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Experimental Therapeutics |
Institute for Drug Development, San Antonio, Texas 78245 [E. I., E. R., D. S., R. A. L., B. E. W., D. D. V. H.]; University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229 [K. K. D]; and Drug Dynamics Institute, College of Pharmacy, The University of Texas, Austin, Texas 78712 [R. T. W., L. H. H.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50 µM. Cytotoxicity of the porphyrins in vitro was assessed in normal human cells (fibroblast and breast) and human tumor cells representing models selected for high telomerase activity and short telomeres (breast carcinoma, prostate, and lymphoma). In general, the cytotoxicity (EC50, effective concentration for 50% inhibition of cell proliferation) against normal and tumor cells was >50 µM. The porphyrins were readily absorbed into tumor cell nuclei in culture. Inhibition of telomerase activity in MCF7 cells by subcytotoxic concentrations of TMPyP4 showed time and concentration dependence at 1–100 µM TMPyP4 over 15 days in culture (10 population doubling times). The inhibition of telomerase activity was paralleled by a cell growth arrest in G2-M. These results suggest that relevant biological effects of porphyrins can be achieved at concentrations that do not have general cytotoxic effects on cells. Moreover, the data support the concept that a rational, structure-based approach is possible to design novel telomere-interactive agents with application to a selective and specific anticancer therapy. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A novel approach toward achieving the net inhibition of telomerase is to target its substrate, the telomere. We used a rational, structure-based approach to the design of telomere interactive agents by considering unique nucleic acid secondary structures associated with the telomerase reaction cycle. One such structure is the G-quadruplex formed by folding of the single-stranded G-rich overhang produced by telomerase activity. The template region of the telomerase RNA has only 1.5 copies of the complementary sequence (3'-CAAUCCCAAUC-5'), so after each extension the end of the DNA must be translocated back to the beginning of the coding region before the next extension (13) . Work by Zahler et al. (14) has shown that potassium ions stabilize the quadruplex and that high concentrations of potassium inhibit telomerase. Furthermore, we (15) have shown that there is an equilibrium between the DNA:RNA heteroduplex and the G-quadruplex that lies in favor of G-quadruplex formation. These two observations point to the involvement of G-quadruplex formation in dissociating the primer from the telomerase RNA template and possibly providing the driving force for the translocation reaction. This led us to hypothesize that the G-quadruplex would indeed be a viable target for drug design, as first suggested by Blackburn (13) , and thus, we have undertaken a study of QIAs.4
The long-term goal of our studies is to identify an effective QIA (with significant concentration differences between telomerase inhibition and the cytotoxic effects), and bring it to clinical trial. Herein we report the inhibition of telomerase by TMPyP4, the related tetraquinolyl porphine QP3, and metal complexes thereof. The cytotoxicity and cellular uptake of this family of porphyrins have been examined in a series of human tumor and normal cell lines. We have demonstrated rapid repression of telomerase activity and cell growth arrest in intact tumor cells by subtoxic concentrations of TMPyP4. This finding suggests that the use of QIAs to directly target telomeres may be a possible therapeutic strategy. Three tumor models (breast, prostate, and lymphoma) are relevant to the future clinical development of telomerase inhibitors. The low cytotoxicity and inhibition of telomerase at low micromolar concentrations combine to make the cationic porphyrins attractive candidates for anticancer drug development.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals and Cell Lines.
All porphyrins were obtained from Midcentury (Posen, IL). The experimental work with porphyrins was performed under minimum exposure to light. All human tumor cell lines and normal human breast cells Hs578Bst were purchased from the American Type Culture Collection. Normal human lung fibroblasts were obtained from Clonetics Corp. The cell lines were grown according to suppliers’ instructions.
Telomerase Inhibition Assay.
Telomerase activity in human tumor cell lines was measured using the non-PCR-based telomerase assay (5)
with 5'-biotinylated d(TTAGGG)3. Extracts were obtained from 1 x 106 cells. The cells were washed once in PBS (400 µl) and pelleted at 10,000 x g for 1 min at 4°C, resuspended in 1.5 ml tubes containing 400 µl of ice-cold washing buffer [10 mM HEPES-KOH (pH 7.5), 1.5 mM MgCl2, 10 mM KCl, and 1 M DTT], then pelleted again at 10,000 x g for 1 min at 4°C. Washed cells were resuspended in 100–400 µl of ice-cold lysis buffer [10 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM ß-mercaptoethanol, 1 mM DTT, 0.5% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate, 10% glycerol, and 40 UI/ml RNase guard]. The suspension was incubated on ice for 30 min, and the lysate was then transferred to polyallomer tubes (Beckman) and spun at 100,000 x g for 1 h at 4°C in a tabletop ultracentrifuge. The supernatants were stored at -80°C in 10% glycerol. Protein concentration was determined by the Bradford assay (Bio-Rad). All tumor cell extracts were normalized to the same protein concentration (1 mg/ml).
Telomerase reaction mixtures were protected from light during the reactions involving porphyrins. In brief, reaction mixtures (20 µl) containing 4 µl of cell lysate, 50 mM Tris acetate (pH 8.5), 50 mM potassium acetate, 1 mM MgCl2, 5 mM ß-mercaptoethanol, 1 mM spermidine, 1 µM telomere primer, 1.5 µM [
-32P]dGTP (800 Ci/mmol), 2 mM dATP, and 2 mM dTTP were incubated at 37°C for 1 h. Reactions were terminated by adding 20 µl of streptavidin-coated Dynabead suspension containing 10 mM Tris-HCl (pH 7.5) and 2 M NaCl. The beads complexed selectively with the 5'-biotinylated DNA. The complex was separated from the suspension using a magnet (Dynal MPC) and washed several times with washing buffer (1 M NaCl) to eliminate [-32P]dGTP background. Telomerase reaction products were separated from the magnetic beads by protein denaturation with 5.0 M guanidine-HCl at 90°C for 20 min. After ethanol precipitation, the reaction products were analyzed by 8% PAGE. Telomerase activity in HeLa cells was used for reference and defined as 100% activity.
Cytotoxicity Assay (MTT).
Exponentially growing cells (1–2 x 103 cells) in 0.1 ml of medium were seeded on day 0 in a 96-well microtiter plate. On day 1, 0.1-ml aliquots of medium containing graded concentrations of the investigational compound were added to the cell plates. On days 7–10, the cell cultures were incubated with 50 µl of MTT, 1 mg/ml in Dulbecco’s PBS for 4 h at 37°C. The resulting formazan precipitate was solubilized with 200 µl of 0.04 M HCl in isopropyl alcohol (17
, 18)
.
Cell Cycle Analysis.
The effects of TMPyP4 on the cell cycle were assessed by flow cytometry by measuring the percentage of cells in G1-G0, S, and G2-M phases, with and without treatment with the porphyrin. Cells were stained with 50 µg/ml propidium iodide in a hypotonic sodium citrate solution with 0.3% NP40 and 1.0 mg/ml RNase A at 1.0 x 106 cells/ml, vortexed, and stained for 30 min at room temperature in the dark. Before flow cytometric measurements, samples were filtered through a 37-µm nylon mesh into 12 x 75-mm tubes and stored at 4°C until analysis within 24 h. All samples were analyzed with an EPICS ELITE flow cytometer (Coulter Cytometry, Miami, FL).
Nuclear Localization of TMPyP4.
MCF7 cells were seeded at 5 x 105 cells per flask and cultured in the presence of 0 or 50 µM TMPyP4 for 7 days with one exchange of medium. The cells were harvested with trypsin-EDTA, washed with PBS, and counted using a Coulter counter. The cells were then pelleted by centrifugation at 700 x g and lysed in a hypotonic solution (0.1x PBS); the resulting lysate was sedimented at 700 x g for 10 min. The supernatant was removed, and the pellet was dissolved in 0.5 ml of 2% SDS. Absorbances of the samples was measured at 423 nm with subtracting the absorbances of the samples without porphyrin. TMPyP4 diluted in 2% SDS containing 1 mg/ml salmon sperm DNA was used for the standard curve.
Metaphase Spreads.
MCF7 cells (at 5 x 105 cells/flask) were grown for 3 days in the presence of 40 µM QP3·InIII (the most fluorescent porphyrin). The cells were trypsinized, harvested by centrifugation, and incubated in Colcimide (Life Technologies, Inc.) at 100 ng/ml for 1 h. The cells were pelleted, and the medium was replaced with 75 mM KCl (hypotonic solution). After incubation for 20 min, the cells were spun at 700 x g for 2 min, all but 1 ml of the KCl solution was removed, and the cells were gently resuspended; 10 ml of freshly prepared methanol:glacial acetic acid (3:1) were added to the cells for fixation. The fluorescence microphotographs were taken with the filters for the excitation wavelength at 563–598 nm (maximum, 582 nm) and emission at 584–620 nm (maximum, 600 nm).
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RESULTS |
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shows the structure of the cationic porphyrin TMPyP4 (Fig. 1A)
and a G-tetrad (Fig. 1B)
. The X-ray crystal structure of this porphyrin with a short duplex has been solved (19)
. This structure has two notable features: the porphyrin only fits halfway into the duplex, and there is extensive disruption of the bases adjacent to the intercalation site. We have built a model consistent with preliminary biophysical data to assess how TMPyP4 molecules may complex with the human telomeric quadruplex. The model shows that TMPyP4 is a good fit for stacking with G-tetrads, where it can be oriented to place each of the cationic N-methylpyridine groups into each of the four grooves of the quadruplex. A section through the minimized model is shown in Fig. 1c
, and further biophysical evaluation of the interaction of TMPyP4 with quadruplexes is under way.
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illustrates the dependence of telomerase activity on porphyrin concentration. The 32P signal intensity, due to incorporation of [-32P]dGTP into the ladder produced by the action of telomerase, was quantitated in each lane. An IC50 (porphyrin concentration that inhibited 50% of the telomerase activity in the control) was determined from a plot of relative activity against porphyrin concentration (Fig. 2B)
. The interpolated IC50s were in the range of 50 µM.
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Within each tumor type, cell lines were selected to represent a spectrum of telomerase activities. In breast carcinoma cell lines, telomerase activity relative to HeLa cells was high in MCF7 (40%), intermediate in MDA-231 (30%), and very low in BT20 (<1% HeLa control). Some of these cell lines were estrogen sensitive (MDA-231), and the others were estrogen resistant (BT20, MCF7, and HS578t). Prostate carcinoma cells were androgen receptor positive (LNCaP) and negative (DU145, PC3), and both had intermediate telomerase activity (30%). Raji lymphoma cell line expressed intermediate telomerase activity (30%), and Daudi lymphoma cells had low telomerase activity (<20%). Most porphyrins showed low cytotoxicity against the tumor cell lines examined and against normal human fibroblasts (Table 1)
. No apparent correlation was observed between cytotoxic effects of porphyrins and the endogenous levels of telomerase activity or the steroid hormone receptor status in the tumor cell lines.
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. Thus, the general toxicity of porphyrins appears not to be telomerase related because telomerase inhibition occurs at concentrations below the toxic threshold for the cells.
Effects of TMPyP4 in Intact MCF7 Human Breast Carcinoma Cells.
To determine the effects of TMPyP4 on whole cells, MCF7 breast carcinoma cells were cultured in the continuous presence of 1, 10, and 100 µM TMPyP4. The porphyrin solution was freshly added to the medium from the concentrated stock at each passage of the cells (every 3–4 days). On days 4, 8, and 15, the cells were lysed, and telomerase activity was measured in the extracts. A clear concentration-dependent loss of telomerase activity in the presence of TMPyP4 was observed at days 4 and 8 (Fig. 3A)
. The results were quantitated and expressed as a percentage of control telomerase activity (Fig. 3B)
. Inhibition of telomerase activity was also time dependent (Fig. 3C)
. Even at 1 and 10 µM TMPyP4 (below the cytotoxic EC50 for MCF7 cells), the inhibition of telomerase activity showed a concentration dependence. The inhibition of telomerase activity by 1, 10, and 100 µM TMPyP4·PtII was also concentration dependent at days 4 and 8, but TMPyP4·PtII was a less potent inhibitor than TMPyP4 (data not shown). The extent of inhibition of telomerase by the two porphyrins in whole MCF7 cells thus parallels the relative potency of telomerase inhibition in the HeLa cell-free system.
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Nuclear Localization of Porphyrins.
Several reports indicate that porphyrins localize specifically in tumor tissue, although sites of subcellular localization may vary widely with porphyrin structure and net charge (23)
. Porphyrin TMPyP4 has been shown to rapidly accumulate (within minutes) in the nuclei of cultured human dermal fibroblasts (23)
. To gain insight into the subcellular distribution of cationic porphyrins, we have cultured MCF7 cells with 0 or 50 µM TMPyP4 for 7 days. When MCF7 cells were grown in the presence of 50 µM TMPyP4, porphyrin concentration in the nuclear pellet was 1.39 nmol/106 cells, and the cytoplasm contained 0.047 nmol/106 cells (3.3% of the TMPyP4 concentration measured in the nuclear fraction). These measurements did not differentiate between free and bound porphyrin; consequently, the measured values are likely to be an underestimate of the local porphyrin concentration associated with the DNA fraction. Assuming that the volume of the nucleus is one-tenth of the cell and that the volume of 106 cells is 10 µl, the calculated concentration of TMPyP4 in the nucleus (1.4 mM) greatly exceeds that of the cytoplasm (4.7 µM) and can readily approach the telomerase IC50 seen in the cell-free system.
To examine whether porphyrin in the nucleus was associated with chromosomal DNA, we examined fluorescence in metaphase spreads prepared from cells cultured in the presence of the cationic porphyrin. From a preliminary assessment of intrinsic fluorescence in solution, porphyrin QP3 · InIII was selected as the strongest fluorophore in the TMPyP4 and QP3 series. MCF7 cells were cultured in the continuous presence of 40 µM QP3 · InIII for 3 days. After that time, the cells were washed, fixed, and used for preparation of metaphase spreads. Direct evidence for the chromosomal localization of QP3 · InIII porphyrin is shown in Fig. 4
. These findings are consistent with the known affinity of the cationic porphyrins for DNA and indicate that porphyrins can accumulate in the nuclei of intact tumor cells at concentrations possibly exceeding those in the surrounding medium.
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DISCUSSION |
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We have shown that cationic porphyrins inhibit telomerase and cause G2-M arrest. These effects, happening in a time frame (<15 days) comparable with that of the cytotoxicity studies, might be expected from the G-tetraplex interactive agents interfering with the correct functioning of telomere structures.
The cationic porphyrins, particularly TMPyP4, are telomerase inhibitors at low micromolar concentrations. Furthermore, these porphyrins are relatively nontoxic to cells (both tumor and normal) at levels that can inhibit telomerase. Therefore, with this class of G-quadruplex interactive compounds, a selective inhibition of telomerase activity can be achieved at concentrations that do not have general toxic effects on cells. The uptake and accumulation data demonstrate that the cationic porphyrins can accumulate at useful levels (i.e., inhibitory to telomerase) in the nuclei of intact cells. Structure-activity relationships for the inhibition of telomerase by cationic porphyrins will be discussed elsewhere. Further development of cationic porphyrins as QIAs is under way.
Our results suggest that the short-term exposure of the human tumor cells to TMPyP4 leads to repression of telomerase activity. The repression was paralleled by a time-dependent increase in cell arrest at the G2-M phase. Telomerase activity may thus be different at various stages of the cell cycle, as proposed (20) . However, with no more than 32% of the porphyrin-treated MCF7 cells arrested in G2-M, only residual telomerase activity was measured. We did not see an apparent correlation between G2-M arrest and telomerase activity because in separate control experiments, using untreated cells with varying percentages of the cells in G2-M, we still measured high telomerase activity. These results would suggest that although TMPyP4 causes G2-M arrest, G2-M arrest alone does not necessary lead to inhibition of telomerase. Therefore, a variation of telomerase activity in the cell cycle is not a plausible explanation for the observed decrease of telomerase activity. Another possibility is an indirect effect of TMPyP4 on down-regulation of telomerase expression, recently postulated as a part of the mechanism of cisplatin action against human testicular cancer cells (12) .
The first indication that TMPyP4 induces chromosomal abnormalities has been found in sea urchin embryos. Recently, we found that TMPyP4 significantly decreased proliferative rates in the developing embryos, increased the percentage of the cell in mitosis, and led to formation of largely elongated mitotic chromosomes.5 A similar phenotype has been described in Tetrahymena with telomerase RNA template mutation (24) and in cells expressing dominant-negative TRF2 allele (25) . In HeLa cells cultured in the presence of 3 µM TMPyP4 for 2–4 days, the number of fused chromosomes was increased in comparison with the untreated controls.6 These chromosomal aberrations may be sufficient to inhibit cellular proliferation even in the absence of repression of telomerase activity.
Although our results indicated that TMPyP4 may induce chromosome destabilization, telomere shortening was not likely to occur under the experimental conditions. Telomeres shorten by 
65–100 bp/cell division (26)
, and 3'-azido-3'-deoxythymidine effects on telomere length have been demonstrated in long-term human cell line cultures (10)
. At this rate of telomere loss, even with relatively short telomeres in MCF7 cells, a long time in culture (>20 population doubling time) in the presence of the porphyrins may be required to achieve significant effects on telomere length.
Telomeres have been shown to play a direct role in mitosis: a physical block in anaphase chromosome separation is caused by telomerase template mutation (25) . One implication of this finding is that porphyrins (as telomere-interactive agents) may arrest the cells in mitosis. We have already observed an increase of the G2-M fraction in MCF7 cells treated with TMPyP4. A thorough investigation of the antiproliferative effects of porphyrins in tumor cells is in progress.
Telomere shortening may not be a paradigm for all telomerase inhibitors. Highly variable telomere lengths in two immortalized cell lines treated with reverse transcriptase inhibitors were supposedly the result of telomerase-dependent and telomerase-independent mechanisms for telomere maintenance (10) . Therefore, agents like TMPyP4 that repress telomerase activity and that can directly interact with telomeres may be effective in situations where telomere length in tumor cells is maintained by telomerase-independent mechanisms (27) . In the present work, we show that the QIAs directly interact with telomeres and rapidly evoke antiproliferative effects in tumor cells. Telomerase repression may be a secondary event to tumor growth arrest. Thus, the QIAs that represent a different mechanism of action than other telomerase inhibitors may be most effective in tumors with low telomerase activity. Ongoing studies address the issue of the best clinical models for QIAs.
In summary, our work provides proof of principle for the development of small-molecule telomerase inhibitors targeting telomeric DNA rather than telomerase per se. The rapid cell response to QIAs indicates greater significance and multiple roles for G-quadruplex in the whole telomere system. Recent reports describing telomere shortening and tumor formation by mouse cells lacking telomerase RNA (28 , 29) raised multiple questions regarding the validity of telomerase as a target for anticancer agents. In the light of these findings, our alternative strategy focusing on telomere interactive agents is strongly justified.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by National Cooperative Drug Discovery Group Grant CA67760 from the National Cancer Institute. Preliminary reports of this work were presented at the 88th Annual Meeting of the American Association for Cancer Research, San Diego, CA, in Proceedings of The American Association for Cancer Research, 38: 637, 1997. 
2 To whom requests for reprints should be addressed, at the Institute for Drug Development, 7979 Wurzbach Road, Suite 337, San Antonio, TX 78229. Phone: (210) 616-5892; Fax (210) 616-5948; E-mail eizbicka{at}saci.org
3 Present address: School of Pharmacy, The University of Bradford, Bradford, West Yorkshire BD7 1DP, United Kingdom.
4 The abbreviations used are: QIA, G-quadruplex interactive agent; TMPyP4, 5,10,15,20-tetra(N-methyl-4-pyridyl)porphine chloride; QP3, 5,10,15,20-tetra(N-methyl-3-quinolyl)porphine chloride; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
5 E. Izbicka, unpublished data.
6 B. E. Windle, unpublished data.
Received 8/ 6/98. Accepted 12/ 2/98.
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S. M. Gowan, J. R. Harrison, L. Patterson, M. Valenti, M. A. Read, S. Neidle, and L. R. Kelland A G-Quadruplex-Interactive Potent Small-Molecule Inhibitor of Telomerase Exhibiting in Vitro and in Vivo Antitumor Activity Mol. Pharmacol., May 1, 2002; 61(5): 1154 - 1162. [Abstract] [Full Text] [PDF]
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J.-L. Mergny, J.-F. Riou, P. Mailliet, M.-P. Teulade-Fichou, and E. Gilson Natural and pharmacological regulation of telomerase Nucleic Acids Res., February 15, 2002; 30(4): 839 - 865. [Abstract] [Full Text] [PDF]
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 J. F. Riou, L. Guittat, P. Mailliet, A. Laoui, E. Renou, O. Petitgenet, F. Megnin-Chanet, C. Helene, and J. L. Mergny Cell senescence and telomere shortening induced by a new series of specific G-quadruplex DNA ligands PNAS, February 14, 2002; (2002) 52698099. [Abstract] [Full Text] [PDF]
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W. Duan, A. Rangan, H. Vankayalapati, M.-Y. Kim, Q. Zeng, D. Sun, H. Han, O. Yu. Fedoroff, D. Nishioka, S. Y. Rha, et al. Design and Synthesis of Fluoroquinophenoxazines That Interact with Human Telomeric G-Quadruplexes and Their Biological Effects Mol. Cancer Ther., December 1, 2001; 1(2): 103 - 120. [Abstract] [Full Text] [PDF]
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S. M. Gowan, R. Heald, M. F. G. Stevens, and L. R. Kelland Potent Inhibition of Telomerase by Small-Molecule Pentacyclic Acridines Capable of Interacting with G-Quadruplexes Mol. Pharmacol., November 1, 2001; 60(5): 981 - 988. [Abstract] [Full Text] [PDF]
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A. Adams, J. M. Guss, C. A. Collyer, W. A. Denny, and L. P. G. Wakelin A novel form of intercalation involving four DNA duplexes in an acridine-4-carboxamide complex of d(CGTACG)2 Nucleic Acids Res., November 1, 2000; 28(21): 4244 - 4253. [Abstract] [Full Text] [PDF]
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S. Y. Rha, E. Izbicka, R. Lawrence, K. Davidson, D. Sun, M. P. Moyer, G. D. Roodman, L. Hurley, and D. Von Hoff Effect of Telomere and Telomerase Interactive Agents on Human Tumor and Normal Cell Lines Clin. Cancer Res., March 1, 2000; 6(3): 987 - 993. [Abstract] [Full Text]
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J. F. Riou, L. Guittat, P. Mailliet, A. Laoui, E. Renou, O. Petitgenet, F. Megnin-Chanet, C. Helene, and J. L. Mergny Cell senescence and telomere shortening induced by a new series of specific G-quadruplex DNA ligands PNAS, March 5, 2002; 99(5): 2672 - 2677. [Abstract] [Full Text] [PDF]
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M. Bennett, A. Krah, F. Wien, E. Garman, R. Mckenna, M. Sanderson, and S. Neidle A DNA-porphyrin minor-groove complex at atomic resolution: The structural consequences of porphyrin ruffling PNAS, August 15, 2000; 97(17): 9476 - 9481. [Abstract] [Full Text] [PDF]
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