells.

Not only angiotensin II (Ang II) but also other angiotensin metabolites such as angiotensin I (Ang I), angiotensin III (Ang III), angiotensin IV, or angiotensin 1-7 have recently been reported to have various activities. Few data, however, are available on the regulation of thrombus formation. In this study, we investigated the effects of angiotensin metabolites on the mRNA expression of tissue factor (TF), tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor-1 (PAI-1), and tissue type plasminogen activator (TPA) in cultured rat aortic endothelial cells. None of the used angiotensin metabolites altered TFPI or TPA mRNA expression levels. Ang I, Ang II, and Ang III made TF and PAI-1 mRNA inductions which were inhibited by an selective antagonist of angiotensin II type 1 receptors. These metabolites made TF predominant to TFPI or PAI-1 to TPA, and could render endothelial cells thrombogenic.

Adrenomedullin inhibits spontaneous and bradykinin-induced but not oxytocin- or prostaglandin F(2alpha)-induced periodic contraction of rat uterus.

In isolated rat uterine strips, adrenomedullin (AM) inhibited the spontaneous periodic contraction in a concentration-dependent manner (IC(50)=22.3+/-0.7 nM). The inhibitory effect of AM was prevented by either AM(22-52), a putative antagonist for AM receptors, or calcitonin gene-related peptide (CGRP)(8-37), a putative antagonist for CGRP receptors. AM also attenuated bradykinin (BK)-induced periodic uterine contraction, which was blocked by AM(22-52) or CGRP(8-37), whereas AM had no effect on the periodic contraction caused by oxytocin or prostaglandin F(2alpha) (PGF(2alpha)). RT-PCR analysis showed that mRNAs for calcitonin receptor-like receptor (CRLR), receptor-activity-modifying protein (RAMP)1, RAMP2 and RAMP3 were expressed in the rat uterus. These results demonstrate that AM selectively inhibits spontaneous and BK-induced periodic contraction via activating receptors for AM and CGRP.

Production of interleukin (IL)-6 and IL-8 by a choriocarcinoma cell line, BeWo.

To clarify the biological and pathological features of choriocarcinoma, we evaluated the in vitro production of cytokines by a choriocarcinoma cell line, BeWo. We measured the concentration of interleukin (IL)-6 and IL-8 in the culture media of BeWo cells after stimulation with various modulatory agents of cytokine expression by enzyme-linked immunosorbent assays. Northern blot analysis was used to examine the expression of IL-6 and IL-8 mRNA in these cells. A weak expression of IL-6 and IL-8 was detected in unstimulated BeWo cells by both methods. IL-6 transcription and secretion were dose-dependently enhanced by stimulation with IL-1beta, tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1 and 12-O-tetradecanoyl phorbol-13-acetate (TPA). Forskolin, lipopolysaccharide and interferon-gamma had no effect on these cytokines production. The TNF-alpha-induced secretion of IL-6 was inhibited by dexamethasone. The TPA-induced production of IL-6 was inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and sphyngosine, suggesting the involvement of a protein kinase C-dependent pathway. Levels of IL-8 mRNA and protein were also dose-dependently increased by stimulation with IL-1beta, TNF-alpha and TPA. In contrast to IL-6, the expression of IL-8 was not affected by TGF-beta1. It is suggested that, in addition to the production of steroidal and non-steroidal hormones, these cytokines may serve as part of a cytokine network that modulates the proliferation and angiogenesis of choriocarcinomas.

Nuclear accumulation of p53 protein and apoptosis induced by various anticancer agents, u.v.-irradiation and heat shock in primary normal human skin fibroblasts.

Relationship between nuclear accumulation of p53 protein and apoptosis induced by both DNA damaging agents (ultraviolet-irradiation and anticancer agents) and heat shock in normal human skin fibroblasts was investigated. Nuclear accumulation of p53 protein and apoptosis were induced dose-dependently by u.v. -irradiation and anticancer agents. Regarding heat shock treatment, intensive nuclear accumulation of p53 protein was induced by treatment at 43 degrees C for 45 min, but, apoptotic cells were never detected. These results indicated that accumulation of p53 protein and apoptosis induced by DNA damaging agents were considered to have a close relationship and the functions of induced p53 protein are different depending on the stimulating factor.

Identification of a specific protein factor defective in group A xeroderma pigmentosum cells.

A protein factor which corrects the defect in xeroderma pigmentosum cells belonging to complementation group A (XP-A cells) was detected in a cell extract prepared from calf thymus. The activity of this factor was measured as the amount of unscheduled DNA synthesis (UDS) reappearing in UV-irradiated XP-A cells after microinjection of the extract. The native molecular mass of this factor was estimated to be 80 kDa by gel-filtration and 25 kDa by glycerol gradient centrifugation. The activity was, however, recovered at a position corresponding to 43 kDa after renaturation on an SDS-PAGE gel. The isoelectric point was determined to be approximately 7.5 by measuring the activity after renaturation on an IEF gel. These values were obtained with a partially purified sample. A spot corresponding to these values was detected on two-dimensional gel electrophoresis with a highly purified sample recovered from an SDS-PAGE gel. The purified protein stimulated UDS specifically in the XP-A cells and endowed the cells with a normal level of UV-resistance. The XP-A cells injected with the factor also showed a normal level of UDS after treatment with either 4HAQO or psoralen plus UV-A. This factor (XP-A complementing factor; XP-ACF) may be involved in the repair of DNA damage induced by various agents.

Oxidative phosphorylation in brown adipose tissue mitochondria from rats kept under normal environmental conditions.

Based on criteria such as the ADP/O ratio and respiratory control by ADP, the energy-coupling efficiency of brown adipose tissue mitochondria isolated from rats kept under normal environmental conditions for a long time decreased remarkably. The presence of bovine serum albumin, GTP, or ATP plus carnitine in the reaction medium markedly increased the efficiency of oxidative phosphorylation of brown adipose tissue mitochondria. Pre-treatment of brown adipose tissue mitochondria with 2% bovine serum albumin, GTP, or ATP plus carnitine caused a decrease in the amount of free fatty acids bound to the mitochondria from 13.1 to 7.0, 9.0, or 8.2 mug per mg protein, respectively. Removal of the free fatty acids by means of these pre-treatments resulted in restoration of efficient oxidative phosphorylation; there was a correlation between the amount of free fatty acids removed and the degree of recovery in the respiratory control ratio. The elimination of only a fraction of the free fatty acids, as little as 4 mug per mg protein, was sufficient to ensure respiratory control by ADP. It appears that the free fatty acids which lie mainly outside the inner mitochondrial membrane are responsible for the decrease in the efficiency of oxidative phosphorylation in brown adipose tissue mitochondria isolated from rats kept under normal environmental conditions.

Intracellular redox state and control of gluconeogenesis in perfused chicken liver.

The role of the cellular redox state in the control of gluconeogenesis was studied in hemoglobin-free perfused chicken liver, by fluorimetric measurement of the redox states of intracellular pyridine nucleotides. The aminotransferase inhibitor, aminooxyacetate, completely inhibited gluconeogenesis from lactate in the perfused rat liver and to a small extent in the perfused chicken liver. In chicken liver, the highest rate of glucose production was seen with lactate, followed by fructose, pyruvate, and glycerol. When compared at 5 mM, the rate of glucose production from pyruvate was only 10% of that from lactate. Glucose production from a pyruvate/lactate mixture decreased with increasing proportions of pyruvate, together with redox changes of pyridine nucleotides to a more oxidized state. Increased reduction of pyridine nucleotides upon infusion of ethanol was associated with an increased glucose production from pyruvate, and the increase was abolished during octanoate infusion. This abolishment was accompanied by an increase in the acetoacetate to beta-hydroxybutyrate ratio with an oxidation of pyridine nucleotides. The octanoate-inhibited gluconeogenesis occurred at the higher lactate concentration (10 mM) with a transient oxidation of pyridine nucleotides. No significant inhibition was observed at 1 mM lactate, although an instant reduction of pyridine nucleotides was taking place. The rate of beta-hydroxybutyrate generation during octanoate infusion was 2.2 times higher at 1 mM than at 10 mM lactate. The inhibitory effect of octanoate on glyconeogenesis was completely relieved by the addition of NH4Cl. The results demonstrate that the regeneration of NADH in the cytosol is limited in chicken liver, and that gluconeogenesis is regulated, in part, by alteration in the redox states of mitochondria and cytosol.

Stimulation of gluconeogenesis by glucagon and norepinephrine in the perfused chicken liver.

The effects of glucagon and norepinephrine on gluconeogenesis by perfused chicken liver were studied with fluorimetric monitoring of the redox states of the intracellular pyridine nucleotides. Glucagon stimulated glucose production from precursors entering the pathway both above and below the level of triose phosphates without causing a detectable effect on the redox states of pyridine nucleotides. Glucagon stimulation was not abolished by subsequent infusion of octanoate or ethanol. The presence of a pyruvate/lactate mixture plus NH4Cl resulted in a maximum efficacy of glucagon. Glucose production from lactate and fructose was stimulated by norepinephrine. Norepinephrine always caused a change towards increased reduction of pyridine nucleotides with an increase in the beta-hydroxybutyrate/acetoacetate ratio, but displayed no stimulation of glucose and lactate production from pyruvate. As a result of octanoate infusion with lactate, the changes induced by norepinephrine were reversed. The responses to norepinephrine and phenylephrine were decreased markedly in liver perfused with a calcium-free medium and/or with phentolamine. Infusion of calcium into the calcium-deficient liver caused an abrupt elevation of glucose production together with a reduction of pyridine nucleotides, and the original response to norepinephrine was recovered. The results demonstrate that the effects of glucagon and norepinephrine on gluconeogenesis are not identical, and that norepinephrine stimulation is mediated through an alpha-adrenergic and calcium-dependent mechanism in which redox changes of mitochondrial pyridine nucleotides are involved.