Inactivation of Rb and p53 Pathways

Telomere erosion is an important - but not the only - block against immortalization in human cells. In the culture dish most cell types (with the exception offibroblasts) cease division within few population doublings, irrespective of telomere length. This arrest is termed M0 [31], and is observed in human and mouse cells, although the underlying mechanism of the two species is different.

Multiple stress factors associated with the cultivation of primary cells in vitro result in activation of the CDKN2A locus in both systems. The locus codes for independent genes via alternative spicing, P16 INK4a and ARF. These two gene products link the main pathways in cell cycle control and tumor protection: P16 (INK4a) inhibits cyclin-dependent kinases CDK4 and CDK6 to maintain the Rb protein in a hypophosphorylated state.

As phosphorylation of Rb is required for cell cycle progression, the expression of P16 (INK4a) therefore locks cells into G1 of the cell cycle. The ARF protein prevents p53 degradation and thus is responsible for p53-dependent halt of the cell cycle and induction of apoptosis.

Although p16 and ARF genes are induced simultaneously in mouse cells, it is only the activation of the ARF/p53 axis that prevents immortalization in mice. In contrast, mainly p16 is activated in human cells. However, even in the presence of active telomerase it is not sufficient only to inhibit p16: deactivation of p16 causes cell cycle progression as Rb is allowed to be phosphorylated. This in turn triggers release ofthe pleiotropic E2F transcription factor which is normally sequestered by Rb. The release of E2F induces cell cycle progression and activation of ARF, and this leads to elevated levels of p53. As p53 accumulates, cell cycle progression is halted, and if p53 activity increases further then apoptosis is induced. In summary, the immortalization of human cells requires not only telomerase expression but also inactivation of two additional pathways, Rb and p53.

As a logical consequence, viral oncogenes from small DNA tumor viruses known to inactivate both Rb and p53 pathways are frequently employed in immortalization protocols, the most popular of which is SV40 large T antigen. Most human cells can be immortalized with SV40 large T antigen in the presence of telomerase, including keratinocytes, endothelial cells, neuronal cells, and even hepatocytes. It is important to use the spliced T antigen gene instead of the complete SV40 early region, which also encodes small T antigen because its coexpression induces full transformation via interaction with protein phosphatase 2A [32].

Instead ofSV40 large T antigen, E6 and E7 genes from human high-risk papillomaviruses (HPV16 and 18) may be used, which interact separately with either p53 or Rb, respectively. The genes of the E1 region from human nontumorigenic C group adenoviruses represent yet another alternative. In addition to E1A, which binds to and inactivates Rb and E1B 55k which converts p53 from a transcriptional activator to a repressor, a third protein is coexpressed: the E1B 19k protein is an analogue of Bcl2 and blocks cytochrome c release from the mitochondrial membrane, thus preventing apoptosis.

The oncoproteins from SV40, HPV and adenovirus fulfill complex functions not limited to the binding of Rb and p53. In fact, they act as master regulators of the viral life cycle and prepare the cell metabolism for virus replication. It is therefore not surprising that the interaction with additional targets will modulate the protein's capacity to immortalize or transform. For instance, the effects of SV40 T antigens in transgenic mice range from hyperplasia to invasive carcinoma accompanied by metastasis, depending on the tissue in which they are expressed [33]. HPV E6 and E7 genes efficiently immortalize keratinocyte epithelial and endothelial cells, while adenovirus genes transform neuronal cells best. The generation of HEK293 (a cell line used extensively in biotechnology applications) illustrates this phenomenon: starting from embryonic kidney a vast number of transfections resulted in a single cell line (HEK293) with clear neuronal characteristics such as expression of p75NGFR ([34] and own data).

In this regard, we generated cell lines from fetal brain, liver and mesenchymal material using adenovirus type 5 E1 genes. The expression pattern of the resulting cell lines deviate from that of their respective ancestors (see Fig. 7.1). Depending on the immortalizing gene, these changes may either eliminate or preserve a tissue-specific pattern. The E1 genes were found to direct an immortal cell towards epithelial characteristics: vimentin, a mesenchymal marker strongly expressed in the primary source, is gradually lost during immortalization while EpCAM, an epithelial marker, is only present in the E1 cell line. In contrast, endothelial differentiation is well supported by E1 [35], making it a suitable tool to derive functional endothelial cell lines.

The pleiotropic effects of the viral oncoproteins may even overcome the need to co-introduce teleomerase into human cells. Myc, a downstream target of Rb, strongly activates the telomerase promoter [36]. Although E1A does not bind DNA directly, it participates in transcription complexes and also activates tert. However, transactivation depends on the accessibility of the tert-locus. This explains why embryonic tissue enriched with stem cells is better suited in immortalization approaches that do not require exogenous tert: although already depressed, the tert promoter is not yet extensively methylated and can therefore be more easily reactivated. Furthermore, E1A, T antigen and HPV E6 also interact with p300, a histone acetylase which is part of the polymerase II complex and induces or fixates global changes in gene expression.

In order to preserve features of the differentiated cell, one should limit the interference with cell metabolism to required pathways. While such attempts have been made, for example by dissecting functions in SV40 T antigen [37], a specific block of RB or p53 pathways by small interfering RNA has not yet found broad application, perhaps because the role of additional pathways is underestimated.

Cytokeratin

Cytokeratin

Fig. 7.1 Immortalization with E1A/E1B induces changes in the expression pattern of tissue-specific markers.

(a) Maintained expression of cytokeratin in primary human mesenchymal cells after immortalization with adenovirus E1.

(b) Gain of EpCAM marker expression.

(c) Loss of vimentin expression.

The primary cells are on the left side of each panel, and immortalized cells on the right side.

Fig. 7.1 Immortalization with E1A/E1B induces changes in the expression pattern of tissue-specific markers.

(a) Maintained expression of cytokeratin in primary human mesenchymal cells after immortalization with adenovirus E1.

(b) Gain of EpCAM marker expression.

(c) Loss of vimentin expression.

The primary cells are on the left side of each panel, and immortalized cells on the right side.

0 0

Post a comment