Wolfgang Mikulits

Molecular aspects of epithelial cell plasticity: implications for local tumor invasion and metastasis

Carcinomas arising from epithelial cells represent the most prevalent malignancies in humans, and metastasis is the major cause for the death of carcinoma patients. The breakdown of epithelial cell homeostasis leading to aggressive cancer progression has been correlated with the loss of epithelial characteristics and the acquisition of a migratory phenotype. This phenomenon, referred to as epithelial to mesenchymal transition (EMT), is considered as a crucial event in late stage tumorigenesis. Here we summarize the multitude of EMT models derived from different tissues, and review the diversity of molecular mechanisms contributing to the plasticity of epithelial cells. In particular, the synergism between activation of Ras, provided by the aberrant stimulation of receptor tyrosine kinases, and transforming growth factor (TGF)-beta signaling plays a pivotal role in inducing EMT of various epithelial cell types. Cytokines such as TGF-beta and extracellular matrix molecules are thought to fundamentally contribute to the microenvironmental interaction between stromal and malignant cells, and provide the basis for a broad repertoire of epithelial differentiation. Investigations of EMT tumor models, which represent in vitro correlates to local invasion and metastasis in vivo, facilitate the identification of diagnostic markers for a more accurate and faithful clinical and pathological assessment of epithelial tumors. In addition, the analysis of molecular mechanisms involved in EMT might yield novel therapeutic targets for the specific treatment of aggressive carcinomas.

Immortalized p19ARF null hepatocytes restore liver injury and generate hepatic progenitors after transplantation

Primary hepatocytes are blocked in mitotic activity and well‐defined culture conditions only allow the limited expansion of these cells. Various genetic modifications have therefore been employed to establish immortalized hepatic cell lines, but, unfortunately, proper hepatocyte cultures conducting a faithful hepatic gene expression program and lacking malignancy are hardly available. Here we report the immortalization of primary hepatocytes isolated from p19ARF null mice using the rationale that loss of p19ARF lowers growth‐suppressive functions of p53 and bypasses cellular senescence without losing genetic stability. The established hepatocytes, called MIM, express liver‐specific markers, show a nontumorigenic phenotype, and competence to undergo Fas‐mediated apoptosis. Intrasplenic transplantation of GFP‐expressing parental MIM cells into Fas‐injured livers of SCID mice revealed liver‐reconstituting activity. In the regenerated liver, MIM cells localized in small‐sized clusters and showed presence in structures comparable to canals of Hering, the site of oval cells. Transplantation of MIM‐Bcl‐XL cells, which are protected against apoptosis, and successive Fas‐induced liver damage, enhanced donor‐derived liver repopulation by showing differentiation into cholangiocytes and cells expressing markers characteristic of both fetal hepatocytes and oval cells. In conclusion, these data indicate that long‐term cultivated p19ARF null hepatocytes are capable of generating hepatic progenitor cells during liver restoration, and thus represent a highly valuable tool to study the differentiation repertoire of hepatocytes. (HEPATOLOGY 2004;39:628–634.)

Overexpression of Thymidine Kinase mRNA Eliminates Cell Cycle Regulation of Thymidine Kinase Enzyme Activity

Expression of thymidine kinase (TK) enzyme activity and mRNA is strictly S phase-specific in primary cells. In contrast, DNA tumor virus-transformed cells have enhanced and constitutive levels of TK mRNA during the whole cell cycle. Their TK protein abundance, however, still increases at the G-S transition and stays high throughout G until mitosis. Therefore, post-transcriptional control must account for the decoupling of TK mRNA from protein synthesis in G. To characterize the underlying mechanism, we studied the consequences of TK mRNA abundance on the cell cycle-dependent regulation of TK activity in nontransformed cells. Constitutive as well as conditional human and mouse TK cDNA vectors were stably transfected into mouse fibroblasts, which were subsequently synchronized by centrifugal elutriation. Low constitutive TK mRNA expression still resulted in a fluctuation of TK activity with a pronounced maximum in S phase. This pattern of cell cycle-dependent TK activity variation reflected the one in primary cells but is caused by post-transcriptional control. Increasing overexpression of TK transcripts after hormonal induction compromised this regulation. At the highest constant mRNA levels, regulation of enzyme activity was totally abolished in each phase of the cell cycle. These data indicate that post-transcriptional regulation of TK is tightly coupled to the amount of mRNA; high concentrations apparently titrate a factor(s) required for repressing TK production during G and presumably also G.

New cellular tools reveal complex epithelial–mesenchymal interactions in hepatocarcinogenesis

To enable detailed analyses of cell interactions in tumour development, new epithelial and mesenchymal cell lines were established from human hepatocellular carcinoma by spontaneous outgrowth in culture. We obtained several hepatocarcinoma (HCC)-, B-lymphoblastoid (BLC)-, and myofibroblastoid (MF)-lines from seven cases. In-depth characterisation included cell kinetics, genotype, tumourigenicity, expression of cell-type specific markers, and proteome patterns. Many functions of the cells of origin were found to be preserved. We studied the impact of the mesenchymal lines on hepatocarcinogenesis by in vitro assays. BLC- and MF-supernatants strongly increased the DNA replication of premalignant hepatocytes. The stimulation by MF-lines was mainly attributed to HGF secretion. In HCC-cells, MF-supernatant had only minor effects on cell growth but enhanced migration. MF-lines also stimulated neoangiogenesis through vEGF release. BLC-supernatant dramatically induced death of HCC-cells, which could be largely abrogated by preincubating the supernatant with TNFβ-antiserum. Thus, the new cell lines reveal stage-specific stimulatory and inhibitory interactions between mesenchymal and epithelial tumour cells. In conclusion, the new cell lines provide unique tools to analyse essential components of the complex interplay between the microenvironment and the developing liver cancer, and to identify factors affecting proliferation, migration and death of tumour cells, neoangiogenesis, and outgrowth of additional malignancy.

Mouse thymidine kinase stability in vivo and after in vitro translation

Using a combination of centrifugal elutriation and recultivation of synchronised cell populations we could show that murine thymidine kinase (TK) is rapidly degraded during mitosis in polyoma virus-transformed mouse fibroblasts, in parallel to the time-course for loss of cyclin A. Transformation is no prerequisite for the instability phenotype since artificial overexpression of TK under the control of a constitutive promoter in normal mouse fibroblasts also resulted in rapid turnover of TK during mitosis. The decay of TK protein could be partially mimicked in vitro with enzymatically active protein translated in a rabbit reticulocyte lysate: full length polypeptide was lost slightly more rapidly in the presence of G2/M cytosolic extracts than with G1/S preparations. In addition, an enzymatically active C-terminal truncation of 37 amino acids at Gln-196 was completely stable under the conditions tested, confining the instability domain between residues 196 to 233. These experiments also indicated the border for intact TK since translation products up to Tyr-189 or less were completely inactive. This was also confirmed by a mutant TK protein from mouse F9tk- teratocarcinoma cells which harboured a similar deletion.