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.

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.

Material analysis end-station of the Hyogo-ken beamline at SPring-8.

Plans to construct surface-analysis equipment which will be placed on beamline BL24XU of SPring-8 are presented. There are three experimental hutches in BL24XU, which are available simultaneously by using diamond monochromators as beam splitters. The purpose of the surface-analysis equipment is the simultaneous measurement of fluorescent and diffracted X-rays in grazing-incidence geometry. The instrument is equipped with a solid-state detector (SSD) and a flat position-sensitive proportional counter (PSPC) combined with analysing crystals for X-ray fluorescence (XRF) analysis. A curved PSPC and the goniometer that mounts the SSD used for XRF are also installed for X-ray diffraction. X-ray fluorescence holography and polarized X-ray emission spectroscopy modes are available, so three-dimensional images of atomic configurations and also the anisotropic structure of materials will be studied.