Figure 2. Barriers to Human Cellular Immortalization. Normal passage of cells is halted at MI unless this barrier is bypassed by p53 and RB inactivation or hTERT expression. These cells can then continue dividing until their telomeres become critically short at M2. hTERT expression or ALT allows telomere length stabilization and cellular immortalization.
senescence is still unclear, although there are a variety of stimuli that have a role in this process (28).
Pre-senescent cells can be experimentally manipulated to bypass replicative senescence through ectopic expression of certain genes. Expression of hTERT, the catalytic subunit of telomerase is capable of bestowing some but not all primary cells with immortality (29,30). Another mechanism of bypassing this first proliferative barrier is through simultaneous abrogation of the P53 tumor suppressor and retinoblastoma (RB) pathways (28). Expression of viral oncoproteins, such as SV40 large T antigen (31) or human papillomavirus E6 and E7 oncoproteins (32), which bind to and inactivate p53 and RB (33), respectively, offer experimental methods of achieving this dual inactivation.
Cells that lack telomerase overexpression but that express the above mentioned viral oncoproteins, may then undergo another 10-20 population doublings before they encounter mortality stage 2 (M2) or crisis. Here the vast majority of cells have short telomeres (34), display karyotypic abnormalities (35), and die by apoptosis (36). Since in culture telomeres shorten by 50-100 base pairs during each cell replication (37), ongoing passage allows telomeres to shorten to a critical length. This results in an inability to protect the ends of chromosomes, leading to genomic instability and triggering crisis (38).
Rare variants, approximately 1 in 10 million cells, emerge from crisis, and have infinite proliferative capability (31). These cells typically exhibit stable telomere lengths and express the hTERT gene with preserved activity (38), which is felt to be expressed at low levels in normal cells (39). These findings have been corroborated by observations in post-senescent, pre-crisis cells that avoid crisis and proliferate indefinitely after transduction with hTERT (40-42). However, a subgroup of cells may become immortal without significant hTERT or telomerase expression (43,44). These cells have a separate mechanism of telomere length maintenance, termed alternative lengthening of telomeres (ALT), which likely involves recombination (45).
The observations that suggested that human cell immortalization is more complex than rodent cell immortalization also complicated attempts for experimental transformation of human cells. From this set of observations, Sager and her colleagues postulated that the senescence program is a barrier to cancer development (46,47). Recent work, however, has begun to identify combinations of genetic alterations that suffice to confer human cellular transformation.
Thus, specifically targeting each of the barriers of immortalization by introduction of the SV40 Early Region, which encodes the large T oncoprotein, in combination with the hTERT gene into normal human fibroblasts and kidney cells suffice for immortalization (48,49). Since the SV40 Early Region also encodes for small t onco-protein, subsequent transduction with oncogenic RAS results in the ability to develop tumors in immunocompromised mice, hence transformation. Additional studies have revealed this combination of genetic alterations to be sufficient to transform multiple cell types, including cells of mammary (50), lung (51), prostate, ovarian, mesothelial (52), endothelial, and neuroectodermal (53) origin. Thus, it is necessary to understand the roles of these basic genetic elements involved in transformation in regards to the critical pathways that they effect. For example, the large T oncoprotein may functionally inactivate the p53 and RB pathways, but the inactivation of these two pathways may in sum not equal the effects of the oncoprotein alone, as there may be additional functions gained with large T. Thus, a myriad of other genetic mutations that lead down similar or parallel paths may also bestow specific "acquired capabilities," leading to similar functional endpoints or the neoplastic phenotype.
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