Cell Immortalization: In Vivo Molecular Bases and In Vitro Techniques for Obtention

Abstract

Somatic human cells can divide a finite number of times, a phenomenon known as the Hayflick limit. It is based on the progressive erosion of the telomeric ends each time the cell completes a replicative cycle. Given this problem, researchers need cell lines that do not enter the senescence phase after a certain number of divisions. In this way, more lasting studies can be carried out over time and avoid the tedious work involved in performing cell passes to fresh media. However, some cells have a high replicative potential, such as embryonic stem cells and cancer cells. To accomplish this, these cells express the enzyme telomerase or activate the mechanisms of alternative telomere elongation, which favors the maintenance of the length of their stable telomeres. Researchers have been able to develop cell immortalization technology by studying the cellular and molecular bases of both mechanisms and the genes involved in the control of the cell cycle. Through it, cells with infinite replicative capacity are obtained. To obtain them, viral oncogenes/oncoproteins, myc genes, ectopic expression of telomerase, and the manipulation of genes that regulate the cell cycle, such as p53 and Rb, have been used.

Keywords: Hayflick limit; alternative telomere elongation; immortalization; telomerase; telomeres.

Figure 1

Structure of telomeres in humans. (a) Schematic representation of telomeric DNA in humans, composed of tandem repeats of the hexanucleotide sequence TTAGGG (G chain) and its complementary sequence in chain C. The G chain can extend 3–12kb in length (G-strand overhang). (b) Adjacent to the telomeres are the sub telomeric regions, also rich in repetitive DNA. Schematic representation of the shelterin or telosome complex. The TRF1 and TRF2 proteins have a binding domain for double-stranded DNA, whereas POT1 can only bind to single-stranded DNA. RAP1 exerts its function on the telomere through a TRF2-binding domain. TIN2 has binding domains for both TRF1 and TRF2, and through another domain, it binds to the TPP1-POT1 complex.

Figure 2

Telomerase activity. Schematic representation of telomere replication by telomerase, as well as the catalytic subunit with reverse transcriptase action (TERT) and the subunit containing the RNA template for telomere replication (Terc). Once the template RNA hybridizes with the telomeric DNA sequence of the G chain, at the 3′-OH end, the telomere polymerization process begins. Once the hexanucleotide sequence is added, the enzyme performs a translocation movement, and telomere elongation continues.

Figure 3

BIR mechanism. Schematic representation of the telomere replication process induced by dsDNA damage. When the dsDNA is damaged, repair mechanisms are activated at the ends of the telomeres. The damaged strand invades the telomeric region of the donor and serves as a primer for the replicative process initiation.

Figure 4

Role of p53 and Rb proteins in senescence and immortalization. Reprinted with modifications from [66] under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0, accessed on 25 January 2023).