Current Medicinal Chemistry - Anti-Cancer Agents - Volume 2, Issue 5, 2002

Telomerase inhibitors have been touted as a novel cancer specific therapy, as most tumor cells have high expression of telomerase, whereas most normal somatic cells express low or undetectable levels of telomerase. Continued proliferation of tumor cells requires activation of telomerase to maintain chromosomal stability and extend life span, because telomerase elongates telomere length and rewinds the cellular mitotic clock. Conversely, shortening of telomeres by inhibition of telomerase activity induces growth arrest (senescence) and apoptosis in tumor cells. Moreover, it has been reported that inhibition of telomerase increases the susceptibility of tumor cells to apoptosis induced by anticancer agents. Thus, telomerase inhibitors could be used as an adjuvant with conventional therapy. However, there are also several potential limitations of telomerase inhibition as a therapeutic strategy. For example, there is a lag phase between telomerase inhibition and telomere shortening, with growth arrest and cell death. In this review, we will discuss the basic biology of telomeres and telomerase as a platform for the development of treatments based upon inhibition of telomerase activity.

Telomerase is a specialized RNA template-containing reverse transcriptase that mediates telomere repeat synthesis at chromosome ends. The maintenance of telomere length and integrity is essential for cell survival. Telomerase is active in most immortal and tumor cells, whereas the majority of normal human cells demonstrate no detectable activity and undergo telomere shortening. The identification of a possible role for telomerase in cellular aging and cancer has led to numerous studies designed to characterize this ribonucleoprotein enzyme. Inhibiting telomerase activity in immortal human cells reduces cellular proliferative capacity and can lead to cell death. Identifying mechanisms to specifically inhibit telomerase activity in malignant cells could thus be of great therapeutic value in the treatment of cancer. In this review, we summarize the current understanding of the mechanism of action of human telomerase. The biochemical characterization of telomerase is necessary for the design and evaluation of antitelomerase therapies. Different strategies are currently under investigation to design inhibitors that target the reverse transcriptase and RNA components of the telomerase complex. Recent advances in the design of these inhibitors and their properties are discussed.

Human telomeres are several kilobases of repeated (TTAGGG)n sequences at the ends of chromosomes, a short fragment of which is lost with each cell division. This shortening serves as a “mitotic clock” which limits the number of divisions that a normal somatic cell can undergo. Cells undergoing continuous division need some method of bypassing this clock. One such method is the expression of telomerase. This ribonucleoprotein is an enzyme that rebuilds the lost portion of the telomeres. Between 80- 95% of tumors are telomerase-positive, including ovarian carcinoma, hepatocellular carcinoma, neuroblastoma, leukemia / lymphoma, and cancers of the breast, prostate, lung, kidneys and bladder, as well as many immortalized cell lines . While absent in most normal tissues, this enzyme is expressed at higher levels in germline tissues, bone marrow, and lymphocytes.Due to the expression of telomerase in most tumor cells and its absence in most normal tissues, telomerase inhibitors are being investigated as possible anticancer agents. This review focuses on non-reverse transcriptase inhibitor, non-oligonucleotide and non-G-quartet interactive agent telomerase inhibitors. These agents include: differentiating agents, kinases and phosphatases, cell cycle and apoptosis regulating agents, immunotherapeutic agents, antibiotics, steroids, bisindole derivatives, and a variety of other compounds. These agents hold much promise for the future treatment of malignancies.

Human telomerase is a ribonucleoprotein enzyme complex that enables cells to maintain telomere length, allowing indefinite replicative capacity. The notion that telomerase is reactivated in 80-90% of human cancers has led to the proposal of telomerase as a promising therapeutic target for novel anticancer interventions. Due to its inherent accessibility to nucleic acids, telomerase appears an ideal target for strategies based on the use of antisense oligonucleotides and ribozymes that target its RNA template. In this review a summary of the different antisense- and ribozyme-based approaches used thus far to inhibit telomerase activity in human cancer cells is provided. All these strategies significantly inhibited the enzyme's catalytic activity in in vitro and in vivo tumor models. However, while in some studies tumor cell growth arrest was observed as a consequence of telomere shortening after prolonged telomerase inhibition, other studies have shown that antisense- and ribozyme-based treatments targeting telomerase induced rapid loss (i.e. within a few days) of tumor cell viability with concomitant apoptosis. In the latter case it is unlikely that cell death was related to telomere erosion since the cells would not have undergone enough divisions to significantly shorten their telomeres. A possible explanation is that telomerase inhibitors may induce apoptosis in cancer cells directly by interfering with the capping function of the enzyme. Overall, the available results indicate antisense oligonucleotides and ribozymes as good tools to inhibit telomerase and suggest that abrogation of telomerase activity may affect tumor cell proliferation also through pathways that are not dependent on telomere erosion.

Telomerase is a target for anticancer research because telomerase activity is closely correlated with malignancy. Inhibition of telomerase activity should increase telomere shortening, which destabilizes chromosomes, thus leading to cellular senescence and death. Extensive investigations have addressed the molecular mechanisms of telomerase activation in cancers. Based on results from these studies, various attempts have been made to inhibit telomerase activity using molecular techniques in cancer cells. Antisense oligonucleotides directed to human telomerase RNA, the dominant negative form of human telomerase reverse transcriptase (hTERT), hammerhead ribozymes that cut hTR and agents that interact with quadruplex DNA represent potential telomerase inhibitors. This review includes a summary of recent attempts to inhibit telomerase activity in cancer cells and a discussion of how these tools can be applied to cancer therapy, especially in combination with established anti-cancer agents.

DNA is prone to structural polymorphism: its three-dimensional structure can differ markedly from the classical double helix. Nucleic acid structures composed of more than two strands have also been observed. The guanine-rich sequence of both the telomere and centromere can form a quadruplex based on G-quartets while the complementary cytosine-rich strand can fold into an intercalated tetramer called the i-motif. The G-quartet is a gold mine for structural biologists and the telomere has become a target for anti-cancer drug design since it was observed that deregulation of telomerase favors proliferation of certain tumors. Other DNA sequences may adopt unusual confor-mations. Polypurine-polypyrimidine sequences capable of forming a triple-stranded structure called H-DNA are found abundantly in the eukaryotic genome and may play a significant role in DNA metabolism, transcription and replication. Triplex-forming oligonucleotides are currently being developed as “anti-gene”agents. Unusual DNA structures may therefore be implicated in fundamental processes such as gene expression and represent unique targets for both structural-specific and sequence-specific agents. In this review, we present work characterizing some of these unusual conformations in terms of structure, stability and formation kinetics and discuss their biological implications.