Telomerase: Target of Immortality
An important step in oncogenesis is the establishment of immortality. Mortality is built in to somatic cells through a number of growth barriers, which includes the senescence pathway. Normal somatic cells undergo a limited number of cell divisions and then senesce. While many aspects of this pathway are not well understood, there is one clear player in the process, telomeres. A simple repeated DNA sequence of TTAGGG is found at the ends of our linear chromosomes called telomeres. The telomere protects the ends of chromosomes keeping them from recombining with each other. It also serves as a buffer between the requisite genes in each chromosome and the natural erosion of chromosome ends that occurs with each round of DNA replication. Human telomeres shorten about 50 basepairs with each cell division. A threshold length of telomere is required for a cell's admittance to the next cell division. There is still substantial length to the telomeres at the point cells reach this threshold, well before exposing any unprotected chromosome end. Cells senesce once they have shortened their telomeres to this threshold point. The precise signaling process that occurs is the target of extensive investigation. Cancer cells must evade or overcome this natural growth barrier.
Escape From Senescence
There are two independent events that frequently occur allowing cells to evade or overcome senescence, mutation of p53 and the activation of telomerase. p53 mutation is one step in the transformation of cells promoting cells to undergo unregulated cell growth. It is also a step in the evasion of senescence in which cells no longer respond to the senescence signals and thus continue to grow. However, telomeres continue to shorten with each cell division past the senescence barrier. Because all cells, even cancer cells, require telomeres, this evasion is not a permanent solution to becoming immortal, but only a stalling mechanism. Once these cells loose all telomere sequence or the telomeres become too short to function properly, the cells reach another growth barrier, called crisis.
Immortality At Last
The most frequent event that allows cells to maintain their telomeres and continue to grow past crisis is the activation of telomerase. 90% of human cancers express telomerase while normal human cells express essentially no telomerase. Telomerase is expressed in germline cells (ie. they are immortal) but is turned off in somatic cells during embryonic development. The cancer specificity of telomerase and its apparent requirement for immortality makes it a viable candidate for therapeutic targeting to treat cancer.
This enzyme is unique in that it is the only known reverse transcriptase endogenous to mammalian cells. Telomerase synthesizes the telomeric sequence TTAGGG using an RNA template within the telomerase complex. The mechanism of telomere synthesis involves telomerase first recognizing the 3' overhanging telomeric sequence that exists at the chromosome ends. The telomerase RNA template sequence basepairs with the terminal TTAGGG repeat to initiate elongation of the 3' DNA end. The RNA template has only 11 bases that match the TTAGGG repeat sequence, such that only one repeat of the sequence can be added in a single elongation. Synthesis terminates with the circularly permuted sequence GGTTAG. Telomerase can continue to synthesize telomeric repeats on the same DNA strand by unwinding the DNA from the DNA-RNA hybrid, holding the DNA end while the RNA slides down 6 bases to allow proper alignment and basepairing. Telomerase continues elongation of the telomere sequence adding DNA in 6-base units. An assay for telomerase activity reveals its ability to add the 6-base repeats, resulting in a 6-base ladder of DNA products when electrophoresed on a gel.
Telomerase Inhibitors And Their Effects
There have been numerous strategies to blocking telomerase leading to a variety of intriguing agents of potentially therapeutic value. Most agents fall into the category of substrate analogs. The nucleoside analogs are the most straightforward. AZT, known for its inhibition properties of the HIV reverse transcriptase, can block telomerase, though it is not a particularly potent inhibitor. Typical concentrations obtained in HIV patients would be insufficient to have significant effect on telomerase. Other more potent nucleoside analogs have also been identified and investigated for their ability to block telomerase, reverse immortality and make cancer cells mortal. The inhibition of telomerase in cancer cell lines grown in culture appears to have the desired effect initially. Telomeres begin to erode or shorten and approach the point where telomere loss would result in blocking cell growth. However, the cells eventually curtail the telomere erosion and the telomeres stabilize while telomerase appears to still be blocked. The ability to maintain telomeres without telomerase has been associated with another pathway for telomere elongation independent of telomerase (the ALT pathway). Other inhibitors of telomerase-mediated telomere synthesis, identified by high-throughput screening assays of chemical libraries, also show similar effects on cancer cells. Therefore, the bad news is that mere inhibition of telomere synthesis by telomerase may not ultimately result in the desired outcome for treating cancer.
The good news is that there are experiments that suggest that telomerase serves another role in addition to synthesizing telomeres that is vital to cancer cells. Targeting this specific property of telomerase or eliminating telomerase all together represents another therapeutic strategy. Telomerase is speculated to provide a telomere capping activity through its ability to bind the telomeric terminus. Thus, blocking this interaction, as opposed to blocking synthesis, could uncap telomeres resulting in telomere dysfunction and crisis. One type of telomerase inhibitor that is capable of blocking telomerase binding to telomeres is an oligonucleotide that competes with the endogenous telomeric termini and competes for the template region of the telomerase RNA. This oligonucleotide functions as an antisense to the RNA and potentially can block the RNA from assembling into the telomerase complex or it can occupy the template region in the telomerase complex preventing interaction with the telomeres. These types of telomerase inhibitors have shown some success in blocking telomerase and resulting in cells entering crisis.
A third strategy for targeting telomerase for treating cancer does not strive to block telomerase but to use the telomerase activity to kill the cell. Nucleoside analogs are being designed that are good substrates for telomerase and can be incorporated into telomeric sequence. These nucleosides would be designed to alter the telomere's ability to form the complexes needed for protecting the chromosome ends. One of these complexes is called the quadruplex. The quadruplex and quadruplex-interactive agents will be a topic of discussion at a later date.