Reduced stability of retinoblastoma protein by gankyrin, an oncogenic ankyrin-repeat protein overexpressed in hepatomas.

Hepatocellular carcinoma (HCC) is one of the most common cancers in Asia and Africa, where hepatitis virus infection and exposure to specific liver carcinogens are prevalent. Although inactivation of some tumor suppressor genes such as p53 and p16INK4Ahas been identified, no known oncogene is commonly activated in hepatocellular carcinomas. Here we have isolated genes overexpressed in hepatocellular carcinomas by cDNA subtractive hybridization, and identified an oncoprotein consisting of six ankyrin repeats (gankyrin). The expression of gankyrin was increased in all 34 hepatocellular carcinomas studied. Gankyrin induced anchorage-independent growth and tumorigenicity in NIH/3T3 cells. Gankyrin bound to the product of the retinoblastoma gene (RB1), increasing its phosphorylation and releasing the activity of the transcription factor E2F-1. Gankyrin accelerated the degradation of RB1 in vitro and in vivo, and was identical to or interacted with a subunit of the 26S proteasome. These results demonstrate the importance of ubiquitin-proteasome pathway in the regulation of cell growth and oncogenic transformation, and indicate that gankyrin overexpression contributes to hepatocarcinogenesis by destabilizing RB1.

Enhanced expression of multiple protein tyrosine phosphatases in the regenerating mouse liver: isolation of PTP-RL10, a novel cytoplasmic-type phosphatase with sequence homology to cytoskeletal protein 4.1.

To elucidate the role that protein tyrosine phosphatase (PTPs) may play in liver regeneration, PTPs expressed in the mouse liver after partial hepatectomy (PH) were investigated by a PCR-based cloning method. Sequencing of 115 cDNA clones identified 10 different sequences including MPTP (T cell PTP), PTP-1B, PTP-P19, mR-PTP mu, R-PTP alpha, PTP NE-3 (PTP-P1), R-PTP-kappa and the murine homologue of human LAR. The remaining two sequences, PTP-RL9 and PTP-RL10, encoded novel PTPs. PTP-RL10 cDNA contained an open reading frame of 1176 amino acids with no apparent membrane-spanning region. The amino-terminal region had sequence homology to those of human erythrocyte protein 4.1 and ezrin, cytoskeletal proteins. In the regenerating liver, the levels of five PTP gene mRNAs (MPTP, PTP-P19, R-PTP alpha, LAR homologue, and PTP-RL9) increased within 6 h, decreased to the normal level by 24 h, and increased again at 48 to 72 h after PH. The levels of PTP-1B and R-PTP-kappa mRNAs peaked within 6 h, decreased gradually, and returned to the normal level by 168 h after PH. In contrast, the levels of two PTP mRNAs (mR-PTP mu and PTP-RL10) peaked at 48 to 72 h, and returned to the normal level by 168 h after PH. No expression of PTP NE-3 was detected in the liver by Northern blotting. The differential expression of multiple PTPs during the pre-replicative and post-replicative stages of liver regeneration suggests that PTPs are involved in the regulation of growth and differentiation of liver cells.

Presence of alternative 5' untranslated sequences and identification of cells expressing ctk transcripts in the brain and testis.

From a mouse brain cDNA library, two species of the Csk-type tyrosine kinase (ctk) gene containing different 5' untranslated sequences were cloned. Using the common as well as specific nucleotide sequences of the two clones as probes, we examined the expression of ctk in various mouse tissues by Northern blot analysis. The results indicated that both species of ctk were expressed in the brain, testis and bone marrow. By in situ histochemistry of the brain, ctk transcript was detected in neurons throughout the entire brain, especially those of the cortex, the hippocampus and the cerebellum. This distribution pattern is similar to that of the Src family kinases including Yes, Src, Fyn and Lyn. In the testis, the major transcript (0.7 kb) was shorter than that expressed in the brain and the bone marrow (2.0 kb). A subsequent Northern blot analysis of fractionated germ cell populations and in situ histochemistry revealed that the short and long transcripts were expressed in germ cells and somatic cells, respectively, and that the expression level was quantitatively regulated during germ cell development. These results suggest that Ctk is involved in the regulation of neural function and differentiation of male germ cells through interactions with member(s) of the Src family kinases.

Cloning and characterization of human CIRP (cold-inducible RNA-binding protein) cDNA and chromosomal assignment of the gene.

Cold stress induces in microorganisms the synthesis of several proteins that are involved in various cellular processes such as transcription, translation and recombination. Recently, the cold-inducible RNA-binding protein (Cirp) was found to be induced in rodent cells by mild cold stress (32 degrees C). Cirp consists of an N-terminal RNA-binding domain and a C-terminal Gly-rich domain, and plays an essential role in cold-induced suppression of cell proliferation. We report here the cloning of a cDNA encoding an 18-kDa protein with 95.3% identity in an amino-acid sequence to that of mouse Cirp. The human CIRP gene has been mapped to the chromosomal locus 19p13.3 by fluorescence in-situ hybridization. CIRP mRNA is constitutively expressed in all cell lines examined, including K562, HepG2, NC65, HeLa, T24, and NEC8 cells. In all of them, the levels of CIRP mRNA and protein were increased within 12 h after a temperature down-shift from 37 degrees C to 32 degrees C. These results demonstrated that CIRP is a cold-shock protein in human cells. Identification of CIRP may provide a clue to the regulatory mechanisms of cold responses in human cells.

Effects of ischemia and H2O2 on the cold stress protein CIRP expression in rat neuronal cells.

Expression of CIRP (cold-inducible RNA-binding protein) is inducible at 32 degrees C in cultured fibroblasts. Because ischemia is known to induce expression of heat shock proteins, its effect on the CIRP expression was examined using the rat transient forebrain ischemia model. The isolated rat CIRP cDNA encoded amino acids 100% identical in its sequence to mouse CIRP. Northern blot analysis revealed that the CIRP transcripts were ubiquitously expressed in various tissues. In situ hybridization histochemistry of normal rat brain revealed the expression of CIRP in neurons in the hippocampus and the cerebral cortex among others. In the hippocampus of postischemic rats, CIRP mRNA level decreased from 3-6 h after the onset of reperfusion, while it did not change in the cerebral cortex. When PC12 pheochromocytoma cells were cultured at 32 degrees C, the CIRP mRNA level was increased. The presence of H2O2 in the culture media inhibited dose dependently this induction as well as constitutive expression, suggesting that the effect of brain ischemia on CIRP expression is related to generation of reactive oxygen species. Further studies are necessary to clarify the roles played by cold shock proteins in the hypothermic therapy of brain damages.

Pathogenic role of Kupffer cell activation in the reperfusion injury of cold-preserved liver.

The present study was designed to investigate the possible participation of Kupffer cells in the development of reperfusion injury of the cold-stored liver graft. In the cold preservation of Kupffer cells with Euro-Collins solution, the proportion of asialo-GM1-positive cells was significantly increased at 12 and 24 hr of storage, and the TNF alpha-producing activity in these cells was approximately fivefold greater than control. Northern blot analysis demonstrated that TNF alpha mRNA was remarkably elevated in the reperfusion of the cold-preserved liver, although that of the prereperfused graft was only slightly induced. The reperfusion experiments of the cold-stored liver graft showed that addition of anti-TNF alpha antibody to the perfusate suppressed the elevation of the effluent levels of GOT and LDH significantly, and that pretreatment with a Kupffer cell inhibitor, gadolinium chloride, inhibited the increase of these enzymes in the effluents almost completely. Histological study revealed deposition of a fibrinlike substance in the sinusoid and the central veins extensively in the reperfused liver graft, whereas no apparent deposition was observed in the gadolinium-pretreated liver. Thus, the present study showed that Kupffer cells were primed by cold preservation with Euro-Collins solution, and then activated when the reperfusion was done. It seems likely that the Kupffer cell activation induced by cold preservation/reperfusion plays a major role in reperfusion injury with sinusoidal microcirculatory disturbance, and that TNF alpha is responsible for the impairment of the reperfused liver graft.

Decreased expression and rare somatic mutation of the CIP1/WAF1 gene in human hepatocellular carcinoma.

CIP1/WAF1, a critical downstream effector of tumor suppressor p53, encodes a cyclin-dependent kinase inhibitor. By Northern blot analysis, the CIP1/WAF1 mRNA level in the tumor was significantly lower than that in the corresponding normal liver from 19 Japanese patients with hepatocellular carcinoma (P < 0.05). In the tumor from only one out of 19 patients (5%), somatic mutations of the CIP1/WAF1 as well as that of p53 gene were identified by RT-PCR/SSCP analysis. These results suggest that the decreased CIP1/WAF1 expression is involved in the carcinogenesis or the progression of hepatocellular carcinoma.

Chemical mediator release and surface marker expression of hepatic macrophages in rats with CCl4-induced liver cirrhosis.

The present study was performed to analyze possible functional alterations of hepatic macrophages (HM phi) in rats with carbon tetrachloride (CCl4)-induced liver cirrhosis. HM phi from rats injected with CCl4 for 13 weeks and cultured for 24 hours released less than normal amounts of prostaglandin E2 (PGE2) and tumor necrosis factor (TNF) and very large amounts of interleukin-1 (IL-1). In rats injected with CCl4 for 9 weeks, only PGE2 production was reduced. Interleukin-2 receptor (IL-2R), Ia antigen and asialo GM1 antigen expressions on HM phi from both the 9- and 13-week groups were significantly decreased. IL-2R and Ia antigen expressions showed larger decreases in the 13-week group. Thus, it is concluded that HM phi derived from CCl4-induced cirrhotic livers show a functional alteration in the release of cytokines (except for IL-1) and a decrease in surface marker expression, as cirrhosis advances. These results should provide a basis for further investigation into the host-compromised status in the presence of liver cirrhosis.

Tumoricidal activity of Kupffer cells augmented by anticancer drugs.

The effect of Mitomycin-C (MMC) and Adriamycin (ADM) on the antitumor-associated function of Kupffer cells was examined. MMC and ADM enhanced the production of superoxide by Kupffer cells in cultures at low concentrations likely to occur in clinical use. The expression of interleukin-2 receptor, Ia antigen and asialoGM1 antigen, measured by flowcytometry, was increased by contact with MMC. Growth inhibition of AH130, rat ascites hepatoma, and P815, murine mastocytoma, by Kupffer cells treated with anticancer drugs was greater than that by Kupffer cells alone or anticancer agent alone. These results show that MMC and ADM activate Kupffer cells, leading to synergistic antitumor activity. The results suggest that some anticancer agents act through immunological mechanisms as well as through direct antineoplastic activity.

Submandibular salivary pH as a diagnostic aid for prognosis of facial palsy.

The present paper aims at introducing a new method of testing the function of the chorda tympani with the aid of submandibular salivary pH. This method has several advantages: (1.) it enables the measurement of the function of only the paralyzed side; (2.) it accurately predicts the outcome of facial palsy at an early stage; and (3.) the method is simple and inexpensive.

[Use of AmpliSensor to quantitate gene expression in small amounts of samples: comparison with the quantitative RT-PCR method using CCD imaging system].

Quantitative PCR methods are potentially useful for determining levels of specific gene expression and gene dosage. Previously we developed a non-radioisotopic quantitative RT-PCR method by utilizing the PCR amplification kinetics and CCD image analyzer. Recently, based on the principle of fluorescence energy transfer, the AmpliSensor system that can quantitate the PCR products concurrent to amplification in a single reaction vessel has been described. Herein, we compared the results obtained by both methods. cDNAs were synthesized from 10 human endometrial biopsy specimens. Aliquots of cDNA were used for quantitation by gel/image analyzer or AmpliSensor assay system. For estimation of the initial amount of the template(I), regression equations of the form:y = I x Ex, where y is the amount of PCR products and x is the number of cycles, were fitted to the data in the linear portion of the semi-logarithmic graphs. The relative levels of beta-actin cDNA estimated by AmpliSensor assay system was in good agreement (r = 0.91) with those of the gel/image analyzer assay system, which is cumbersome, time-consuming and needs post-amplification procedures. The AmpliSensor assay is suitable for a quantitative PCR assay based on PCR kinetics, and is applicable for diagnostic testings.

A novel hsp110-related gene, apg-1, that is abundantly expressed in the testis responds to a low temperature heat shock rather than the traditional elevated temperatures.

We isolated a novel hsp110-related gene, apg-1, from a testis cDNA library. The apg-1 transcripts were constitutively expressed in the testicular germ cells and, in some degree, most tissues examined. In a mouse TAMA26 Sertoli cell line, apg-1 transcripts were induced in 2 h by a temperature shift from 32 to 39 degrees C, but not by a shift from 37 to 42 degrees C, the traditional heat stress, or a shift from 32 to 42 degrees C. The heat response pattern of hsp110 expression was similar to that of apg-1. Although induction of a hsp70 transcript was observed in 2 h by a shift from 32 to 39 degrees C, the induction was more apparent by a shift from 37 to 42 degrees C or from 32 to 42 degrees C. Essentially similar differential response patterns were observed among these genes in NIH/3T3 fibroblasts as well. The nuclear run-on assay and the native gel mobility shift assay demonstrated that, by the 32 to 39 degrees C temperature shift, the apg-1 gene was transcriptionally activated, and heat shock factor 1 bound to the heat shock elements in the 5'-flanking region of the apg-1 gene. These results demonstrated that expressions of apg-1, hsp110, and hsp70 could be heat-induced at a temperature lower than the traditional elevated temperatures in somatic cells of both testis and nontestis origin and suggest that the mechanisms regulating the transcript levels of apg-1 and hsp110 are different from those of hsp70. Furthermore, the constitutive expression in germ cells suggests that APG-1 plays a specific role in spermatogenesis as well as in stress response.

Burn perforation as a method of middle ear ventilation.

Eardrum perforation caused by burn is well known to have a tendency to resist closure. This unfavorable tendency to resist closure. This unfavorable tendency can be turned to the benefit of patients with severe, Eustachian tube dysfunction. Pressure equalization of sufficient duration can be produced by making a burn perforation of the tympanic membrane. Multiple, small perforations placed closely or a large perforation of about one eighth of the tympanic membrane will remain patent and equalize pressure across the tympanic membrane for a period greater than three months. The procedure is of a minor nature comparable with myringotomy or with the removal of a plugged clot from a tube.

Superoxide release by primary-cultured human hepatic macrophages and peripheral monocytes from patients with normal and cirrhotic livers.

We analysed superoxide anion (O2-) release from primary-cultured human hepatic macrophages (HHM phi) and peripheral blood monocytes (MO) derived from patients with normal and cirrhotic livers. Primary cultured human hepatic macrophages and MO from cirrhotic patients released less O2- than cells from normal patients. Superoxide anion release by HHM phi showed a significant, positive correlation with the serum glutamate pyruvate transaminase (GPT) level, whereas O2- release by MO showed only a weak correlation with the GPT level. In conclusion, a significant reduction in O2- release was observed in HHM phi and MO cultured from cirrhotic patients. Primary-cultured human hepatic macrophages are probably more susceptible to environmental changes within the cirrhotic liver than MO, since they are found locally within the liver and correlate with serum GPT levels.

Enhancement and hepatocyte-modulating effect of chemical mediators and monokines produced by hepatic macrophages in rats with induced sepsis.

We investigated the production of chemical mediators by hepatic macrophages from rats with sepsis and the modulation of hepatocyte function by these hepatic macrophages. The chemical mediators we measured were superoxide (O2-), TNF, IL-1, and PGE2. Production of these mediators by hepatic macrophages from rats with sepsis was significantly increased. Furthermore, protein synthesis by cultured hepatocytes was inhibited in a co-culture system of hepatocytes and hepatic macrophages from rats with sepsis, and it was even inhibited by the supernatant of cultured hepatic macrophages from septic rats. These results demonstrate that hepatic macrophages are activated in sepsis and may play a role in inducing hepatic dysfunction in sepsis.

Depressed function of Kupffer cells in rats with CCl4-induced liver cirrhosis.

In the present study, the Kupffer cell function of rats with CCl4-induced liver cirrhosis was tested by analyzing the changes in the host defense system. In rats without liver cirrhosis injected with CCl4 for 3 weeks concomitant with the high opsonic activity the endocytic index was significantly increased. Rats treated for 9 and 13 weeks developed cirrhosis, and their endocytic indices were not increased despite the rise in their opsonic activity. Particularly, the endocytic index of 13-week-treated rats with advanced liver cirrhosis was significantly lower than that of the other groups. The organic distribution of 51Cr-endotoxin injected intravenously exhibited characteristic changes in 9-week- and 13-week-treated rats: decreased hepatic uptake and increased splenic uptake. In contrast, pulmonary uptake was increased in all CCl4-treated rats. The superoxide production by Kupffer cells from 13-week-treated rats was greatly reduced, accompanied by the decreased superoxide dismutase activity of liver homogenate. Thus, results of this study suggest that Kupffer cell dysfunction is one of the main factors affecting host defenses in liver cirrhosis.

Chemical mediators released from hepatic macrophages in primary culture--basic characteristics of human hepatic macrophages and changes in liver cirrhosis.

Chemical mediators released from human hepatic macrophages (HHM phi) in primary cultures were analyzed for their secretory function and probable contribution to the modulation of the host defense system and metabolism in liver cirrhosis. In our basic studies, HHM phi increased dose dependently the release of superoxide (O2-) and interleukin-1 (IL-1) when stimulated by opsonized zymosan, up to 1000 micrograms/dish. PGE2 production showed a relatively narrow range of dose dependency, and larger doses led to a reduction of PGE2 yield in some samples. Next, we compared the mediator release from the HHM phi of patients with liver cirrhosis with that from HHM phi in normal liver. O2- released from HHM phi of 8 patients with liver cirrhosis was significantly decreased (controls, n = 20) (P < 0.01). IL-1 released from the HHM phi of 6 cirrhotic patients tended to be higher than that from the HHM phi of 10 control patients, but the difference was not statistically significant (P < 0.10). PGE2 production, however, was about the same in the two groups. These results suggest that cultured HHM phi have certain basic characteristics in releasing mediators with highly potent specific activities and also that these secretory abilities may change in liver cirrhosis. In conclusion, the analysis of cultured HHM phi may be a very practical way to clarify their inherent abilities and participation in the complicated clinical features of liver cirrhosis.

Increased transcript level of RBM3, a member of the glycine-rich RNA-binding protein family, in human cells in response to cold stress.

Although the cold-shock responses of microorganisms have been extensively investigated, those of mammalian cells are just beginning to be understood. Recently, CIRP, a member of the glycine-rich RNA-binding protein (GRP) family, has been identified as the first cold-shock protein in mammalian cells. Here, we report that RBM3, another member of the GRP family, is induced in human cells in response to cold stress (32 degrees C). RBM3 transcripts were constitutively expressed in all cell lines examined including K562, HepG2, NC65, HeLa, and T24 cells. In all of them, the transcript levels of RBM3 were increased at 24 h after the 37 to 32 degrees C temperature down-shift. In NC65 cells, the kinetics of RBM3 induction was different from that of CIRP. Protein synthesis inhibitors cycloheximide and puromycin induced RBM3 transcripts, but cadmium chloride, H2O2, ethanol, and osmotic shock had no effect. Combined with the different tissue distribution of expression, these results suggest that RBM3 and CIRP play distinct roles in cold responses of human cells.

Augmented local immunity in the liver by a streptococcal preparation, OK432, related to antitumor activity of hepatic macrophages.

The aim of this study was to investigate the augmentative effect of a streptococcal preparation, OK432, on the immunological competence of hepatic macrophages. We found that OK432 was distributed predominantly to hepatic macrophages after intravenous injection, and Northern blot analysis revealed that OK432 induced the gene expression of IL-1 alpha, beta, and TNF alpha in the liver. The induction of mRNAs was evident 1 h after the intravenous injection of OK432 and their accumulation reached a maximal level at 3 h. TNF production of hepatic macrophages was also increased by the intravenous injection of OK432. Furthermore, OK432 significantly increased the proportion of IL-2 receptor-positive hepatic macrophages. As for antitumor activity in the liver being augmented by OK432, the cytotoxic and cytostatic activity of hepatic macrophages from OK432-treated rats against tumor cells was significantly increased and OK432 markedly reduced the number of tumor nodules in the liver after the inoculation of tumor cells via the portal vein. These findings, which indicate that OK432 has various immuno-stimulating actions on hepatic macrophages, leading to the augmentation of antitumor activity in the liver, suggest that OK432 may be of some benefit in helping to prevent hepatic metastasis, at least in part, via its activation of hepatic macrophages.

Reduced expression of kan-1 (encoding putative bile acid-CoA-amino acid N-acyltransferase) mRNA in livers of rats after partial hepatectomy and during sepsis.

We isolated a cDNA clone, kan-1, from a rat liver cDNA library using a reverse transcriptase PCR cloning method. The kan-1 cDNA encoded a polypeptide of 420 amino acids, and was 70 and 69% identical in nucleotide and amino acid sequences respectively with human liver bile acid-CoA-amino acid N-acyltransferase (BAT). Thus Kan-1 is probably a rat homologue of human BAT (rBAT). Kan-1/rBAT mRNA was mainly expressed in the livers of adult rats and rats immediately after, but not before, birth. It was expressed in the hepatocytes, the sinusoidal endothelial cells and the Kupffer cells of the liver. An anti-Kan-1/rBAT polyclonal antibody detected a protein of molecular mass 46 kDa in the liver. After partial hepatectomy, the levels of Kan-1/rBAT mRNA decreased at 6 and 12 h in the regenerating liver. In a sepsis model, hepatic expression of Kan-1/rBAT mRNA decreased at 6 and 12 h after caecal ligation and puncture. The kinetics of Kan-1/rBAT mRNA expression suggests that it may play a role in acute-phase reactions.