The ends of linear eukaryotic chromosomes contain specialized structures called telomeres.
Human telomeres consist of tandem repetitive arrays of the hexameric sequence TTAGGG, with overall telomere sizes ranging from ~15 kb at birth to sometimes 55 kb in chronic disease states.
The telomeric repeats help maintain chromosomal integrity and provide a buffer of potentially expendable DNA.
The ends of telomeres are protected and regulated by telomere-binding proteins and form a special lariat-like structure called the t-loop.
This packaging or protective cap at the end of linear chromosomes is thought to mask telomeres from being recognized as broken or damaged DNA, thus protecting chromosome termini from degradation, recombination and end-joining reactions.
The inability of DNA polymerase to replicate the end of the chromosome during lagging strand synthesis ("end replication problem") coupled with possible processing events in both leading and lagging daughters, results in the losses of telomeric repeats each time a cell divides and ultimately leads to replicative senescence.
The ability to bypass replicative senescence is thought to be one critical rate-limiting step in the evolution of most malignancies.
While there is substantial correlative evidence that there is telomere attrition in pre-cancerous tissues, the direct evidence that most pre-cancerous cells senesce, and that senescence is a potent tumor suppressor pathway, remains elusive.
Most malignant tumors must have a mechanism for bypassing senescence to have the unlimited proliferative capacity that appears to be required for advanced cancers.
The loss of cellcycle checkpoint pathways leads to an extended lifespan but continued telomere losses.
This eventually leads to crisis or the M2 stage of replicative senescence.
To escape M2, a rare human cell (about 1 in 10 million) can reactivate or upregulate telomerase activity, even though in certain cancer types up-regulation of telomerase can occur at an earlier stage.
Even more rarely, a cell may engage an alternative to telomerase for maintaining telomeres that appears to involve DNA recombination between telomere sister chromatids.
Telomerase is a cellular ribonucleoprotein enzyme responsible for adding telomeric repeats onto the 3' ends of chromosomes.
It has two major components (protein and RNA): an enzymatic human telomerase reverse transcriptase catalytic subunit, hTERT, and an RNA component (hTR or hTERC).
Telomerase uses its integral RNA component (which contains an 11-bp sequence complementary to the telomeric single stranded overhang) as a template in order to synthesize telomeric DNA (TTAGGG)n, directly onto the ends of chromosomes.
After adding six bases, the enzyme pauses while it repositions (translocates) the template RNA for the synthesis of the next 6 bp repeat (i.e. telomerase is processive).
This extension of the 30 DNA template eventually permits additional replication of the C-rich strand, thus compensating for the end-replication problem.
The enzyme is expressed in embryonic cells but the hTERT gene undergoes silencing and the enzyme activity is repressed.
Telomerase is present in adult male germline cells, but is undetectable in most normal somatic cells except for proliferative cells of renewal tissues where there is regulated telomerase activity (e.g. hematopoietic proliferating stem-like cells, activated lymphocytes, proliferative transit amplifying cells of the epidermis, proliferative endometrium and intestinal crypt cells).
In normal somatic cells, even including stem-like cells expressing telomerase, progressive telomere shortening is observed, eventually leading to greatly shortened telomeres and to a limited ability to continue to divide.
This implies that the functional telomerase activity in these stem-like cells may be enough to slow but not prevent telomere shortening.
Direct evidence linking telomere shortening to replicative senescence can be demonstrated by producing telomerase activity in telomerase-negative cells following the introduction of only the hTERT catalytic component (normal cells constitutively express the RNA component of telomerase).
Normal human cells stably expressing transfected telomerase can divide indefinitely, providing direct evidence that telomere shortening controls replicative senescence.
Furthermore, elongating telomeres with telomerase and then excising the exogenous gene results in a greatly extended lifespan, demonstrating that it is the length of the telomere rather than telomerase itself that is responsible for the proliferative limits.
The introduction of hTERT either before M1 or in between M1 and M2 results in direct immortalization, thus demonstrating the importance of telomeres in both stages of replicative senescence (see Figure 1).
Cells with introduced telomerase maintain a normal chromosome complement for a considerable period and continue to grow in a normal manner.
These observations provide direct evidence for the hypothesis that telomere length determines the proliferative capacity of human cells.
The ectopic expression of the catalytic subunit of hTERT results in immortalization of human cells if telomeres are rate-limiting for continued cell proliferation.
Telomeres are thus important in both senescence (M1) and crisis (M2) as hTERT introduction either before M1 or after M1 results in cell immortalization.
If the introduction of hTERT does not result in immortalization this reflects another type of growth arrest that is telomere-independent and is likely to reflect inadequate culture conditions leading to STASIS or what has been termed premature senescence or culture shock.