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We hypothesize that telomerase inhibition (telomere shortening) can sensitize human tumor cells to existing anticancer drugs. During the period of the grant we made the following findings-: 1) 2'-methoxy ethyl oligonucleotides inhibit - -telomerase in prostate cancer cells, cause telomeres to shorten, and cause cell- proliferation to decrease; -2) Cell proliferation in culture is -more pronounced when cells are grown under conditions that mimic tumor growth; 3) Cell proliferation is dramatically reduced in a xenograft model using LNCAP cells, and 4) We did not observe significant synergy with standard chemotherapy agents. The ability of relatively short-term treatments with telomerase inhibitors to slow tumor growth in vivo suggests that telomerase inhibitors are a reasonable approach to prostate cancer therapy.
Malignant prostate cells must divide many times to form a tumor, metastasize or recur after therapy. Critical for this type of endless cell division is an enzyme named telomerase. This enzyme is usually dormant in most normal tissues, but is resurrected in prostate cancer cells by yet unknown genetic changes. Since the enzyme is required for survival of cancer cells but is not present in most normal cells, inhibition of telomerase may specifically target prostate cancer cells. We have shown that impeding the function of telomerase by expressing a mutated version of this protein kills prostate cancer cells in an experimental setting. We now wish to translate this finding into a more practical application by searching for smaller molecules capable of inhibiting telomerase activity. The approach we propose to take is one that capitalizes on reagents already in hand. First, we have mapped parts of telomerase that are extremely sensitive to even small disruptions. We now wish to find molecules that bind to these sensitive regions, as such regions represent the most attractive parts of the protein to target for inhibition. We will therefore screen millions upon millions of small molecules, termed peptides, for those few that adhere to these regions of the telomerase enzyme. Such peptides could serve as a blueprint to develop even more clinically relevant inhibitors of telomerase.
Telomerase, an enzyme that maintains telomeres and endows eukaryotic cells with immortality, was first discovered in tetrahymena in 1985. In 1990s, it was proven that this enzyme also plays a key role in the infinite proliferation of human cancer cells. Now telomere and telomerase are widely accepted as important factors involved in cancer biology, and as promising diagnostic tools and therapeutic targets. Recently, role of telomerase in “cancer stem cells” has become another attractive story. Until now, there are several good books on telomere and telomerase focusing on biology in ciliates, yeasts, and mouse or basic sciences in human, providing basic scientists or students with updated knowledge.
The mechanism for genistein-induced activation of STAT3 was then determined. A physiological concentration of genistein activated signaling proteins upstream of STAT3 including JAK2, Akt, Src, and MAPK p44/42 in human prostate cancer cells. Specific shRNA inhibition of these signaling proteins in stable STAT3 reporter prostate cancer cells reduced STAT3 reporter activity and cancer cell proliferation. Genistein treatment of cells with the signaling proteins inhibited did not increase STAT3 reporter activity and cancer cell proliferation to the level observed in genistein treated control cells, indicating that JAK2, Akt, Src, and MAPK p44/42 are necessary for genistein-induced activation of STAT3. The data collectively suggests that physiologically achievable concentrations of genistein enhance cancer cell proliferation by increasing telomerase activity via STAT3 activation mediated by upstream signaling proteins. The result that physiological concentrations of genistein promote prostate cancer cell growth indicates that prudence should be exercised when genistein is considered for chemotherapeutic purposes as unfavorable effects in individuals with prostate cancer is possible.
This volume presents a compendium of the most recent and advanced methods applied to the rapidly expanding field of telomerase inhibition. The techniques described provide the researcher with a diverse and comprehensive set of tools for the study of telomerase inhibition. The volume is aimed at biochemists, molecular biologists, cancer researchers, and geneticists.
In studies to define the mechanisms involved in the progression of immortal, non-tumorigenic prostate cells to a tumorigenic state, we have found that molecular chaperones are elevated, which causes increased telomerase activity. In order to determine the importance of the chaperone increase during prostate cancer progression, we have taken a 2-pronged approach, using both genetic and pharmacologic approaches: 1-define whether ectopic chaperone expression results in transformation, and 2-determine whether chaperones are targets for prostate cancer therapy. The hsf-1 transcription factor has been over-expressed in non-tumorigenic prostate cells, resulting in increased hsp90 and hsp70 expression, an upregulation of telomerase, and no effect on tumorigenicity. However, preliminary data suggests that hsf-1 may directly effect telomerase expression, which would further define the regulation of telomerase during cancer progression. Using both a pharmacologic (radicicol, a specific hsp90 inhibitor) and genetic (siRNA to hsp90) approaches, prostate cancer cell lines exhibit only a transient decline in telomerase activity but a significant decrease in telomeres, which we have shown to be directly damaged by free radicals produced as a result of deregulation of the nitric oxide synthase pathway.
Telomerase, a ribonucleoprotein enzyme minimally composed of an RNA template (hTR) and a catalytically active protein subunit (hTERT), synthesizes telomeric repeats onto chromosome ends and is obligatory for continuous tumor cell proliferation, as well as malignant progression of breast cancer cells. Telomerase is an attractive anticancer therapeutic target because its activity is present in over 90% of human cancers, including more than 95% of breast carcinomas, but undetectable in most somatic cells. Traditions chemo- and radio-therapies lack the ability to effectively control and cure breast cancer, in part because residual cells are or become resistant to DNA damaging modalities. While various telomerase inhibition strategies cause cancer cells to undergo apoptosis car senescence, there is often a lag period between administration and biologic effect (Corey, 2002). Our goal in this study was to compare the efficacy of different telomerase inhibition strategies in concert with standard chemotherapeutic agents at triggering senescence and/or apoptosis in cultures of breast cancer cells. We hypothesized that telomerase inhibition strategies will sensitize breast cancer cells to traditional chemotherapies, potentially reducing the lag phase, allowing for more potent anti-tumor effects at lower doses, and therefore ultimately imparting less toxicity to the patient. We blocked telomerase by targeting hTR and hTERT, individually and collectively utilizing synthetic short interfering RNA (siRNA), short hairpin RNA (siRNA), and a dominant negative form of hTERT (DN-hTERT) in MCF-7 breast cancer cells. We analyzed the efficiency of telomerase inhibition for each strategy alone and then treated the cells with two mainstay chemotherapeutic agents, Adriamycin (AdR) and Taxol. The most effective telomerase inhibition strategies were synthetic siRNA and DN-hTERT, individually. After treatment with various concentrations of AdR or Taxol, breast cancer cells with inhibited telomerase grew significantly slower and exhibited widespread senescence or apoptosis within a much shorter time period and at a dose that is insufficient to trigger cytostasis. In addition, we provide evidence that cells in which telomerase was inhibited were more sensitive to anti-cancer agents, whether the drug inhibited topoisomerase II resulting in DNA damage (AdR) or blocked mitosis via protracted microtubule stabilization (Taxol). Collectively, our data indicate that alone, anti-telomerase inhibition strategies differ in their efficacy. However, when used in the adjuvant setting with diverse acting chemotherapeutic agents, there is a potent synergy resulting in chemotherapeutic sensitization characterized in part by widespread senescence and/or apoptosis.
Telomerase, a ribonucleoprotein enzyme minimally composed of an RNA template (hTR) and a catalytically active protein subunit (hTERT), synthesizes telomeric repeats onto chromosome ends and is obligatory for continuous tumor cell proliferation, as well as malignant progression of breast cancer cells. Telomerase is an attractive anti-cancer therapeutic target because its activity is present in over 90% of human cancers, including more than 95% of breast carcinomas, but undetectable in most somatic cells. Traditional chemo- and radiotherapies lack the ability to effectively control and cure breast cancer, in part because residual cells are or become resistant to DNA damaging modalities. While various telomerase inhibition strategies cause cancer cells to undergo apoptosis or senescence, there is often a lag period between administration and biologic effect (Corey, 2002). Our goal in this study was to compare the efficacy of different telomerase inhibition strategies in concert with standard chemotherapeutic agents at triggering senescence and/or apoptosis in cultures of breast cancer cells. We hypothesized that telomerase inhibition strategies will sensitize breast cancer cells to traditional chemotherapies, potentially reducing the lag phase, allowing for more potent anti-tumor effects at lower doses, and therefore ultimately imparting less toxicity to the patient.
Telomerase, a ribonucleoprotein enzyme composed of an RNA template (hTR) and a catalytically active protein subunit (hTERT), synthesizes telomeres after cell divisions and is obligatory for continuous tumor cell proliferation as well as malignant progression of breast cancer cells. Telomerase is an attractive anti-cancer therapeutic agent because telomerase activity is present in over 90% of human breast cancers but is undetectable in most normal somatic cells. Traditional therapies (surgery, chemotherapy, radiotherapy, etc.) lack the ability to effectively control and cure breast cancer, primarily because residual cells are or become resistant to DNA damaging modalities including standard chemo- and radio-therapies. Since telomerase requires its associated hTR for repeat synthesis, we have chosen to use RNA interference as a method to inactivate hTR and hence telomerase. RNA interference (RNAI) has become a powerful tool for the analysis of gene function in that RNAI allows sequence specific inhibition of gene expression. Another protein we targeted is p21, which has long been established as a requirement for senescence. We wanted to further examine its relationship to senescence and apotosis, in an attempt to sensitize breast tumor cells more effectively.