Preoperative estimation of remnant hepatic function using fusion images obtained by (99m)Tc-labelled galactosyl-human serum albumin liver scintigraphy and computed tomography.

BACKGROUND: Assessment of hepatic functional reserve is important in hepatic resection. The aim of this study was to evaluate the role of hepatic asialoglycoprotein receptor (ASGP-R) analysis in the preoperative estimation of remnant liver function in liver surgery. METHODS: One hundred and one patients undergoing hepatic resection for liver tumours were studied. Seventeen patients had preoperative percutaneous transhepatic portal vein embolization (PTPE). Function of the hepatic remnant was estimated before surgery using radioactivity in fusion images of both liver single-photon emission computed tomography and computed tomography scans using (99m)Tc-labelled diethylene triamine penta-acetate-galactosyl-human serum albumin. RESULTS: All three patients with an ASGP-R concentration below 400 nmol/l and preoperative total amount of receptor in the future remnant liver (R0-remnant) of less than 53.0 nmol per liver died. Two patients with chronic hepatitis and R0-remnant values between 53.0 and 65.0 nmol per liver and a receptor concentration lower than 600 nmol/l developed liver dysfunction. The incidence of liver failure decreased inversely with increasing R0-remnant value. CONCLUSION: A combination of receptor concentration and the amount of hepatic receptor in the future liver remnant as detected on fusion images is useful in evaluating the risk of postoperative liver failure.

[Lung abscess which ruptured during the medical treatment of lung abscess; report of a case].

63-year-old man was admitted to our hospital with fever and cough for about 2 months. Laboratory data showed marked inflammatory changes, and chest computed tomography (CT) scans revealed right-sided hydrothorax, atelectasis of the right middle lobe, and a cystic mass in the right middle lobe. We diagnosed the patients as having lung abscess and empyema. Following the intravenous antibiotic chemotherapy, symptoms and laboratory data showed the improvement, however, on the 11th hospital day, he developed high fever again. A chest CT showed pneumopyothorax suggesting the rupture of lung abscess. Since the chest tube drainage was ineffective, open chest surgery was performed. Curettage of both thoracic and abscess cavity with closure of air leakage successfully cured the pyothorax.

Actual invasive potential of human hepatocellular carcinoma revealed by in situ gelatin zymography.

BACKGROUND: The matrix-degrading proteinases are believed to play an important role in the invasion and metastasis of hepatocellular carcinoma (HCC), but no one has ever seen the in situ matrix-degrading activity in HCCs. PURPOSE: To demonstrate the cellular localization of actual gelatinolytic activity and to investigate the invasive potential of human HCC. EXPERIMENTAL DESIGN: HCC cases (30) were subjected to in situ gelatin zymography and SDS-gelatin gel zymogram. RESULTS: In situ gelatin zymography revealed a heterogeneous gelatinolytic activity in HCC cells, as well as stromal cells of noncancerous livers. The gelatinolytic intensity was stronger in 15 HCC nodules than in the corresponding noncancerous livers and was significantly associated with the cancer invasion to the capsule of the HCCs and to the portal veins. An intense gelatinolytic activity was detected in HCC cells in the front of tumor invasion. SDS-gelatin gel zymogram revealed gelatinases A and B that were mostly in latent forms. CONCLUSIONS: The present study demonstrates high gelatinolytic activity at the invasive front of HCCs at a cellular level and that HCC has an invasive potential with the gelatin (matrix)-degrading metalloproteinases. Furthermore, it suggests the importance of the activation mechanism of gelatinolytic enzymes in the invasion and metastasis of HCCs.

The formation of capsule and septum in human hepatocellular carcinoma.

The formation of fibrous capsule around the cancer nodule and of the septum in the tumor is frequently observed with the development of hepatocellular carcinoma (HCC). We aimed to clarify how the capsule and septum were formed during the growth of HCC. Liver samples surgically resected from 25 patients with HCC were studied with in situ hybridization for type-I, -III, and -IV procollagen. Type-I and -III procollagen-expressing cells, mostly alpha-smooth muscle actin (SMA)-positive, were increased in the fibrous capsule and in the septum between HCC nodules. These cells were also found at the invasion front of HCC and around the necrotic cancer tissues. Type-IV procollagen gene expression was mainly observed in mesenchymal cells localized in both HCCs and non-cancerous liver. Cancer cells or hepatocytes did not express any of these procollagen genes. The present study reveals that the capsule and septum are mainly formed by alpha-SMA-positive mesenchymal cells at the interface between two different tissues (e.g., cancer nodule vs non-cancerous liver or another cancer nodule). The wound healing occurs even in HCC. The capsule formation may result from interaction between tumor and host liver and interfere the growth and invasion of HCC.

Effects of zinc deficiency/zinc supplementation on ammonia metabolism in patients with decompensated liver cirrhosis.

Hepatic encephalopathy is one of the major complications in decompensated liver cirrhosis. The current study was conducted to clarify the mechanisms of zinc deficiency in liver cirrhosis and its involvement in hepatic encephalopathy via ammonia metabolism. Ten patients each with compensated or decompensated liver cirrhosis and 11 healthy volunteers were enrolled in the study. Serum zinc levels and its daily urinary excretion were measured, an oral zinc-tolerance test was performed to examine zinc malabsorption, and the effects of diuretics on zinc excretion and of zinc supplementation on ammonia metabolism in the skeletal muscle were studied. The mean serum zinc levels in patients with decompensated liver cirrhosis were found to be significantly lower than the levels in controls and patients with compensated liver cirrhosis. The serum zinc levels were inversely correlated with blood ammonia in the fasting state. In the oral zinc-tolerance test, the percent increase in serum zinc levels 120 and 180 min after ingestion was less in cirrhotic patients than in controls. A diuretic administration resulted in a significant reduction in serum zinc levels. An increased uptake of ammonia by and an increased release of glutamine from leg skeletal muscle after oral supplementation of zinc sulfate were evident. Taken together, zinc deficiency in decompensated cirrhotic patients appears to be due to low absorption and to high urinary excretion, for which excessive diuretic administration is, in part, responsible, and zinc supplementation might play an important role in the prevention of hepatic encephalopathy by activating glutamine synthetase.

Serum interferon-gamma-inducing factor/IL-18 levels in primary biliary cirrhosis.

Primary biliary cirrhosis is an autoimmune disease of the liver in which T helper 1 cytokines predominate over those of T helper 2 in the pathogenesis. Interleukin- 18 (IL-18), for which the gene was recently cloned, is a novel T helper 1 cytokine, which augments interferon-gamma production. We designed this study to clarify the role of IL-18 in primary biliary cirrhosis and to examine whether serum IL-18 level can be a prognostic indicator for the disease. Serum IL-18 levels were measured using an enzyme linked immuno sorbent assay with mouse monoclonal antibodies. Twenty-two healthy volunteers, 31 patients with primary biliary cirrhosis (Scheuer's stage I, 13; II, 10; and IV, 8), 20 patients with autoimmune hepatitis, 11 patients with virus-related liver cirrhosis and six patients with obstructive jaundice were enrolled. Significant differences of serum IL-18 levels were observed between patients with Scheuer's stage IV and those with stage I, or II, virus-related liver cirrhosis and obstructive jaundice (P < 0.05). The IL-18 levels in primary biliary cirrhosis increased according to the disease progression, and fell promptly after living-related liver transplantation. Moreover, serum IL-18 levels in primary biliary cirrhosis were correlated with serum bilirubin concentrations and the Risk scores of the Mayo Clinic prognostic model for the disease. The IL-18 levels observed in patients with autoimmune hepatitis were also elevated, and correlated with the activity of the disease. These results indicate that serum interleukin-18 levels reflect the severity of primary biliary cirrhosis, the activity of autoimmune hepatitis, and may be an additive prognostic indicator in primary biliary cirrhosis.

Expression of MAGE, GAGE and BAGE genes in human liver diseases: utility as molecular markers for hepatocellular carcinoma.

BACKGROUND/AIMS: The MAGE, GAGE and BAGE genes encode tumor antigens recognized by autologous cytotoxic T lymphocytes. The aim of this study was to evaluate the possibility of using these genes as molecular markers and as the targets of specific immunotherapy for human hepatocellular carcinoma (HCC). METHODS: The expressions of MAGE-1, MAGE-3, GAGE1-6, GAGE1-2 and BAGE mRNA in 33 surgically resected HCC samples and 26 of their corresponding non-cancerous samples (11 liver cirrhosis and 15 chronic hepatitis) were studied by a reverse-transcription polymerase chain reaction, and were compared with clinicopathological parameters. The expression of MAGE-1 was also examined in 16 biopsied HCC samples. RESULTS: MAGE-1, MAGE-3, GAGE1-6, GAGE1-2 and BAGE mRNA were expressed in 67%, 39%, 36%, 30%, and 21% of the HCC, respectively. At least one transcript was detected in 88% of the HCC, while no expression was observed in the non-cancerous livers. There was no significant correlation between the expression of any of the tumor antigens examined and the differentiation stage or size of the HCC. Especially, MAGE-1 was highly expressed in small HCC with a diameter of less than 2 cm and in well-differentiated HCC (81% and 70%, respectively), and was also expressed even in alpha-fetoprotein-negative and PIVKA-II-negative HCC (58% and 76%, respectively). The MAGE-1 expression was detected in 69% of biopsied HCC samples and the expression was high in both small and well-differentiated HCC. CONCLUSIONS: These tumor-specific antigens can be useful as molecular markers and as the possible target molecules for the specific immunotherapy of human HCC.

Expression of telomerase-associated protein 1 and telomerase reverse transcriptase in hepatocellular carcinoma.

To know whether two protein components of human telomerase (human telomerase-associated protein 1 (hTEP1) and human telomerase reverse transcriptase (hTERT) are useful markers for telomerase activation in human liver diseases, we examined mRNA levels of these and telomerase activity in human liver samples. Twenty-three human hepatocellular carcinomas (HCCs) and corresponding adjacent livers were analysed for hTEP1 and hTERT expression by semiquantitative reverse transcription-polymerase chain reaction, and for telomerase activity by a telomeric repeat amplification protocol assay. Thirteen liver samples (ten HCCs and three dysplastic nodules) that were biopsied with 21-gauge needles were analysed for hTERT expression. hTEP1 was expressed in all samples examined. No correlation between hTEP1 expression and telomerase activity was observed. hTERT expression significantly correlated with telomerase activity (P< 0.001). The positivity of hTERT for HCC and corresponding non-cancerous liver was 100% and 30.4% respectively (P < 0.001). Seventy-four per cent (17/23) of HCCs showed strong hTERT expression, but none of the non-cancerous liver tissues did. hTERT expression of the 21-gauge needle biopsied specimens showed no significant difference from that of the surgical samples. The present study revealed that hTERT is strongly expressed in most HCCs, and that hTERT but not hTEP1 is a key component regulating telomerase activity in human liver.

Expression of MAGE-1 and -3 genes and gene products in human hepatocellular carcinoma.

MAGE gene family encodes peptides recognized by autologous cytotoxic T lymphocytes in a major histocompatibility complex (MHC) class-I restricted fashion. In the present study, we have performed reverse-transcription polymerase chain reaction (RT-PCR) for the genes, as well as immunohistochemical analysis and Western blotting of MAGE-1 and -3 proteins in 33 surgically resected hepatocellular carcinomas (HCCs). MAGE-1 and -3 mRNAs were constitutively expressed exclusively in 78 and 42% of HCCs respectively. On immunohistochemistry with monoclonal antibodies, 77B for MAGE-1 and 57B for MAGE-3, MAGE-1 and -3 proteins were recognized in cytoplasm of only six among 33 (18%) and two of 29 HCCs (7%) respectively. The distribution pattern was mostly focal in HCC nodules. By contrast, the Western blot analysis revealed that the MAGE-1 (46 kDa) and -3 proteins (48 kDa) were expressed in 80 and 60% of 15 HCCs examined respectively. The proteins of MAGE-1 and -3 were also expressed exclusively in HCCs regardless of the histological grading and clinical staging. Our results indicate that the detection of the genes by RT-PCR or the proteins by Western blotting is useful for differentiating early HCCs from non-cancerous lesions, and that the peptides derived from MAGE-1 and -3 proteins might be suitable targets for immunotherapy of human HCC.

Expression of membrane cofactor protein (MCP, CD46) in human liver diseases.

Membrane cofactor protein (MCP, CD46) is one of the complement regulatory proteins, and is widely distributed in human organs and protects cells from complement-mediated cytotoxicity. We analysed the distribution and the intensities of MCP in liver diseases and evaluated the role of MCP during hepatocarcinogenesis. Western blot analysis revealed that relative densities (density of the sample/density of the standard sample) of MCP in 27 HCC, 18 liver cirrhosis, nine chronic hepatitis and 12 normal liver were 0.63+/-0.23, 0.21+/-0.07, 0.25+/-0.10 and 0.11+/-0.03 (mean+/-s.d.) respectively. MCP expression in hepatocellular carcinoma (HCC) was significantly higher than that in both liver cirrhosis and chronic hepatitis (P < 0.01). The difference in the tumour sizes, the grades of differentiation and viral marker status did not affect the expression. Immunohistological analysis revealed that MCP was distributed mainly in the basolateral membrane of the hepatic cord in non-cancerous liver, along with endothelial cells and bile duct cells. In HCC, the protein was observed on the membrane in a non-polarized fashion. These data suggest that HCC cells acquire the increased MCP expression in a development of HCC and may escape from tumour-specific complement-mediated cytotoxicity.

Expression of alpha-fetoprotein and albumin genes in human hepatocellular carcinomas: limitations in the application of the genes for targeting human hepatocellular carcinoma in gene therapy.

For an approach of gene therapy for hepatocellular carcinoma (HCC), transcriptional regulatory sequence (TRS) of either alpha-fetoprotein (AFP) or albumin has been used for targeting cancer cells. To examine the feasibility of using TRSs of these genes for possible gene therapy of HCCs, the cellular distribution of AFP and albumin gene transcripts was studied in 25 cases of surgically removed human HCCs. AFP gene expression was observed in HCC nodules of 13 cases. The expression in HCC was heterogeneous, and the distribution of the transcripts was mostly sparse and spotty. The higher the serum AFP levels, the larger population of the AFP-expressing HCC cells tended to reflect. In noncancerous liver, a slight AFP expression was found by Northern blot analysis, but the transcripts were not detected in the liver sections. In contrast, albumin expression was found in all HCCs as well as in noncancerous hepatocytes. In HCC, the transcripts for albumin were distributed in cancer cells, and the expression varied with nodules. There were more albumin-expressing cancer cells than the AFP-expressing cells. Albumin expression was retained even in poorly differentiated HCC, although the intensity of the signal was not as strong as in more-differentiated HCCs. Metastatic HCC nodules revealed transcripts for both AFP and albumin genes, and those were clearly recognized in the lung tissue. These results suggest that, for gene therapy for HCCs, neither AFP nor albumin are ideal options for targeting HCC cells. AFP-TRS may be used as a transcriptional regulator in selected cases in which AFP gene expression is observed in the cancer nodules. The serum AFP level appears to be an important indicator in selecting cases. Albumin-TRS in conjunction with retroviral vector might be used in limited cases such as HCCs with no AFP expression. However, careful consideration must be taken, because albumin is constitutively expressed in normal hepatocytes, and AFP-expressing nonmalignant progenitor cells possibly exist.

Proliferating cell nuclear antigen and grade of malignancy in small hepatocellular carcinoma--evaluation in US-guided specimens.

BACKGROUND/AIMS: We investigated the value of the proliferating cell nuclear antigen labeling index ratio (PCNA-LI ratio: PCNA-LI of cancer/PCNA-LI of surrounding non-cancerous liver tissue) to clarify the prognosis of the patients bearing small hepatocellular carcinomas (HCC) less than 20 mm in diameter and treated by percutaneous ethanol injection therapy (PEIT). MATERIAL AND METHODS: Twenty eight HCC patients who had received PEIT were divided into 3 groups. The non-recurrence (NR) group in which no new lesions were observed for at least 18 months after PEIT (13 patients), the early recurrence group (ER) in which lesions recurred within one year (6 patients), and the late recurrence group (LR) in which lesions recurred more than one year after PEIT (9 patients). Immunohistochemical staining of PCNA was done using biopsied specimens. RESULTS: The PCNA-LI ratio in 37 well differentiated, 13 moderately differentiated, 11 poorly differentiated HCC were 1.86 +/- 0.55, 3.33 +/- 0.51, and 4.75 +/- 0.81 (mean +/- SD), respectively. The ratio in ER group (4.57 +/- 0.57) was significantly higher than that in LR (2.04 +/- 0.61) and NR group (1.87 +/- 0.62) and the PCNA-LI ratio tended to correlate with the periods until the development of recurrent lesions in cases of ER group. CONCLUSIONS: These results indicate the PCNA-LI ratio, in conjunction with the histological grade, is a useful marker for evaluating the grade of malignancy, and for predicting the period until recurrence after treatment of small HCC.

Cellular distribution of 92-kd type IV collagenase/gelatinase B in human hepatocellular carcinoma.

To examine the possible involvement of gelatinase B in human hepatocellular carcinoma (HCC), cellular localization of transcripts and protein of gelatinase B were studied by using in situ hybridization and immunohistochemistry. Transcripts for gelatinase B were observed in tumor cells in 22 cases of 27 HCCs and also in dysplastic nodules. However, there was no significant difference in the expression among histological grades of HCC. The expression was mostly homogeneous, but the intensity varied with the nodules. Of 13 cases with capsular invasion, 12 expressed gelatinase B, whereas 10 of 14 without capsular invasion did (P < 0.05). Gelatinase B transcripts were commonly observed in the sinusoidal cells of the hepatic lobules, in mesenchymal cells both in fibrous capsules and around the necrosis, and also in some undefined cells of the portal tracts of noncancerous liver. Localization of gelatinase B protein was mostly similar to but sometimes different from that of the transcripts in cancer nodules. In conclusion, the expression of gelatinase B appears to be an important characteristic of malignant transformation of hepatocytes. The findings suggest that gelatinase B synthesized by cancer cells plays an important role in the growth and invasion of HCC by degrading surrounding extracellular matrices.

Cathepsin B in the growth of colorectal cancer: suppressive effect of leupeptin on the growth of DMH-induced rat colon neoplasm.

Cathepsin B, a thiol protease, has been reported to be involved in cancer progression and metastasis. The suppressive effects of two kinds of protease inhibitors, leupeptin and dietary camostate (FOY-305), on tumorigenesis and progression in 1, 2-dimethylhydrazine (DMH)-induced rat colon neoplasm were examined in relation to tissue cathepsin B activity. Male Donryu rats were treated with leupeptin or FOY-305 during or after the administration of DMH. There were no significant differences in average tumor numbers among all DMH-treated groups. However, the percentage of small tumors was significantly higher in the group in which leupeptin was supplied during DMH administration. This trend was not recognized in the FOY-305-treated groups. The ratio of cathepsin B activity in the tumors to that in the tumor-bearing tissue (T/Tb) was significantly increased with increasing tumor size (P = 0.009). The cathepsin B activity levels in the tumor-bearing mucosa in the groups which received leupeptin or FOY-305 following DMH treatment were both significantly lower than that in the group which received neither protease inhibitor (P = 0.046 and P = 0.0067, respectively). The results obtained indicate that leupeptin may have suppressed tumor growth by lowering the tissue cathepsin B activity.

Cathepsin B in the growth of colorectal cancer: increased activity of cathepsin B in human colorectal cancer.

Cathepsin B, a thiol protease, is involved in cancer metastasis. To clarify the role of cathepsin B in tumor progression in human colorectal cancer, the relationship between its activity, immunohistochemical staining, and clinical tumor progression was investigated. Cathepsin B activity in adenocarcinomas was significantly elevated compared with that in the tumor-bearing tissue. Furthermore, the tumor/tumor-bearing tissue (T/Tb) ratio of the activity was significantly higher than that of colorectal adenoma. Immunohistochemical studies demonstrated intense staining in the cancerous tissue. With respect to the clinical stage of tumors, the activity tended to be higher in tumors that had invaded the serosa or subserosa than in those that invaded the proper muscle. The results suggest that cathepsin B participates in the progression of human colorectal cancer, and its increased expression is a sensitive marker of the differentiation between colorectal adenoma and adenocarcinoma.

Telomere length in human liver diseases.

To determine the role of telomere-mediated gene stability in hepatocarcinogenesis, we examined the telomere length of human liver with or without chronic liver diseases and hepatocellular carcinomas (HCC). The mean telomere restriction fragment (TRF) length of normal liver (n = 13), chronic hepatitis (n = 11), liver cirrhosis (n = 24) and HCC (n = 24) was 7.8 +/- 0.2, 7.1 +/- 0.3, 6.4 +/- 0.2 and 5.2 +/- 0.2 kb, respectively (mean +/- standard error). TRF length decreased with a progression of chronic liver diseases and that in HCC was significantly shorter than that in other chronic liver diseases (p < 0.05). The ratios of TRF length of HCC to that of corresponding surrounding liver of well differentiated (n = 7), moderately differentiated (n = 10) and poorly differentiated (n = 4) HCCs were 0.83 +/- 0.06, 0.75 +/- 0.05 and 0.98 +/- 0.09, respectively. The ratio of poorly differentiated HCC was significantly higher than that of moderately differentiated HCC (p < 0.05). A comparison between the size and telomere length ratio of moderately differentiated HCCs revealed a decrease of the ratio with size until it reached 50 mm in diameter. In contrast, the ratio increased as the size enlarged over 50 mm. These findings suggest that the gene stability of the liver cells mediated by the telomere is reduced as chronic liver disease progresses and that telomerase is activated in poorly differentiated HCC and moderately differentiated HCC over 50 mm in diameter.

Usefulness of indocyanine green injection during ultrasound-guided liver biopsy for the diagnosis of small hepatocellular carcinoma.

To diagnose hepatocellular carcinoma (HCC) functionally and immediately, we examined the usefulness of indocyanine green (ICG) injection during ultrasound-guided liver biopsy. Liver specimens were obtained after intravenous ICG injection by ultrasound-guided biopsy from 251 space-occupying lesions (SOL) in 136 patients. The tissues were immediately examined for ICG uptake using an infrared Vidicon camera and were also subjected to histopathological examinations. Of the 112 ICG-negative biopsy specimens, 105 were histologically diagnosed as HCC, 6 as dysplastic nodules (DN) and 1 as a regenerative nodule (RN). Of the 139 ICG-positive specimens, 18 were diagnosed as HCC, 1 as DN and 120 as RN. The sensitivity of the absence of ICG uptake (SEAIU), the specificity of the absence of ICG uptake (SPAIU), and the positive predictive value of the absence of ICG uptake (PPAIU) for the diagnosis of HCC were 85.3%, 94.5% and 93.8%, respectively. Of the 251 SOLs, 184 were less than 2 cm. SEAIU, SPAIU and PPAIU for the diagnosis of these small HCC were 85.3%, 94.5% and 91.4%, respectively. These results support the reliability of ICG injection during ultrasound-guided liver biopsy to diagnose even small HCC.

Telomerase as a tool for the differential diagnosis of human hepatocellular carcinoma.

BACKGROUND: Telomerase activation is thought to be essential for the immortality of cancer cells. We measured telomerase activity in human liver samples, including hepatocellular carcinoma (HCC), and evaluated this assay as a tool for the diagnosis of HCC using 21-gauge (21-G)-needle biopsy specimens. METHODS: Ninety-four liver samples (27 HCC, 27 liver cirrhosis, 37 chronic hepatitis, and 3 normal liver) that were surgically resected or biopsied with a 12-gauge Silverman needle and 13 HCC samples that were biopsied with a 21-G needle were analyzed for telomerase activation. RESULTS: Eleven of 29 (38%) tumor-bearing liver samples were weakly telomerase-positive, whereas telomerase activity was observed infrequently in nontumor-bearing liver samples (6 of 35; 17%) and in normal liver samples (0 of 3; 0%). The positivity of surgical samples for well differentiated, moderately differentiated, and poorly differentiated HCC was 88% (7 of 8), 87% (13 of 15), and 0% (0 of 2), respectively. In telomerase-positive HCC, 43% (3 of 7) of well differentiated samples were weakly positive, whereas 92% (12 of 13) of moderately differentiated samples were strongly positive. The difference in the tumor sizes and viral marker status did not affect the activity. The telomerase activity of the 21-G-needle biopsied specimens showed no significant difference from that of the surgical samples. The positive incidence of 21-G specimens was 80% (8 of 10) and 100% (2 of 2) in well differentiated HCC and moderately differentiated HCC, respectively. CONCLUSIONS: An incremental positivity of telomerase was observed during hepatocarcinogenesis. The use of this assay in 21-G-needle biopsy specimens may be useful in clinical examination.

Cellular distribution of transcripts for tissue inhibitor of metalloproteinases 1 and 2 in human hepatocellular carcinomas.

The cellular distribution of tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 was studied by using in situ hybridization in surgically removed human hepatocellular carcinomas (HCCs) and cholangiocellular carcinomas (CCCs). The purpose of this study was to characterize the protein involvement of TIMPs in the development of HCCs and CCCs. All HCCs and CCCs expressed TIMPs. The distribution of transcripts for TIMPs in the tumors was mostly homogeneous. Expression of TIMP in cancer cells was more intense than that in the surrounding noncancerous liver (either, cirrhosis, chronic hepatitis, or normal), and expression of TIMP-1 was stronger than that of TIMP-2. Expression of TIMPs varied among HCC nodules, but there was no obvious association between the expression level of TIMPs and differentiation stages or invasiveness of the HCCs. Transcripts for TIMPs were clearly demonstrated in the metastatic HCC nodules in the lung. Expression of TIMP-1 CCC was strong, and small nodules of CCC were recognized in the liver. Immunohistochemical study for TIMP-1 revealed a consistent staining of the TIMP protein with the transcripts. In the peritumoral histologically normal liver, which was not infected with either hepatitis B or C virus, expression of TIMP-1 was found in various cell types, but that of TIMP-2 was weak. Expression of TIMP-1 in hepatocytes revealed clear zonal distribution. These results suggest that TIMPs may act on modulating the matrix/tumor interaction and may play an important role in growth and invasion of HCCs and CCCs. Expression of TIMP-1 can be a marker of HCC metastasis to the lung, and also that of the extent of CCC invasion. Furthermore, the consistent expression of TIMPs in many cell types of the noncancerous liver suggests some unknown functional role that must be clarified.

Apoptosis and proliferation of human hepatocellular carcinoma.

To investigate the contribution of apoptosis, a major mechanism of cell death, in the growth of hepatocellular carcinoma, we analyzed both apoptosis and cell proliferation in human hepatocellular carcinoma. We used the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method and proliferative cell nuclear antigen staining, respectively. Among 21 hepatocellular carcinoma specimens examined, four were well, ten were moderately, and seven were poorly differentiated hepatocellular carcinoma. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells in hepatocellular carcinoma were scattered individually or were sometimes clustered in the tumors. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling indices were 0.35 +/- 0.09, 0.81 +/- 0.29, and 1.9 +/- 0.94 in well, moderately, and poorly differentiated hepatocellular carcinoma, respectively. The proliferative cell nuclear antigen labeling indices were 6.6 +/- 0.9, 13.1 +/- 3.5, and 26.7 +/- 6.3 in hepatocellular carcinoma in the same respective order of differentiation. The differences in both terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling indices and proliferative cell nuclear antigen labeling indices (p < 0.05) were significant between well, moderately and poorly differentiated hepatocellular carcinoma. There was a positive correlation between the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and proliferative cell nuclear antigen labeling indices in hepatocellular carcinoma (r = 0.84, p < 0.001). This study showed that the proliferation rate and the incidence of apoptosis increased as the differentiation grade of hepatocellular carcinoma was lowered, suggesting a rapid turnover of cancer cells in the lower differentiation grades. Apoptosis may thus play an important role in the growth of hepatocellular carcinoma.