Conditional Immortalization

The precise inactivation of a minimal number of genes may only in some cases improve the applicability of designer cell lines in drug testing. In general, re-entry of a resting, terminally differentiated cell into proliferation will inevitably modify expression patterns. This raises the question of whether immortalization can be reversed after the required cell mass has been generated.

Temperature-dependent inactivation of mutant (tsA58) large T antigen represents the classical approach. Cells are grown at the permissive temperature of 32 °C and shifted to 39 °C or even 40 °C for redifferentiation. This approach has been applied to multiple tissues, ranging from thyroid epithelium [38] to osteocytes [39], glomerular epithelial cells [40] and (rat) hepatocytes [41].

The high restrictive temperature limits the value of the approach to generate "normal" cells under physiological conditions. Therefore, tight transcription regulation, for example by the Tet system [42] or excision by site-specific recombi-nases Cre/loxP and the Flp/FRT, yield better results [43-45]. Care must be taken to limit the number of replication cycles, since mutations accumulate with extended passaging due to inactivation of p53-dependent DNA repair control [41]. Moreover, when applied to E7/E1A, chromosomal changes are likely to accumulate because both genes interact with the Ran GTPase uncoupling the centrosome cycle from the cell cycle [46].

Immortal cell lines in some way resemble stem cells from the source tissue. Removal of the immortalizing gene function is just one part on the path to redifferentiation; just as for stem cells, medium supplements or scaffolds that allow a three-dimensional, tissue-like structure may be required to revert an immortal line. Increasing knowledge concerning the molecular events leading to immortalization has helped substantially to fine tune these approaches and to provide a large number of cell lines that may serve as models in the drug screening process. For one highly relevant model of drug-related toxicity, the kidney (proximal tubular epithelium), a human cell line (HK-2) immortalized using HPVE6/E7 [47], has found widespread application (over 60 publications). However, for the most relevant target for drug toxicity - the human liver - no suitable model has yet become available.

As fascinating as these novel substrates are in theory, a reverted previously immortal cell is an artificial product that needs to be compared to the natural tissue in preliminary assays before it can be applied in large screens. While some physiological responses are faithfully reproduced, others may be disturbed, and it is important to keep these limitations in mind.

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