Changing incidence of hepatocellular carcinoma in Japan.

A trend in the incidence of hepatocellular carcinoma (HCC) in Japan was studied from the data of the Osaka Cancer Registry (population, 8,512,351 in 1981) for the period of 1963-1983, the Vital Statistics of Japan, Ministry of Health and Welfare, and the Japan Autopsy Registry which contained 594,132 individually filed cases in the 26-year period from 1958 to 1983. Both cancer registry data and autopsy records showed a more than 2-fold increase in HCC incidence, particularly in the last 10 years or so, among males and a less pronounced increase in females. The same trend was borne out by the cancer registries of Nagasaki City and Miyagi Prefecture and the Vital Statistics. When studied with the autopsy data, it was found that the numbers of autopsies for cirrhosis without HCC and autopsies for HCC (with and without cirrhosis) were about the same in 1958-1961 and that currently (1980-1983) the latter is about 2 times the former. As one of the possible causes of increase in HCC incidence other than prolonged survival of patients with cirrhosis, chronic non-A, non-B hepatitis is discussed.

Association of adenocarcinoma of the lung with cigarette smoking by grade of differentiation and subtype.

A hospital-based case-control study was carried out in order to evaluate the risk of adenocarcinoma of the lung associated with cigarette smoking according to grade of differentiation and subtype. The cases studied were 238 patients with adenocarcinomas of the lung (158 males and 80 females) that were surgically resected at the Center for Adult Diseases, Osaka. For each case, 2 controls were chosen at the same hospital from outpatients who had not been diagnosed as having smoking-related diseases, matched by sex, age, and year of first visit. When the male cases with adenocarcinoma were classified according to the grade of differentiation, the odds ratios (ORs) associated with exsmokers and current smokers were: 1.0, 2.1 for well-differentiated; 4.1, 7.7 for moderately differentiated; and 8.5, 7.9 for poorly differentiated adenocarcinoma. The OR associated with current smokers for poorly and moderately differentiated adenocarcinoma combined was significantly higher than that for well differentiated adenocarcinoma. Approximately the same pattern of ORs was observed in females. For poorly and moderately differentiated adenocarcinoma, a significant dose-response relationship was observed in males. Comparison between the ORs for papillary type and tubular type showed no difference.

Expression of polysialic acid and STX, a human polysialyltransferase, is correlated with tumor progression in non-small cell lung cancer.

Polysialic acid (PSA) is a carbohydrate composed of a linear homopolymer of alpha-2-8-linked sialic acid residues and is mainly attached to the neural cell adhesion molecule (NCAM). Because of the large negative charge of PSA, presence of PSA attenuates the adhesive property of NCAM and increases the cellular motility. PSA expression on NCAM is developmentally regulated, and PSA plays important roles in formation and remodeling of the neural system through regulation of the adhesive property of NCAM. Expression of the polysialated form of NCAM has been also demonstrated in some malignant tumors, such as Wilms' tumor and small cell lung cancer. Despite the possible importance as an onco-developmental antigen, however, significance of PSA expression in most malignant tumors has not been revealed. Therefore, PSA expression in non-small cell lung cancer was assessed in the present study. PSA was expressed only in 5 (20.8%) of 24 pathological stage I cases, whereas it was expressed in most stage IV cases (76.8%, 11 of 14 cases). PSA expression was correlated with nodal metastasis and distant metastasis, but not with local extent of the primary tumor. Next, expression of polysialyltransferase genes (PST and STX genes) which controlled formation of PSA, was examined. The PST gene was constantly expressed in both normal lung tissue and tumor tissue of all cases. In contrast, the STX gene was not expressed in normal lung tissue of any case, and STX gene expression in tumor tissue was closely correlated with tumor progression. The STX gene was expressed only in 1 (4.2%) of 24 stage I cases, whereas it was expressed in most stage IV cases (85.7%, 12 of 14 cases). These results suggested that the PSA and STX genes could be new targets of cancer therapy as well as important clinical markers.

Prognostic significance of polysialic acid expression in resected non-small cell lung cancer.

Polysialic acid (PSA) is a carbohydrate attached mainly to the neural cell adhesion molecule. Because PSA is composed of a linear homopolymer of alpha-2-8-linked sialic acid residues and has a large negative charge, the presence of PSA attenuates the adhesive property of neural cell adhesion molecule and increases cellular motility. In an earlier study, we demonstrated that PSA and STX, a polysialyltransferase, were associated with tumor progression in non-small cell lung cancer (NSCLC) (F. Tanaka et al., Cancer Res., 60: 3072-3080, 2000). Therefore, in the present study, to assess the prognostic significance of PSA in resected NSCLC, a total of 236 patients who underwent complete resection for pathological (p)-stage I-IIIa disease were reviewed retrospectively. PSA was expressed in 44 of 236 (18.6%) patients, and the expression was correlated with p-stage disease. For all p-stage patients, 5-year survival rates for those with PSA-positive and PSA-negative tumors were 52.1% and 71.3%, respectively, demonstrating a significantly worse prognosis for the PSA-positive patients (P = 0.012). Analysis for only p-stage I patients also demonstrated a significantly worse prognosis for the PSA-positive patients; 5-year survival rates of the PSA-positive and the PSA-negative patients were 45.1% and 83.5%, respectively, (P < 0.001). In addition, there proved to be no difference in the postoperative survival among p-stage I, II, and IIIa patients when PSA expression was positive. Multivariate analysis confirmed that PSA expression was an independent factor to predict poor prognosis in resected NSCLC. These results suggested that PSA could be an important clinical marker and that preoperative induction and/or postoperative adjuvant therapies should be performed for PSA-positive NSCLC, even if the disease is classified as p-stage I.

Co-expression of Fas and Fas-ligand on the surface of influenza virus-infected cells.

Influenza virus-infected cultured cells undergo apoptosis after an increment of Fas (APO-1/CD95) on the cell surface. By flow cytometry, cell surface Fas-ligand was detected in virus-infected cells with a time course similar to that of Fas. Moreover, Fas and Fas-ligand were co-expressed in those cells. The mode of induction, however, appeared to be distinct for the two proteins. Influenza virus infection induced the externalization of phosphatidylserine on the cell surface at the early stage of apoptosis, an event that has been observed in cells undergoing Fas-mediated apoptosis. In fact, apoptosis of the virus-infected cells was inhibited in the presence of an antagonistic anti-Fas-ligand monoclonal antibody. These results suggest that influenza virus infection causes augmented expression of both Fas and Fas-ligand and apoptosis is induced when the infected cells come into contact with each other.

Mast cell degranulating (MCD) peptide and its optical isomer activate GTP binding protein in rat mast cells.

The MCD peptide in bee venom induces degranulation in mast cells. The internal calcium concentration of mast cells increased and remained high following MCD stimulation. This calcium increase was blocked by pertussis toxin (Ptx) treatment, suggesting that MCD peptide activates Ptx-sensitive G-protein. Even in the absence of external calcium in the incubation medium, the calcium concentration increased by MCD treatment, but soon returned to the original level. D-MCD, the optical isomer of the MCD peptide, also increased the internal calcium concentration through a Ptx-sensitive pathway. We suggest that cationic clusters at one side of the surface are more important in activating the G-protein than the alpha-helix conformation.

A method of record linkage.

In cancer epidemiology, prospective approaches are very important both in testing etiological hypotheses and in evaluating preventive procedures. Prospective studies, however, are very difficult and expensive, because a large number of people and a long period of observation are necessary for a satisfactory study. As a data source for follow-up studies, population-based cancer registry is very useful. The Osaka Cancer Registry has been in operation since December, 1962. Since 1968 the data processing, including the work of collation, has been semicomputerized. In order to identify cancer patients, we use the following six indices: date of birth, first Chinese character of a person's family name, address a: city, ward, town or village, address b: further details. i.e., street, avenue, section, hamlet etc., site, and sex. When we have data on the collation indices for the subjects to be followed up, we can conduct follow-up studies easily and accurately, using a semicomputerized collation method similar to that in the cancer registration system. Because the master file of the Osaka Cancer Registry contains the data of cancer cases reported and all cancer deaths among the residents of Osaka Prefecture, we can follow up the subjects living in Osaka Prefecture and obtain data about vitually all cancer incidences and deaths among them. In this follow-up method by means of record linkage to the cancer registry, some considerations should be taken into account for the following factors; coverage of cancer data in the Osaka Cancer Registry, reliability of the collation method, and address of the subjects to be followed up. As an example of a study with this method, we present the follow-up study of the screenees of a mass screening program for stomach cancer.

Population-based case-control study on cancer screening.

Matched case-control studies have recently been used to evaluate the effectiveness of cancer screening. They enable us to estimate the odds ratios of dying of cancer or of getting invasive cancer. The study compares people with various patterns of screening history with those who were not screened. Criteria for eligible cases, controls, and screening histories that are compared as exposures are discussed. The results from a case-control study for evaluating screening for cervical cancer are shown as an example. Also, a study design of a case-control study for evaluating lung cancer screening in Japan is discussed, along with biases and applications of case-control studies in evaluating cancer screening.

3-Nitropropionic acid produces striatum selective lesions accompanied by iNOS expression.

Systemically administered 3-nitropropionic acid (3-NPA) that inhibits the mitochondrial oxidative phosphorylation induces selective lesions in the striatum. To investigate the nature of these selective lesions, we administered 3-NPA (20 mg/kg, s.c. daily for 2 or 3 days) to Wistar rats and investigated the behavioral disturbance, striatal lesions and their variations after modulating the activity of nitric oxide synthase (NOS). On the second or third day of 3-NPA administration, half the animals manifested behavioral disturbances (paddling, rolling, tremor, abnormal gait, and recumbence). A strong extravasation of immunoglobulin G (IgG) and a decrease in immunoreaction for glial fibrillary acidic protein (GFAP) were detected, and iNOS-like (iNOS-L) immunoreactive small cells appeared in the lateral and central striatum especially around the vessels. A week later, lesions lacking GFAP-immunoreaction were detected in the striatum in survived animals. Pretreatment with N-nitro-L-arginine methyl ester (L-NAME) along with each injection of 3-NPA did not improve the behavioral disturbances nor the survival rate, but attenuated the extravasation of IgG and iNOS-L immunoreaction. Pretreatment with aminoguanidine or FK506 improved the behavioral symptoms and survival rate. Extravasation of IgG and expression of iNOS-L immunoreactivity were attenuated, and the striatal lesion was reduced. Data indicate the involvement of NO in the high vulnerability of the striatum, and that iNOS, one of inflammatory markers, is induced following exposure to 3-NPA.

Mortality among Koreans living in Osaka, Japan, 1973-1982.

The mortality pattern of Koreans living in Osaka, Japan was surveyed by comparing their age-specific and age-adjusted death rates with those among Japanese during 1973-1982. Cancer was the leading cause of death among Korean males, while cerebrovascular disease was most common among Korean females in Osaka. Mortality rates from tuberculosis, cancer, mental disorder, cerebrovascular disease, chronic obstructive pulmonary diseases (COPD), liver cirrhosis, accidents and suicide were significantly higher for Korean males than for Japanese males. COPD, liver cirrhosis and accidents were more frequent for Korean females than for Japanese females. In cancers, liver cancer was most common among Korean males, followed by stomach and lung cancers. Stomach cancer was most frequent among Korean females, followed by uterine and liver cancers. The ratio of cancer mortality rates for Koreans and Japanese was significantly higher than 1.0 for oesophagus, liver and lung among males, and for liver among females. Koreans had considerably higher levels of liver cancer and liver cirrhosis compared with Japanese. Mortality from stomach cancer was significantly lower in both sexes among Koreans in Osaka and the reduction of this disease among Koreans in Japan occurred more rapidly than among Japanese.

Time trends of the prevalences of hepatitis B e antigen positives among hepatitis B virus carriers in Japan.

In order to examine time trends of the prevalences of HBeAg positives among HBV carriers in Japan, we analysed data on HBeAg of HBsAg positive voluntary blood donors (23,560 males, and 8659 females) at the Osaka Red Cross Blood Centre between January 1977 and March 1984. Age-specific prevalences of HBeAg positives decreased year by year for both sexes, especially for those in their teens and twenties. The prevalences of HBeAg positives decreased with age, but at any given age it was lower for the later than for the earlier birth cohorts. Although reasons for the secular declines are unknown, the findings suggest that the prevalence of HBeAg positives among HBV carriers will continue to decrease in Japan. This, together with the immunization programme implemented this year, may lead to a future reduction in the risk of HBV related liver diseases in Japan.

Isolation and characterization of an N-linked oligosaccharide that is significantly increased in sera from patients with non-small cell lung cancer.

The structures of N-linked oligosaccharides present in human sera from 12 healthy volunteers and from 14 patients with non-small cell lung cancer (NSCLC) were analyzed by our recently developed partially automated systematic method. Thirty different structures of oligosaccharides were deduced, and these accounted for 84.1% of the total N-linked oligosaccharides present in human sera. All of the quantified oligosaccharide levels in healthy human sera were within twice the standard deviation. The amount of a triantennary trigalactosylated structure with one outer arm fucosylation (A3G3Fo) was found to be markedly increased in NSCLC patients in comparison to that in healthy volunteers (p < 0.01). No significant positive correlation with other clinical data was found. Serum A3G3Fo levels can thus be a novel marker for the diagnosis of NSCLC.

Peritoneal fluid cytology and prognosis in patients with endometrial carcinoma.

Cytologic examination was performed on peritoneal fluid collected from a total of 235 consecutive patients with endometrial carcinoma who underwent laparotomy as the initial treatment at the Cancer Institute Hospital from 1971-1985. The rate of cases positive for cancer cells in the peritoneal cytologic examination was 18.7% for all stages (44 of 235 cases). In stage I endometrial carcinoma, the 5-year and 10-year cumulative survival rates with positive peritoneal cytologic findings were 91.6 and 90.0%, respectively; those in cases with negative cytologic findings were 90.2 and 90.2%, respectively, with no significant difference. The recurrence rate in the same stage was 12.0% for cytologically positive cases versus 9.5% for negative cases. In stages II and III, no significant difference was noted in the survival rate between nine cases with positive peritoneal cytologic findings without macroscopic peritoneal metastasis and 47 comparable cases with negative cytologic findings. Therefore, in endometrial carcinoma, the presence of malignant cells in peritoneal fluid is not a useful prognostic factor.

Dopamine-denervation enhances the trophic activity in striatum: evaluation by morphological and electrophysiological development in PC12D cells.

To evaluate the possibility that dopamine (DA) denervation enhances the trophic activity in striatum, normal or DA-depleted striatal tissue extract (N- or L-extract, respectively) was obtained, and their trophic effects on PC12D cells were investigated from the viewpoints of differentiation using morphological and electrophysiological analyses. Treatment with N- or L-extract induced neurite outgrowth in a concentration-dependent manner, and induced the enlargement of cell size. These effects were stronger in L-extract than in N-extract. Cation currents were investigated in whole cell patch-clamp mode. Development of cation current started with delayed-rectifier type K+ current (IK) and transient type K+ current (IA), followed by Ca2+ current (ICa) and tetrodotoxin-sensitive Na+ current (INa). INa was expressed more frequently in L-extract treated cells than N-extract treated cells at D7-9. The larger IK amplitude in L-extract treatment at D7-9 seemed to be related to the expression of INa. Development of IA was similar at any stage for both treatments. ICa development started at D3-5 after treatments, and the amplitude and current density were similar in both treatments. ICa was strongly blocked by omega-conotoxin GVIA (3 microM), indicating that N-type channels were mainly expressed after treatments. The data suggests that L-extract has stronger effects to hasten the differentiation of PC12D cells than N-extract by promoting the neurite outgrowth, cell enlargement and expression of voltage-dependent cation channels, especially INa and IK.

Dopa-producing astrocytes generated by adenoviral transduction of human tyrosine hydroxylase gene: in vitro study and transplantation to hemiparkinsonian model rats.

Astrocytes secreting a large amount of 3,4-dihydroxyphenylalanine (dopa) were generated by adenoviral transduction of the human tyrosine hydroxylase (TH) gene. After characterizing in vitro, the effect of transplantation of these astrocytes to the striatum of hemiparkinsonian model rats was investigated. Subconfluent cortical astrocytes were infected by replication-defect adenovirus type 5 carrying the human TH-1 gene or the LacZ reporter gene under the promoter of the glial fibrillary acidic protein (AdexGFAP-HTH-1, AdexGFAP-NL-LacZ). Dopa secretion was not evident at 3 days after the transduction of the HTH-1 gene but it increased from 7 days up to at least 4 months. The secretion was substrate (tyrosine)-dependent, and was enhanced by loading tetrahydrobioputerin (BH4) concentration-dependently. One-third of the hemiparkinsonian model rats, that were transplanted the HTH-1 gene-transduced astrocytes or introduced the direct injection of the viral vector to the striatum, showed a reduction of methamphetamine-induced rotations for at least 6 weeks. Apomorphine-induced rotation was decreased to the 50% level of the control's, but the reduction was obtained equally by the transplantation of HTH-1 gene-transduced or LacZ reporter gene-transduced astrocytes, or by the introduction of HTH-1 or LacZ gene carrying adenovirus. Treatment with FK506 for 3 weeks improved the late-phase apomorphine-induced rotations following the introduction of the HTH-1 gene carrying adenovirus. Histological examination revealed that, in animals that showed a reduction of methamphetamine-rotation, the TH positive astrocytes-like cells were distributed widely in the host striatum for at least 4 weeks. The number of TH positive astrocytes-like cells and their immunoreactivity decreased after 6 weeks when OX-41 positive microglias/macrophages were infiltrated. Data indicate that the adenoviral transduction of the human TH gene to astrocytes and its introduction to the striatum is a promising approach for the treatment of Parkinson's disease. However, the further technical improvements are required to optimize the adenoviral gene delivery, such as the control of viral toxicity and the regulation of the immune response.

Acute 3-nitropropionic acid intoxication induces striatal astrocytic cell death and dysfunction of the blood-brain barrier: involvement of dopamine toxicity.

Mechanisms underlying the selective vulnerability of the lateral striatal area to the toxic effects of 3-nitropropionic acid (3-NPA) were investigated in rats. A single exposure to 3-NPA (20 mg/kg, s.c.) induced no deficits in behavior and histology, but subsequent injection produced motor symptoms, catalepsy, lip smacking, abnormal gait, paddling, rolling, opisthotonos, tremor, recombence, somnolence and so on, in 30% of the animals within a few hours. Diffusion-weighted magnetic resonance imaging of the brains revealed an area of high signal intensity in the bilateral striata. By this stage (within a few hours), striatal astrocytes had become swollen and disintegrated. Extravasation of immunoglobulin G was detected, indicating blood-brain barrier (BBB) dysfunction. Electron microscopy revealed edema and disorganization of structures inside the astrocytic end-feet around the branches of the lateral striatal artery. Neurons were less vulnerable than astrocytes to the 3-NPA injury. Treatment of the rats with D2 receptor agonist prior to exposure to 3-NPA attenuated the behavioral abnormalities and histological damage whereas pretreatment with D2 antagonist exacerbated these changes. The concentrations of extracellular dopamine (DA) and dihydroxyphenyl acetic acid (DOPAC) were both increased in rats exposed to 3-NPA. In vitro imaging of astrocytes revealed a progressive increase in [Ca2+]i after superfusion with 3-NPA, and the 'ceiling' level was maintained even after extensive washing. DA superfusion also increased the astrocytic [Ca2+]i and this increase was reversible. Data indicate that 3-NPA-induced striatal damage was associated with astrocytic cell death and dysfunction of the BBB. Intracellular edema and extreme Ca2+ overload induced by the toxin were further aggravated by an increase in the level of DA activity. These factors acting either singly or in combination may trigger astrocyte destruction.

GTP-binding protein activation underlies LTP induction by mast cell degranulating peptide.

Mast cell degranulating peptide (MCD) induces long-term potentiation (LTP) in the CA1 region of the hippocampus. MCD has been shown to bind to a voltage-dependent A-type potassium channel with high-affinity (less than 1 nM). However, the concentration necessary to induce LTP is more than 500 nM, suggesting that some other functions of MCD are also fundamental to LTP induction. The concentration of MCD required for LTP induction was greatly reduced by preactivating G proteins. This fact suggests that G protein activation by MCD also plays an important role in LTP induction. MCD-binding proteins were purified from rat brain. G proteins were found to exist in a non-denatured state in this affinity-purified fraction. When reconstituted into a planar lipid bilayer membrane, a potassium-selective and voltage-dependent current could be observed. This channel was blocked by MCD at a high concentration equal to the effective concentration for G protein activation. Addition of GTP-gamma-S significantly blocked the reconstituted current. Thus, we identified a pathway for LTP induction by MCD in which high concentrations of MCD activate G protein which in turns leads to blocking of a potassium channel.

K+ channel involvement in induction of synaptic enhancement by mast cell degranulating (MCD) peptide.

A bee venom, mast cell degranulating peptide (MCD), which induces long-term potentiation (LTP) of synaptic transmission in hippocampal slices, was found to possess multiple functions. They include (1) binding and thereby inhibiting a voltage-dependent K(+)-channel in brain membranes, (2) incorporation in a lipid bilayer to form voltage-dependent and cation-selective channels by itself, and (3) activation of a pertussis toxin (Ptx)-sensitive GTP-binding proteins. In this study, we prepared several derivatives and analogues of MCD and investigated which function is more closely related to the induction of LTP. Another bee venom, apamin, formed ion channels in a lipid bilayer which were indistinguishable from those formed by MCD. D-MCD, an optical isomer of MCD, activated a Ptx-sensitive GTP-binding protein. However, these peptides did not induce LTP in the hippocampal slices. A snake venom, dendrotoxin-I (DTX-I), bound to the same K(+)-channels as MCD and did induce LTP. These results suggest that the most potent aspect of MCD involved in LTP inducibility is its interaction with the voltage-dependent K(+)-channel.

GDNF is a major component of trophic activity in DA-depleted striatum for survival and neurite extension of DAergic neurons.

Extracts from dopamine (DA)-depleted striatal tissue (lesion extract) and from intact striatal tissue (intact extract) were prepared, and trophic activities in these extracts were evaluated using survival and neurite extension of DAergic neurons as indices. Levels of brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), glial cell-line derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) in extracts were measured using enzyme-linked immunosorbent assay (ELISA). The lesion extract exhibited a stronger trophic activity on survival and neurite extension of DAergic neurons than intact extract. In lesion extract, bFGF was slightly and GDNF was significantly increased, while BDNF and NT-3 were the same level in each extract. The peak increase of bFGF and GDNF was during 2 to 3 weeks after DA depletion. Trophic activity of extract was strongly attenuated after immunoprecipitation of GDNF and partly attenuated after immunoprecipitation of bFGF. In parallel immunohistological study, no significant variations were found for striatal microtubule-associated protein-2 (MAP-2)- nor OX-41-immunoreactive cells, while the number of strongly labeled glial fibrillary acidic protein (GFAP)-immunoreactive cells were increased in DA-depleted striatum, suggesting reactive gliosis. Data suggest that bFGF is a minor, while GDNF is a major component of trophic activity for DAergic neurons in DA-depleted striatum, and increased bFGF and GDNF levels may be mediated partly by reactive gliosis.

Tissue extract from dopamine-depleted striatum enhances differentiation of cultured striatal type-1 astrocytes.

The effects of tissue extract from dopamine (DA)-depleted striatum (lesion extract, L-ext) on morphological and electrophysiological natures of cultured striatal astrocytes were investigated. L-ext treatment suppressed the proliferation of type-1 astrocytes. They became fibrous in a concentration-dependent manner. These changes were not observed in type-2 astrocytes. By whole cell patch-clamp recording, two kinetically and pharmacologically distinct voltage-activated potassium currents, A current and delayed rectifier, were identified. L-ext treatment enhanced both currents in type-1 astrocytes, but only A current in type-2. Data suggest that in tissue extract from DA-depleted striatum, there are increased trophic activities that promote the differentiation of type-1 astrocytes.