Frequent genomic imbalances suggest commonly altered tumour genes in human hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is one of the most frequent-occurring malignant tumours worldwide, but molecular changes of tumour DNA, with the exception of viral integrations and p53 mutations, are poorly understood. In order to search for common macro-imbalances of genomic tumour DNA, 21 HCCs and 3 HCC-cell lines were characterized by comparative genomic hybridization (CGH), subsequent database analyses and in selected cases by fluorescence in situ hybridization (FISH). Chromosomal subregions of 1q, 8q, 17q and 20q showed frequent gains of genomic material, while losses were most prevalent in subregions of 4q, 6q, 13q and 16q. Deleted regions encompass tumour suppressor genes, like RB-1 and the cadherin gene cluster, some of them previously identified as potential target genes in HCC development. Several potential growth- or transformation-promoting genes located in chromosomal subregions showed frequent gains of genomic material. The present study provides a basis for further genomic and expression analyses in HCCs and in addition suggests chromosome 4q to carry a so far unidentified tumour suppressor gene relevant for HCC development.

HOPE fixation: a novel fixing method and paraffin-embedding technique for human soft tissues.

We have developed a novel method for tissue fixation, including subsequent paraffin-embedding and sectioning, that allows the complete pathological analysis of all types of human soft tissues. Furthermore, it maintains additional positive features relevant to immunohistochemistry and molecular pathology. The so-called HOPE-technique (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) comprises a protection-solution with an organic buffer, acetone as the only dehydrating agent, and pure paraffin of 52-54 degrees C melting temperature. Although the exact mechanism of protection has still to be elucidated, it seems rather unlikely that chemical bindings occur during the whole process of fixation, which is described and compared with the standard formalin-paraffin technique. Essentially, HOPE-fixed sections show formalin-like morphology. However, the sections are somewhat difficult to handle because of their fragility. This is due to the absence of any type of protein cross-linking and the dynamic processes of immersion and outflow of the HOPE protection solution. HOPE-fixed sections provide an excellent preservation of proteins and antigenic structures for differential analysis by immunohistochemical and/or enzyme histochemical techniques. However, their most remarkable feature is the extremely low degradation of nucleic acids (DNA and RNA) combined with good results obtained by in situ hybridization techniques. In conclusion, HOPE fixation may become a valuable additional tool in modern pathology.

Establishment and characterization of a nontumorigenic cell line derived from a human hepatocellular adenoma expressing hepatocyte-specific markers.

In the present study the establishment and characterization of a nontumorigenic liver epithelial cell line (HACL-1) derived from a human hepatocellular adenoma is described. The HACL-1 cells have a finite life span (i.e., they proliferate for a period of 2 months and then senesce), show cell-cell contact inhibition, do not grow in soft agar, are not tumorigenic when injected in nude mice, and possess a normal diploid karyotype. The cultured cells resemble hepatocytes, but exhibit some features of dedifferentiation. At the ultrastructural level the cells are endowed with round or oval nuclei, abundant cytoplasmic organelles, and varying amounts of glycogen. The rough endoplasmic reticulum is disorganized, while peroxisomes and matrix granules within mitochondria are lacking. HACL-1 cells are cytokeratin 18-positive as well as (transiently) albumin- and alpha-fetoprotein-positive, but do not express cytokeratin 19. Furthermore, no mutations were observed in exons 5-8 of the tumor suppressor gene p53. Taken together these results show that HACL-1 cells are nontumorigenic proliferating liver epithelial cells, which might prove to be of great value in future studies on diverse aspects of human liver cell biology and carcinogenesis.