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Experimental Therapeutics |
Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Kami-Ikebukuro, Toshima-ku, Tokyo 170-8455 [I. N., H. S., T. Y., T. T.], and Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032 [I. N., T. T.], Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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35 µM. Using this alkaloid as a seed compound in COMPARE resulted in the identification of other berberine-like compounds and mitochondria-accumulating agents as highly related to berberine. Among these compounds, MKT077, a rhodacyanine derivative currently under Phase I clinical trials, showed a potent inhibitory effect with 50% inhibition at 5 µM. With MKT077 as an upgraded seed for a new round of COMPARE analysis, we identified rhodacyanine FJ5002, a close derivative of MKT077, as the most potent telomerase inhibitor with 50% inhibition at 2 µM. Long-term cultivation of U937, a human leukemia cell line, with subacute concentrations of FJ5002 resulted in population-doubling dependent changes characterized by progressive telomere erosion (from 10 to 4.0 kb), increased chromosome abnormalities, and senescence/crisis-like features. These results indicated that FJ5002 is a genuine and effective antitelomerase agent. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Detection of Telomerase Activity in Cell Lysates.
TRAP assay was performed as described previously (8
, 16)
with the addition of an internal competitive standard, ITAS (17)
and a modified CX primer, CXext (18)
. Buffered solutions of test compounds were added (5 µl each) to 45 µl of telomerase reaction buffer. Telomerase products were resolved by PAGE and visualized by staining with SYBER Green (Takara, Tokyo). For quantitative measurements in Dixon analysis, the reaction conditions (e.g., primers’ concentrations, PCR cycle number) were optimized in preliminary experiments to be in the dynamic range with the correlation coefficient (r) between the signal (log) and the equivalent number of cells in the extract (log) exceeding 0.99. In all of the assays, a cell lysate equivalent to 103 cells was used in each reaction. Calculations were carried out by densitometric analysis using the NIH Image software version 1.60.
Chromosome Analysis in Metaphase Cells.
Cells were incubated in 0.1 µg/ml colcemide for 1 h, harvested, incubated for 8 min at 37°C in 0.075 M KCl, and then fixed in freshly prepared methanol:acetic acid (3:1 vol/vol) solution. Cells were preserved at -20°C and, when needed, dropped onto cleaned slides and oven-dried at 50°C overnight before use.
Q-banding.
Slides prepared as above were incubated in 50 µg/ml quinacrine mustard in MacIlvaine solution (pH 5.6) for 7 min at room temperature, briefly washed with tap water, and incubated again in MacIlvaine buffer for 7 min. Each slide was carefully wiped, supplemented with a 20 µl of an antifade solution (SlowFade, Molecular Probes, Oregon) per slide, covered with a coverslip (0), and sealed. One hundred metaphases from each treatment were examined for quantification of chromosome abnormalities.
PRINS Analysis.
PRINS labeling of telomeres (19)
was performed according to the instructions provided by the supplier (Boehringer Mannheim, Mannheim, Germany) with minor modifications. Briefly, by using the heating block of a thermal cycler (model 9600, Perkin-Elmer, Norwalk, CT), the slides were heated at 96°C for one min; then, 28 µl of a reaction mixture containing the telomere-specific probe, Taq polymerase, and digoxigenin-labeled deoxynucleotides were added. Each slide was quickly covered with a coverslip and heated for another 4 min at 96°C. The temperature was then regulated down to 62°C and left for 30 min to allow for progress of polymerization reaction. Slides were then treated with a stop buffer [50 mM EDTA, 50 mM NaCl (pH 8.0)], followed by a treatment with FITC-labeled antidigoxigenin antibodies. The slides were then washed and counterstained with 20 µl of propidium iodide (0.03 µg/ml) per slide in an antifade solution (SlowFade), covered with a coverslip (0), and sealed. Twenty-five spreads of each treatment were examined, and the telomeric FITC signals were quantified on equal backgrounds (see "Microscopy" below).
Microscopy.
A Zeiss Axiophot2 fluorescence microscope equipped with a KAF1400-G2 CCD camera (Photometrics) was used. Images visualized with a x60 objective were captured and saved into a Macintosh computer in a PICT format and then noise-filtered, corrected for background, and merged using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA) files.
Telomere Length Analysis.
Telomere length was analyzed by determining the mean length of the TRFs. The genomic DNA was extracted from pooled parallel subcultures [to avoid possible selection due to the wide divergence of telomere lengths (20)
] by an anion exchange column method (QIAGEN). Approximately 4 µg of DNA were digested by HinfI/RsaI and processed for Southern blotting and chemiluminescence detection according to the instructions provided by the manufacturer of the TeloQuant assay kit (PharMingen, San Diego, CA). Films were quantified using an ATTO densitometer/software system.
Flow Cytometry.
DNA contents and cell cycle phases were analyzed as described previously (16)
using a fluorescence-activated cell sorter (FACScan, Becton Dickinson, San Jose, CA).
HPLC.
FJ5002 was extracted from treated cells by ethanol (70%) and sonication (extraction ratio was 100 µl solvent:1 x 106 cells), and the supernatant (100 µl) was injected into an HPLC system (Shimadzu LC-6A) equipped with an UV detector (Shimadzu SPD-6AV) set at 335 nm and with an Intersil ODS-2 reverse-phase column. The mobile phase consisted of 50 mM KCl solution:methanol:triethanolamine:acetic acid [30:70:0.2:0.2 (pH 7.0)].
Determination of Melting Temperature.
Denaturation profiles were obtained for the telomeric sequence d5'-AGGGTTAGGGTTAGGGTTAGGG-3' (10 µM; Ref. 21
) in the presence or absence of FJ5002 in 10 mM sodium cacodylate and 100 mM NaCl buffer (pH 7.0). Solutions were heated for 5 min at 90°C and then slowly cooled to room temperature over 30 min. Solutions were then placed into a quartz cuvette and heated from 20°C to 84°C with absorbance at 295 nm recorded over a 5-min equilibration time at increments of 5°C with a JASCO Ubest-30 spectrophotometer and a JASCO EHC-363 Peltier temperature-control accessory. FJ5002 was added equally to the sample and the blank cuvettes to avoid variances caused by its intrinsic absorbance.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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35 µM (Fig. 1, a and b
; Fig. 3a
).
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. Testing the telomerase inhibitory effect of the top 20 related agents (r > 0.6) identified MKT077, a rhodacyanine that is currently under Phase I/II clinical trials (22)
, as a potent telomerase inhibitor with 50% inhibition at 5 µM (Fig. 2, a and c
; Fig. 3a
).
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2 µM (Table 2
; Fig. 2, b and d
; Fig. 3a
). Using this compound as a new seed for a third round of COMPARE yielded a repeated list of related compounds all of which had already appeared in the first and second rounds, indicating that FJ5002 was the strongest inhibitor that could be detected within the capacity of the available database. It is clear from Fig. 3a
that a more than 17-fold increase in inhibition potency could be achieved through the use of COMPARE analysis.
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1.8 µM (Fig. 3b)
. We also tested whether FJ5002 interferes with the G-quartet structure of telomeres, a possible mechanism for the inhibition of telomerase that uses the G-quartet structure as a driving force (23
, 24)
. Changes in G-quartet melting temperature of the telomeric sequence d5'-AGGGTTAGGGTTAGGGTTAGGG3-' in the presence of FJ5002 were monitored using the method of Mergny et al. (21)
. As shown in Fig. 3c
, FJ5002 had no effect on the G-quartet structure at a molecular stochiometry (substrate/inhibitor) that was similar to the one used for elaborating the inhibitory effect by the TRAP assay. This finding is in agreement with a model in which FJ5002 inhibits telomerase by a direct interaction. The fact that FJ5002 did not inhibit Taq polymerase in the TRAP assay was by itself evidence for its relative specificity against telomerase.
Long-Term Cultivation of Cancer Cells in the Presence of FJ5002.
To examine the long-term effect of FJ5002 on U937 cells, it was necessary to identify the drug concentration window in which telomerase could be inhibited but without extensive inhibition of cell proliferation. On the basis of a series of preliminary experiments and HPLC analyses, we found that FJ5002 could accumulate in U937 cells at an intracellular concentration of 1000 nM (20-fold) when used at an extracellular concentration of 50 nM. This level was approximately one-half of the maximal level (2000 nM) that exhibited cytotoxicity within four to five population doublings. Accordingly, we used 50 nM as the treatment concentration in the long-term cultivation experiments that followed.
During long-term cultivation, cells were monitored by microscopic and flow cytometric analyses and by telomere-length estimation using Southern blotting and PRINS. Within the first 20–30 days of passage, no noticeable morphological differences could be detected between treated and untreated control cells except that the growth rate of treated cells was about 20% slower than that of the control cells (Fig. 4a)
. However, from about day 40 and thereafter, treated cells started to exhibit replicative senescence and/or crisis properties, and a marked increase in cells with enlarged size, cloudy cytoplasm, and ragged plasma membrane was observed. Furthermore, the cell population with positive SA-ß-Gal, a senescence marker (25)
, increased from <10% at about day 4 to >70% at about day 90 (Fig. 4c)
. Although an annexin-binding test of apoptosis showed a 20% increase in the apoptosis rate of the cells at crisis, flow cytometry and DNA fragmentation tests failed to detect a clear pattern of apoptosis in these cells (data not shown). It seems that the cell death at crisis was mediated by both apoptic and nonapoptic pathways. The crisis state observed here closely resembled the M2 crisis state in which the cells show a continuing mitotic activity, a positive staining with SA-ß-Gal, and a coexisting rate of cell death (26)
.
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Fig. 5
shows telomere erosion as determined by estimation of the length of TRF in U937 and NHLF cells. At a cytotoxic concentration (0.25 µM for 96 h, three rounds of cell division), FJ5002 did not induce any detectable loss in telomeres (Fig. 5a)
, indicating that this drug did not actively degrade the telomeric ends by causing DNA damage through intercalation or cross-reaction, a mechanism that was recently observed with cisplatin (27)
. Interestingly, telomere erosion was concentration-responsive, and showed dynamics that were similar to the cell viability dynamics throughout the course of cultivation (Fig. 5b, c, e)
. Flow cytometric analysis of the changes in cell cycle phases revealed a gradual increase in the aneuploidy fraction (Fig. 6a and b)
. This was further confirmed on an individual cell basis using microscopic examination of chromosomal spreads from late passages by Q-banding for general chromosomal abnormality quantification, and by PRINS technique for direct individual telomere length visualization (Fig. 6c and d)
. The frequency of chromosomal abnormalities (such as end-to-end association, circularization, and translocation) markedly increased in treated cells (abnormality rate was 0.4 and 0.8 event/spread in control and treated cells, respectively, at day >116). On the other hand, the telomeric signal in PRINS analysis of treated cells was, in general, weak, and the number of chromosome ends without FITC signal was 3-fold higher than that of control cells at the same passage day (the rate was 1.3 and 3.7 deleted spot/spread in control and treated cells, respectively, at day 153).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our study demonstrated a novel application of the above concept by exploiting the DOS database to define inhibitors for newly suggested targets (like telomerase) in cancer therapy. Our cell panel of 38 different and well-characterized human cancer cell lines from 10 different tissues (lung, ovary, CNS, blood, stomach, colon, kidney, skin, breast and prostate) is more than sufficient for reflecting the chaotic nature of cancer. Similarly, our DOS database, despite being young, contains more than one thousand standard and experimental anticancer agents, and thus, it is likely to cover almost all of the known and unknown pharmacological trends in cancer.
When we tested various chemical modifications on berberine (palmatine, dehydroberberine, 13-propylberberine, 13-methyldehydroberberine, changing the methoxy with a hydroxy or ethoxy groups), none of these modifications provided a satisfactory telomerase inhibitory effect (data not shown). This indicates that the strategy of using DOS and COMPARE is very helpful in facilitating the tedious and laborious screening for effective inhibitors. After the discovery of berberine, a few molecules (20 new molecules) were screened to obtain a 17-fold stronger inhibition. Telomerase inhibitory activity did not seem to be related to the property of accumulation in the mitochondria because rhodamine123, a typical mitochondrial accumulating agent, was almost ineffective in inhibiting telomerase (Fig. 2, a and c)
.
Although the growth response of the cell line panel in the 48-h drug-test period does not reflect the toxicity buildup at a lineage level, a property that is expected in the case of specific telomerase inhibitors, the strategy of screening presented here is still effective because it identifies molecules with intrinsic similarity of interaction with the same spectrum of the chaotic biomolecular divergence in cancer cells. The final inhibitor, FJ5002, was tested on live cancer cells at a proliferation-permissive concentration and found to be effective in inducing telomere erosion and loss of cellular viability. Because in vitro (cell-free) telomerase activity was nearly equal in lysates from treated and untreated cells before entering crisis (data not shown), FJ5002 seems to have a reversible effect, and telomerase can regain activity after an approximate 100-fold dilution during the detergent extraction step of TRAP assay. Although FJ5002 at higher concentrations (0.25–5 µM for 96 h) induced a dramatic growth arrest with marked telomerase inhibition (data not shown), the affected cells did not show any features characteristic of replicative senescence or crisis (e.g., SA-ß-Gal positivity, telomere shortening, increased cell size). This indicates that the loss of cellular viability observed in the prolonged passage experiment was due to a slow buildup of a heritable defect, which is most likely the gradual telomere erosion during cell division. After the adjustment of FJ5002 intracellular concentration to 5001,000 nM, the average telomere lengths showed a steep shortening at a rate of 100 bp per division, consistent with the speculated speed of telomere erosion in the absence of telomerase (because of the DNA end replication problem). That the onset of telomere erosion coincided with a minimal range of intracellular concentrations of FJ5002 has a significant implication on the pharmacodynamics of targeting telomerase in cancer therapy. These findings suggest that the possible future clinical application of telomerase inhibitors should be accompanied by cautious monitoring of the minimal therapeutic dose to ensure persistent telomerase inhibition; otherwise, telomeres would regain their original lengths.
As a negative control, we examined the prolonged passage of adult NHLF cells (which have no telomerase activity) in the presence of 50 nM of FJ5002 and could not find any detectable difference between treated and untreated cells in terms of the rate of telomere erosion (Fig. 5d)
or morphology (data not shown). In addition to the results of telomerase inhibition in a cell-free system, the population doubling-dependent telomere erosion and crisis of U937 cells, failure of FJ5002 to induce similar effects in telomerase-negative NHLF cells, and inability of FJ5002 to interact directly with telomeric DNA (Fig. 3c
, and data not shown), altogether suggest that FJ5002-induced telomere shortening is mediated by a telomerase-inhibitory mechanism. It is noteworthy that the TRF values shown in Fig. 5e
represent the average of the total lengths of all of the telomeric and subtelomeric regions. The actual telomere shortening observed here could reach far more critical levels (1 kb), particularly if we considered a cutoff value at the lower part of the TRF lane in Southern blotting. This is in agreement with previous findings that showed that transformed human cells enter crisis once TRF reaches a length of 4 kb (14)
. Strahl and Blackburn (29)
have shown that some nucleoside analogues (dideoxyguanosine and azidothymidine at 10 and 100 µM, respectively) inhibit telomerase and induce telomere shortening in some T- and B-cell cultures but without detectable loss of cell viability. Although their study lacked in-depth investigations on inhibition mechanisms and cellular viability, they attributed the telomere-shortening resistance of these cells to the presence of telomerase-independent mechanisms for chromosome healing. In the present study, there was also a remaining subpopulation of U937 cells that continued to proliferate despite the critical level of telomere shortening. In these cells, telomerase remained sensitive to FJ5002 in vitro (data not shown). Recently, two mechanisms were proposed for explaining the survival of telomerase-deleted fission yeast, one by chromosome circulization and the other presumably by recombination (30)
. We are presently investigating these two possibilities for explaining the survival mechanism of the final lineages of U937 leukemia cells observed in the present study. Given that U937 lymphoblastoid cells, similar to cells in the immune system, are equipped with advanced DNA recombination systems for receptors and antibody production, the possible existence of a recombination mechanism for survival is highly probable. Like many other cancer cells, U937 cells are homozygously mutant for the tumor suppressor gene p53 (data not shown); thus, they could survive for a long period with short telomeres because p53 (and possibly other suppressors) senses the early events of telomere erosion as an irreparable DNA damage and induces growth-arrest before reaching a marked shortening of telomeres (31)
. It would also be interesting to test the long-term effect of FJ5002 on p53-positive cell lines.
In conclusion, our novel application of COMPARE informatics for exploiting the database generated from our cell-panel screening program enabled the identification of FJ5002 as a potent telomerase inhibitor. This compound could be potentially useful as a lead molecule for additional in vivo and clinical studies aimed at telomerase targeting.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported in part by a special grant for Advanced Research on Cancer and a Grant-in-Aid for Cancer Research from the Ministry of Education, Science, Sports and Culture, Japan. Dr. I. Naasani is supported by a grant from the Organization for Pharmaceutical Safety and Research (OPSR). 
2 To whom requests for reprints should be addressed, at Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Kami-Ikebukuro, Toshima-ku, Tokyo 170-8455. Fax: 81-3-3918-3716; E-mail: inaasani{at}ns.jfcr.or.jp
3 The abbreviations used are: DOS, disease-oriented screening program; PRINS, primed in situ hybridization; TRAP, telomeric repeat amplification protocol; TRF, terminal restriction fragment; NHLF, normal human lung fibroblast; HPLC, high-performance liquid chromatography; TS, telomerase substrate; SA-ß-Gal, senescence-associated ß-galactosidase; Q-banding, quinacrine banding.
Received 2/22/99. Accepted 6/16/99.
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