The objective of this literature review project is to investigate current therapeutics involved in the treatment of cancer, particularly telomerase inhibitors. One current hypothesis regarding cancer growth is that it requires activation of telomerase to replicate. One way that cancer cell growth may be controlled is by inhibiting telomerase, an enzyme involved in shortening the DNA telomeres. This project reviews several clinical trials which show a promising telomerase inhibitor that is specific to cancer cells: 2’-O-methoxyethyl oligonucleotide (MOE), which binds to the telomerase RNA template, hTR. Certain telomerase inhibitors are more specific towards the telomerases that extend the telomere oligonucleotides on the oncogenes that control the cell cycle, such as the Ras and p53 genes. Currently, the amount of chemotherapy that is given to a cancer patient is limited by the toxic side effects of the chemotherapy. The current research being reviewed is aimed at inhibiting telomerase which is specific to cancer cells. This in effect will kill the cancer cells while having little or no effect on the normal surrounding cells. Because telomerase inhibitors specifically kill cancerous cells and not the normal surrounding cells, they are a new promising class of chemotherapeutic agents.
Cancer is defined as any tumor or malignant growth that invades other tissues due to abnormal cell proliferation (Royal Society of Chemistry, 2003). Cancer cells lose their ability to undergo apoptosis (programmed cell death) and divide uncontrollably. When the DNA of normal somatic cells are damaged, proteases and nucleases destroy the cell. This destruction decreases the chance of mutated cells from surviving. Cancer cells bypass senescence (non-dividing state) and divide without restraint. Growth factors and other extracellular factors regulate cell growth in non-cancerous cells (Royal Society of Chemistry, 2003). In cancerous cells these factors no longer correctly regulate cell growth. In cancerous cells, telomerase activity exists because the cell is dividing. Although stem cells and germ-line cells experience ongoing cell division, normal (non-dividing) somatic cells lack active telomerase because telomerase is inactive.
Telomerase is a specialized ribonucleoprotein that is present replicating cells such as cancerous cells. Telomerase is an enzyme that catalyzes the attachment of telomeres, or repeat bases TTAGGG, at the end of every chromosome (Figure 1). Telomeres protect the DNA of the cell and prevent mutations from occurring. The presence of telomerase during DNA replication is vital to the proliferation of cancer cells because telomeres need to be added to the end of the newly formed DNA (Bearss, 2002).
The end of the telomere forms a t-loop and a d-loop which help control telomere length. The t-loop and d-loop have binding sites where telomere-associated proteins, such as TRF1 and TRF2 specifically bind to control division. As the telomere increases in length, a t-loop forms and inhibits telomerase, from extending the telomere. During DNA replication, telomeres decrease in length by 25-200 base pairs of DNA (Bearss, 2002). Cancer cells elongate their shortened telomeres by telomerase activity.
Human telomerase (Figure 2) consists of the catalytic subunit, hTERT and an RNA template hTR. hTERT catalyses telomere lengthening by serving as a template for the second telomerase component (hTR) to bind. The hTR component of telomerase is approximately 445 nucleotides with its 11 nucleotides, sequence CAAUCCCAUC identified as the active binding site for hTR. Here base A-U pairing G-C interactions elongate the telomere (Royal Society of Chemistry, 2003).
Human telomerase is active in sperm, ova, and developing embryos: “Once the body is formed and their telomeres of sufficient length have been synthesized, the telomerase activity is shut off in the majority of cells and their telomeres begin to shorten as they reproduce” (Royal Society of Chemistry, 2003). The research article entitled, “Telomere maintenance mechanisms as a target for drug development”, telomerase is shown to be active in 85% of human malignancies, and only 0.5% of normal cells (Bearss, 2002). This finding is significant because inhibiting telomerase would specifically target cancerous cells and have little effect on normal cells.
The presence of telomerase is vital to the proliferation and differentiation of cancer cells because it adds the telomere to the end of replicating DNA (Figure 3 and 4). By inhibiting telomerase each subsequent cell generation will have shorter telomeres until cell proliferation ceases. Oligonucleotides that inhibit telomerase are shown to decrease proliferation in cancer cells. Since “telomeres in cancer cells are hundreds or thousands of bases long, and if telomerase is fully inhibited, telomeres may erode at a rate of 50 – 200 bases per population doubling, with variability likely caused by genetic background and growth conditions” (Chen, 2003). Observation of telomerase activity in tumor cells has become a significant and ever-growing approach to cancer therapeutic development. To develop a better understanding and approach to cancer therapeutics, several experiments were conducted to examine the effects of telomerase inhibition by using powerful MOE (2’-O-methoxyethyl) RNA oligomers. Significant antiproliferative effects were seen (Chen, 2003).
Because there was an increase in telomerase activity in human tumors, a hypothesis was formed, “that tumor growth requires reactivation of telomerase and that telomerase inhibitors represent a class of chemotherapeutic agents” (Herbert et al., 1999). In this experiment, progressive telomere shortening by 2'-O-MeRNA caused “cell death” in cancer cells. They also set the criteria for telomerase dependent mechanisms of cell death: i) cell growth rates would not be immediately reduced because the telomeres would not be shortened immediately, ii) rather the telomeres would be shortened with every division until iii) cell growth would stop.
Additional research by Elayadi et al., 2001 discussed the “Inhibition of telomerase by 2'-O-(2 methoxyethyl) RNA oligomers” and observed the effects of length, phosphorothioate substitution and time of the oligomer inside cells” (Elayadi, 2001). Once telomerase inhibitors were shown to end cell life cycle in vitro, tests were performed to see what combination of nucleotides would produce a stable oligonucleotide in vivo. Elayadi et al. 2001 treated DU145 prostate cancer cells with different RNA sequences to determine which one would be most stable in vivo testing. A 13 base phosphodiester 2'-MOE RNA oligonucleotide called ISIS 24691 was found to be most pharmacokinetically stable. Two agents were found to inhibit cancer cell growth for up to 7 days with one dose (ISIS 24691 and ISIS113749), but because ISIS24691 is a shorter RNA oligonucleotide, it is more cost effective and practical to produce.
An oncogene is defined as a cancer “causing” gene which is simply a “normal” gene involved in growth or cell division that is mutated: leading to cancer. Two genes involved in cell proliferation that are commonly mutated in cancer cells are the Rb and p53 genes. If the gene is mutated it is followed by a (-) sign (Example: p52-). If it is not mutated it is known as wild type and is followed by a + sign. Telomerase inhibitors work only when the telomeres are completely reduced in size. Complete telomere shortening does not occur until many cell generations later. This led researchers to conclude that telomerase inhibition would have no effect on cell proliferation until the telomeres would be completely shortened (Chen et al, 2003). In their research Chen et al, 2003 demonstrate that there is no significant loss in cell proliferation with short term treatment. However, with long term treatment telomerase inhibitor 2’-0-methoxyethyl oligonucleotide binds to the RNA template on telomerase and acts as a competitive inhibitor. The results of their experiment show that 2’-0-methoxyethyl RNA is effective on the tested DU145 cell line, which has Rb- and p53- genes, but not effective on the tested LNCaP cell line, which has Rb+, p53+ gene. This is significant because the antiproliferative effects of the 2’-O-methoxyethyl RNA were specific for the mutated gene, but did not inhibit proliferation of the wild type cells. This allows for a higher dose of telomerase inhibitors which is specific for destroying cancer cells but not showing antiproliferative effects on normal non-mutated cells. (Chen et al, 2003)
The objective of this experiment conducted by Nair et al. was to determine whether immunization against hTERT (catalytic reverse transcriptase protein subunit) generated a cytotoxic T lymphocyte (CTL) immune response against tumors that were distinct from one another (Nair, 2000). The researchers tested and found that the effects of inoculating TERT RNA-transfected dendritic cells (DC) into mice stimulated CTL. These lymphocytes destroyed and lysed “melanoma and thymoma tumor cells” and inhibited proliferation of three unrelated tumors in mice of different genetic backgrounds (Nair, 2000). The purpose in using hTERT RNA-transfected dendritic cells (DC) was to activate a CTL response, in vitro and in vivo, specific to TERT. “TERT RNA-transfected human DC stimulated TERT-specific CTLin vitro that lysed human tumor cells, including Epstein Barr virus (EBV)-transformed B cells as well as autologous tumor targets from patients with renal and prostate cancer” (Nair, 2000). Researchers concluded that, evidently, cytotoxic T cells destroyed tumor cells taken from renal cancer patients that were placed in culture.
Currently, Geron Corp. of Menlo Park, California developed a potential telomerase vaccine which is in Phase II trials for prostate cancer (Figure 5). Blood that is cultured from a cancer patient is surged with telomerase RNA into its dendritic cells. The telomerase vaccine operates in a way where the injected telomerase RNA dendritic cells are reinjected into the patient. The newly reinjected dendritic cells signal an immune response (that of cytotoxic T cells) in pursuit of telomerase. If Phase II trials of telomerase vaccine are successful, the vaccine may be used to treat many cancers due to active presence of telomerase in cancer cells (Jaffe, 2004).
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