In vivo expression of soluble Fas and FAP-1: possible mechanisms of Fas resistance in human hepatoblastomas.

Many tumour cells express both Fas and its ligand (FasL) on their surface and it has remained a mystery why such cells do not simply kill themselves. It remains to be determined whether Fas and FasL are expressed in human hepatoblastomas and if so, what is responsible for the possible Fas resistance of these tumours. In this study, the expression of Fas and FasL was examined in 23 cases of human hepatoblastoma by immunohistochemical staining. To elucidate possible Fas resistance in hepatoblastomas, Fas-resistance pathways including the expression of bcl-2 and Fas-associated phosphatase-1 (FAP-1), and the expression of soluble Fas (sFas) mRNA, were analysed by immunohistochemistry and in situ reverse transcription-polymerase chain reaction (in situ RT-PCR). Fas gene mutation in the death domain was also examined. Fas and FasL were expressed in all hepatoblastomas analysed. Twenty (87 per cent) and 18 (78 per cent) cases of hepatoblastoma were positive for sFas mRNA and FAP-1, respectively, but none of the hepatoblastomas expressed bcl-2. Mutation in the death domain of the Fas gene was not found in hepatoblastomas. Taken together, these findings demonstrated that Fas, a death receptor, and its ligand are co-expressed in hepatoblastomas in vivo, but some inhibitors of Fas-mediated apoptosis are also expressed in these tumours. These results suggest that it is probably due to the action of inhibitory molecules of the Fas pathway that the tumour cells of hepatoblastomas do not kill themselves in an autocrine-driven cycle and that in this manner hepatoblastomas avoid apoptosis.

Somatic mutations in the kinase domain of the Met/hepatocyte growth factor receptor gene in childhood hepatocellular carcinomas.

The MET protooncogene encodes a transmembrane tyrosine kinase identified as the receptor of a polypeptide known as hepatocyte growth factor/scatter factor. We performed PCR-based single-strand conformational polymorphism and sequencing analysis of the tyrosine kinase domain of the MET gene (exon 15-19) in 75 primary liver cancers. Three missense mutations were detected exclusively in 10 childhood hepatocellular carcinomas (HCCs), while no mutations were detected in 16 adult HCCs, 21 cholangiocarcinomas, or 28 hepatoblastomas. The extremely short incubation period from hepatitis B virus infection to the genesis of childhood HCC as compared with the adult HCC suggests that there may be an additional mechanism that accelerates the carcinogenesis of childhood HCC. Our results indicate that mutations of the tyrosine kinase domain of the MET gene may be involved in the acceleration of the carcinogenesis in childhood HCC.

Fas ligand is expressed in normal skin and in some cutaneous malignancies.

Fas, a cell surface receptor and member of the tumour necrosis factor receptor superfamily, induces apoptosis upon oligomerization by its ligand (Fas ligand: FasL). Detailed studies have revealed that Fas is broadly expressed in normal human tissues, but relatively little is known about the range of cell types capable of expressing FasL. The aim of this study was to determine the in vivo patterns of expression of Fas and FasL in human skin tissues. Immunohistochemistry was performed using paraffin-embedded samples of normal and neoplastic skin tissues. In normal skin, FasL was expressed in the epidermis, sebaceous glands, sweat glands and outer root sheath of the hair. In squamous cell carcinomas (SCC), all cases analysed expressed FasL at high levels, whereas 60% of basal cell carcinomas (BCC) were positive for FasL. Expression of Fas in normal skin was observed in the basal and spinous layers of the epidermis, the outer root sheath of the hair, and the sebaceous glands. Expression of Fas was observed in all the SCC tested and none of the BCC tested. Expression of FasL by normal cells and tumour cells in skin tissue, demonstrated for the first time in the present study, may provide an important clue to understanding skin physiology, and immune evasion of skin tumours.

Frequent somatic mutations in serine/threonine kinase 11/Peutz-Jeghers syndrome gene in left-sided colon cancer.

We analyzed somatic mutation and loss of heterozygosity (LOH) in the serine/threonine kinase 11 (STK11)/Peutz-Jeghers syndrome gene in 49 colorectal tumors in three different stages of a dysplasia-carcinoma sequence. We detected LOH in 10 of 19 (52.6%) informative colorectal cancers at loci D19S886 and/or D19S883, but no LOH was observed in 25 informative adenomas. We detected a total of 9 somatic mutations [7 of 13 (53.8%) left-sided colon cancers and 2 of 7 (28.6%) left-sided adenomas with high-grade dysplasia], but no mutations were detected in right-sided colon tumors. Of the nine mutations, one was a frameshift mutation (the same mutation detected in Peutz-Jeghers syndrome family previously), and the other eight were missense mutations. This results indicate that STK11 is a tumor suppressor gene and that genetic changes of STK11 play an important role in left-sided colon cancer carcinogenesis.

Detection of soluble Fas mRNA using in situ reverse transcription-polymerase chain reaction.

Fas protein (Fas) is known to induce cell death by apoptosis in susceptible cells. Alternative splicing of the Fas gene produces soluble Fas protein (sFas), which is considered to block the function of Fas. The serum level of sFas is elevated in patients with various malignancies in a manner reflective of disease stage and tumor burden, but the precise cellular origin of sFas in vivo has not yet been clarified. To identify the cells that synthesize sFas mRNA on histologic specimens, we applied in situ reverse transcription-polymerase chain reaction (in situ RT-PCR) in 11 cases of gastric adenocarcinoma/metastatic lymph node. Furthermore, we studied the distribution of Fas using immunohistochemistry and Fas mRNA using in situ RT-PCR. In all primary tumors and 10 of 11 metastatic tumors, tumor cells expressed both Fas- and sFas mRNA. Lymphocytes infiltrated in the tumor tissues and the lymph nodes also revealed both mRNA signals. A clear correlation between the tissue distribution for Fas and its mRNA was also observed. These observations demonstrated that solid tumors in vivo can synthesize sFas mRNA and suggest that tumor cells are responsible in part for elevated sFas in human malignancies. However, the additional expression of sFas mRNA in tissue lymphocytes indicates the complex regulatory mechanisms of Fas-mediated apoptosis pathway in tumor pathogenesis and host defense. We also demonstrated that in situ RT-PCR can be a suitable method for in situ detection of alternatively spliced mRNA.

A distinct region of chromosome 19p13.3 associated with the sporadic form of adenoma malignum of the uterine cervix.

Adenoma malignum (AM) is known to be one of the malignant tumors that is commonly associated with Peutz-Jeghers syndrome. Recently, the genetic locus of Peutz-Jeghers syndrome was mapped to the telomeric region of chromosome 19p. We analyzed nine sporadic cases of AM with high-density loss of heterozygosity to study the region of chromosome 19p13.2-13.3 using eight microsatellite markers. Our deletion mapping data revealed a distinct region with 100% loss of heterozygosity frequency at marker D19S216. This result indicates that a putative tumor suppressor gene for AM is located at D19S216 on chromosomal band 19p13.3 and plays an important role in AM tumorigenesis.