Tetsu Watanabe

Changes of proliferative activity and phenotypes in spontaneous differentiation of a colon cancer cell line

We examined the alterations of proliferative activity and c‐myc expression of a colon cancer cell line (Caco‐2) during its spontaneous differentiation. Caco‐2 cells were cultured in various types of media and the degree of differentiation was monitored in terms of dome formation in cell monolayers and expression of alkaline phosphatase (ALP) activity. In Caco‐2 cells cultured with Eagles minimum essential medium (EMEM) containing 10% fetal calf serum (FCS), dome formation was demonstrated and ALP activity was markedly increased after the cells reached confluence. Five‐fold reduction of c‐myc mRNA and a marked decrease in S‐phase cells were observed in the differentiated cells. These changes were not induced in FCS‐free EMEM. The addition of insulin and transferrin to FCS‐free EMEM did not induce cell differentiation or reduction of c‐myc mRNA expression. When Caco‐2 cells were cultured with three different serum‐free media, the induction of dome formation and the increase of ALP activity were observed to varying degrees. Expression of c‐myc mRNA in the cells cultured with one serum‐free medium decreased to a level similar to that in fully differentiated cells cultured with EMEM containing 10% FCS. These results suggest that a spontaneous switch from a proliferative state with high c‐myc expression to differentiated phenotype occurs after cells reach confluence and depends on the culture conditions.

A randomized, controlled trial of weekly administration of lymphoblastoid interferon in patients with chronic hepatitis C

To evaluate the efficacy of a treatment of weekly interferon administration in patients with chronic hepatitis C, 36 patients were randomly assigned to two groups. In one group lymphoblastoid interferon was given at a dose of 6 million units, intramuscularly, once per week for 24 weeks, and no treatment was given to the other. Serum alanine aminotransferase levels in the treated group were significantly lower during therapy than in the control group, although there was no significant difference between these two groups before therapy. The normalization of serum alanine aminotransferase levels at the end of therapy was observed in 50% of the treated group, and in 11.1% of the control group. This difference was statistically significant (p < 0.03). Response to interferon was better in patients with chronic persistent hepatitis or with chronic active hepatitis than in patients with chronic active hepatitis with cirrhosis. Relapse after the end of therapy was observed in 83.3% of the responders. These results indicate that the weekly administration of 6 million units of lymphoblastoid interferon is effective in decreasing serum alanine aminotransferase levels in patients with type C chronic persistent hepatitis or chronic active hepatitis.

Decrease of transplantability by the immunopotentiators, OK-432 and interleukin-2: experiments on a human hepatoma cell line in nude mice.

The relationship between nonspecific cytotoxic activity of spleen cells and the resistance against the graft challenge of a human hepatoma cell line (HCC-M) was investigated in nude mice. Two administrations of an immunopotentiator, OK-432 or human interleukin-2, prior to the subcutaneous inoculation of HCC-M cells, which was performed 24 h after the last administration, significantly inhibited the tumor development in terms of rate of tumor take and tumor size. This effect was abrogated by simultaneous administration of an anti-asialo GM1 (ASGM1) antiserum. There was a significant inverse correlation between tumor volume and spleen cell cytotoxicity which was determined at the time of HCC-M cell inoculation against a YAC-1 or HCC-M target. Spleen cell cytotoxicity enhanced by these immunopotentiators could not completely be abolished by in vitro treatment with ASGM1 and complement. This result suggests that effector cells of the enhanced cytotoxicity consist of heterogeneous cells including both ASGM1+ natural killer cells and other nonselective cytotoxic cells. These results suggest that nonspecific cytotoxic cells play crucial roles in the resistance against tumor cell challenge and that the total level of cytotoxic activity of these cells at the time of tumor cell challenge is a key factor which determines tumor development.

Impaired T cell function and decreased natural killer activity in patients with liver cirrhosis and their significance in the development of hepatocellular carcinoma.

SummaryIt is well known that the incidence of hepatocellular carcinoma (HCC) in patients with liver cirrhosis (LC) is very high. We investigated the immunological state in patients with LC and HCC.T cell population of peripheral blood lymphocytes (PBL) and blast transformation of PBL by phytohaemagglutinin (PHA) were significantly decreased in patients with LC. Natural killer activity against HeLa cell was also significantly decreased in these patients.These results suggest that immunological surveillance is impaired in patients with LC and this may be one of the aetiological factors in genesis of hepatocellular carcinoma in patients with LC.

Relationship between hepatitis C virus subtypes and clinical features of liver disease seen in alcoholics

The influence of hepatitis C virus and its subtypes on the clinical course of liver disease in alcoholics was assessed. Hepatitis C virus infection was confirmed by a reverse transcription and polymerase chain reaction method for the hepatitis C virus NS-5 region in the sera of alcoholics with various stages of histologically proven liver disease. The frequency of hepatitis C virus was significantly higher in alcoholics with chronic hepatitis (73%) than in those with liver fibrosis (18%), alcoholic hepatitis (17%), and fatty liver (0%). Hepatitis C virus subtypes, namely K1 and K2, were determined by dot-blot hybridization analysis of the polymerase chain reaction products with specific probes, and their frequencies were 68% and 32%, respectively. The proportion of patients whose serum transaminase levels returned to normal following 4 weeks of abstinence in hospital was significantly lower in alcoholics with hepatitis C virus viremia (glutamic oxaloacetic transaminase: 53.8%; glutamic pyruvic transaminase: 42.3%) than in those without viremia (glutamic oxaloacetic transaminase: 86.2%, p < 0.01; glutamic pyruvic transaminase: 89.7%, p < 0.01). When alcoholics with the K1 and K2 subtypes of hepatitis C virus were compared, normalization of transaminase levels was less frequent in alcoholics with K1 (glutamic oxaloacetic transaminase: 42.8%; glutamic pyruvic transaminase: 28.6%) than in those with K2 (glutamic oxaloacetic transaminase: 88.9%, p < 0.05; glutamic pyruvic transaminase: 77.8%, P < 0.05). These data indicate that hepatitis C virus infection is associated with a reduced rate of recovery of serum transminase levels following abstinence in subjects with alcoholic liver disease, more so in the K1 subtype than in the K2 subtype.

Transformation of NIH/3T3 cells by DNA from a human hepatoma cell line with integrated hepatitis B virus DNA

We have studied by means of DNA-mediated gene transfer the transforming activity of the DNA of the human hepatoma cell line HCC-M, which contains genomes of hepatitis B virus (HBV) in integrated form. DNA from HCC-M induced transformed foci on transfection of NIH/3T3 cells. DNAs from primary transformants were capable of inducing secondary transformants. Most of the DNAs of these transformants were demonstrated to contain both human repetitive sequences and HBV DNA, indicating that the transformants had incorporated exogenous human DNA and HBV DNA as well. These results suggest that transformation occurs as the result of the transfer of oncogene which might be closely associated with HBV genome.

New micro-glass-tube leukocyte adherence inhibition assay assessing cell adherence of mononuclear cell subpopulations defined by monoclonal antibodies

A new micro-glass-tube leukocyte adherence inhibition (LAI) assay which is appropriate for detecting delayed type hypersensitivity in vitro has been developed for human leukocytes. Enumeration of adherent cells is replaced by a cellular radioimmunoassay determining antibody binding of the monoclonal reagents, OKT4, OKT8 and OKM1, to glass-adherent cells, fixed by glutaraldehyde or formaldehyde. An LAI reactivity to purified protein derivative of tuberculin (PPD) was detectable in donors giving a positive PPD skin test with OKT4 reagent, but not with the other two reagents.

Inhibitory effect of sera from patients with liver cirrhosis on natural killer activity

The effect of sera from patients with liver cirrhosis (LC) and from healthy donors on natural killer (NK) activity of normal peripheral blood mononuclear cells (PBMC) was investigated. PBMC from a healthy donor were pre‐incubated for 3 h at 37°C in media containing 50% test sera and washed twice with phosphate‐buffered saline (PBS). Cytotoxic activity was determined using 51Cr‐labelled K‐562 cells as target. It was demonstrated that sera from both healthy donors and patients with LC suppressed the NK activity of normal PBMC compared with a PBS control. But the level of inhibition caused by patients’ sera (n= 30) was significantly higher than that caused by normal sera (n= 30; P < 0.001, Wilcoxon rank sum test). Sera from a patient with LC and a healthy subject were fractionated by a Sephacryl S‐300 column and the effect of five fractions on NK activity of normal MNC was assessed. Fractions 3 and 4 from both sera had inhibitory effects although Fraction 5 revealed an enhancing effect. These results suggest that human sera contain factors which both inhibit and enhance NK activity and that their net effects are inhibition. Since it is known that IgG is eluted in Fraction 4, IgG was separated from sera with a DEAE‐Sephacel column and its effect on NK activity was examined. IgG from patients’ sera was observed to suppress NK activity more often than those from healthy donors at the same concentrations.