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¹ Cancer Research UK Biomolecular Structure Group, School of Pharmacy, University of London, London, United Kingdom; ² Institute of Child Health, London, United Kingdom; and ³ Antisoma Research Laboratories, St. George's Hospital Medical School, London, United Kingdom

Requests for reprints: Stephen Neidle, Cancer Research UK Biomolecular Structure Group, School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, United Kingdom. Phone: 44-207-753-5969; Fax: 44-207-753-5970. E-mail: stephen.neidle{at}ulsop.ac.uk

	Abstract

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The trisubstituted acridine derivative BRACO-19 has been designed to interact with and stabilize the quadruplex DNA structures that can be formed by folding of the single-stranded repeats at the 3' end of human telomeres. We suggest that the BRACO-19 complex inhibits the catalytic function of telomerase in human cancer cells and also destabilizes the telomerase-telomere capping complex so that cells enter senescence. Here, we present evidence showing that the inhibition of cell growth caused by BRACO-19 in DU145 prostate cancer cells occurs more rapidly than would be expected solely by the inhibition of the catalytic function of telomerase, and that senescence is accompanied by an initial up-regulation of the cyclin-dependent kinase inhibitor p21, with subsequent increases in p16^INK4a expression. We also show that treatment with BRACO-19 causes extensive end-to-end chromosomal fusions, consistent with telomere uncapping.

	Introduction

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Telomeres are repetitive DNA sequences that together with associated proteins are present at the ends of all eukaryotic chromosomes. Their primary role is to prevent loss of coding DNA via recombination, end-to-end fusions, or exonuclease action. Telomeric DNA progressively shortens in somatic cells as a consequence of the inability of DNA polymerase to fully replicate the ends. Thus, normal diploid cells have a finite replicative life span and ultimately enter a state of senescent growth arrest (M1) once a critical telomere length has been reached. By contrast, telomeres in tumor cells are maintained in length, the overwhelming majority by the action of the enzyme telomerase (1). Telomerase activity is detectable in only a subset of normal tissues but is present in >80% of tumor cells (2). Several observations, notably that inhibition of telomerase limits the growth of human tumor cells (3, 4), have led to proposals that telomerase can be a target for cancer chemotherapy.

Telomeres may be in either an open conformation, where the telomere termini are available for telomerase to act on them, or a closed, capped conformation. In the latter state, the telomere is folded back into a loop motif (5), is associated with additional telomere-associated proteins and telomerase, and is likely to have the extreme 3' end of the telomere (which is single stranded), incorporated into the loop, in a manner that has not been defined in detail. Uncapping of the telomere end leads to exposure of the critical 3' single-stranded overhang and then the induction of senescence and apoptosis (6, 7). Although many cellular pathways act to maintain senescence, two are thought to be primarily involved in its induction. These involve p53 and p16^INK4a, respectively, both of which act to inhibit the progression of the cell cycle, via the inhibition of cyclins and cyclin-dependent kinases. Wild-type p53 up-regulation activates G₁ cell cycle arrest and DNA damage repair. Increased p16^INK4a expression leads directly to the inhibition of cdk4 and cdk6 and the prevention of retinoblastoma protein phosphorylation. The p16^INK4a pathway can act independently of p53 status (8).

One approach for telomerase inhibition involves the induction of folding the 3' single-stranded telomeric DNA overhang into a four-stranded G-quadruplex structure (9). This interferes with the initial step in the elongation of telomeric DNA by telomerase (10) because effective recognition of the telomere overhang by the RNA template domain of telomerase requires its 3' end to be single stranded. Ligands that stabilize quadruplex DNA and inhibit hybridization of the 3' end with the RNA template are effectively acting as inhibitors of telomerase. Several such ligands have been reported (e.g., refs. 11, 12), with several having low micromolar or nanomolar inhibitory activity and showing a range of cellular effects consistent with telomerase inhibition. We have designed previously by molecular modeling a series of trisubstituted acridine derivatives that have high potency against telomerase (13, 14), and one compound from this series, BRACO-19 (Fig. 1), has also been shown to induce long-term growth arrest and replicative senescence in the 21NT breast carcinoma cell line and has also shown some in vivo activity against a tumor xenograft (15).

View larger version (8K): [in this window] [in a new window]   Figure 1. Structure of the G-quadruplex ligand BRACO-19.

We suggest that induction of a quadruplex DNA structure at the end of the 3' single-stranded telomeric overhang results in uncapping of the end from associated proteins. The overhang can then be sensed as a DNA damage signal. Consistent with this is the observation that BRACO-19 is a potent inducer of senescence, and increases in the expression of p21 and p16^INK4a proteins are found, which are associated with the activation of the pathway to the senescent phenotype. The effects of BRACO-19 on chromosomal integrity at metaphase have been examined, which show pronounced end-to-end chromosomal fusions, consistent with this hypothesis.

	Materials and Methods

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The G-quadruplex binding ligand BRACO-19 was synthesized in this laboratory as reported previously (13, 14). A stock was supplied as the HCl salt in aqueous solution and stored at –70°C prior to use. Pure water was used as the vehicle control.

Tissue Culture
The DU145 prostate cancer and murine 3T3 cell lines (European Collection of Cell Cultures, Salisbury, United Kingdom) were maintained in DMEM containing 10% (v/v) fetal bovine serum (Life Technologies, Paisley, United Kingdom), 0.5 µg/mL hydrocortisone (Acros Chemicals, Loughborough, United Kingdom), and 2 mmol/L L-glutamine and nonessential amino acids (Invitrogen, Groningen, Netherlands) and was incubated at 37°C and 5% CO₂. Medium was changed twice weekly. Cells were harvested by washing with PBS (Dulbecco's PBS solution A, Invitrogen), incubating in trypsin-EDTA (0.05% trypsin in 0.02% EDTA, Life Technologies) at 37°C, neutralizing with medium, and seeding at appropriate concentrations into tissue culture flasks (Costar, Corning, Corning, NY). All cells were tested for Mycoplasma.

Sulforhodamine B Growth Inhibition Assay
Growth inhibition was measured using the sulforhodamine B assay as described previously (14). Briefly, between 3,000 and 6,000 cells were seeded into the wells of 96-well microtiter plates and allowed to attach overnight. BRACO-19, as the HCl salt was dissolved at 500 µmol/L in water and immediately added to wells in quadruplicate at final concentrations of 0.05, 0.25, 1, 5, and 25 µmol/L. Following an incubation period of 96 hours, remaining cells were fixed with ice-cold 10% (w/v) trichloroacetic acid (30 minutes) and stained with 0.4% sulforhodamine B in 1% (v/v) acetic acid (15 minutes). Mean absorbance at 540 nm for each drug concentration was expressed as a percentage of the control untreated well absorbance and an IC₅₀ value (the concentration required to inhibit cell growth by 50%) was determined for the compound.

Long-term Treatment with BRACO-19
In initial experiments, varying numbers of cells were seeded in 125 mL tissue culture flasks to determine the optimum number of cells for the experiment. Treatment was planned to take place twice weekly and cells were to be counted once weekly. If cells were seeded at a very high concentration, then confluence would be achieved before 7 days and cells would be unable to grow any further. If cells were seeded too sparsely, cell growth would be reduced as cells made new cell-cell adhesions and gave false results. The optimum number of DU145 prostate cancer cells was found to be 1 x 10⁵. This number reached 75% confluence after 7 days and were growing strongly in exponential phase at this point. Cells (1 x 10⁵) were seeded in T80 tissue culture flasks (Costar, Corning) in 10 mL of medium and an appropriate concentration of BRACO-19 (as the HCl salt) or the vehicle control was added. Cells were incubated for 3 days before medium was aspirated and the cells were washed in PBS and medium plus drug was reapplied. Three days later, medium was removed and the adherent cells were trypsinized as normal and pelleted. The pellet was resuspended in medium and the cells were counted before centrifuging and storing at –80°C for extraction of DNA and protein; 1 x 10⁵ of the counted cells were reseeded in a new flask and were treated as previously. Cells were counted and reseeded once weekly. The same procedure was used for both DU145 and 3T3 cells.

Western Blotting and Senescence Staining
Western blot analysis of proteins was carried out using asynchronous cells in exponential growth phase. Bands were detected using enhanced chemiluminescence (Perkin-Elmer Life Sciences, Boston, MA). Antibodies were obtained from Santa Cruz Biochemicals [Santa Cruz, CA; p16^INK4a (C-20) and ß-tubulin loading control (D-10)] and Oncogene Research Products [San Diego, CA; p21 (Ab-1)]. Secondary antibodies were purchased in each case from Amersham Pharmacia Biotech (Little Chalfont, United Kingdom). Cells were assessed for the onset of senescence by staining for the expression of senescence-associated ß-galactosidase using a commercial kit as per the manufacturer's instructions (Invitrogen).

Chromosome Banding and Metaphase Spreads
Optimally dividing DU145 cells were incubated in RPMI 1640 with 20% FCS at 37°C. Cells were exposed to colcemid (0.05 µg/mL) for 45 minutes at 37°C and harvested routinely. Metaphase chromosomes were stained and GTG banded using a conventional trypsin-Giemsa technique and visualized using an Axioskop light microscope (Zeiss, Welwyn Garden City, United Kingdom).

	Results

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Treatment with BRACO-19 Leads to Rapid Inhibition of DU145 Cell Growth and the Onset of Senescence
A 96-hour exposure of DU145 cells to BRACO-19 showed only a modest cytotoxic effect, with an IC₅₀ value of 22.3 ± 0.8 µmol/L. Exposure of cells to the quadruplex binding ligand at a 2 µmol/L concentration would therefore not produce acute cytotoxic kill. This value exceeds by >200-fold the concentration at which there is 50% in vitro telomerase inhibition, as determined by the telomerase repeat amplification protocol assay, with a ^telEC₅₀ value for this compound of 100 ± 18 nmol/L (15). Figure 2A shows that BRACO-19 profoundly inhibits DU145 cell growth, with effects apparent after <7 days. Population doublings cease altogether after 21 days. Cells treated with vehicle control exhibit normal growth characteristics. BRACO-19-treated DU145 cells were stained for the expression of the senescence-associated marker ß-galactosidase at each time point (Fig. 2B). Western blotting was also done to measure any changes in the expression of the p21 and p16^INK4a proteins. The presence of senescence-associated marker ß-galactosidase staining in the DU145-treated cells showed that BRACO-19 treatment led to rapid induction of senescence in these cells but not in the vehicle control ones. After 7 days of treatment, almost 50% of the cell population were senescent (Fig. 3A). The cells also became very large, flat, and dense during treatment, morphology typical of senescence (observed by light microscopy; data not shown). Some increase in p16^INK4a expression was observed at 7 days of BRACO-19 treatment (Fig. 3B), with a 5.5-fold increase compared with control cells increase in expression being observed at 21 days. A sustained 3-fold increase in p21 expression was observed from days 7 to 14 (Fig. 3B). This decreased by 20% at day 21 of treatment. Few cells remained after 28 days of treatment to harvest sufficient amounts of protein for Western blots.

View larger version (12K): [in this window] [in a new window]   Figure 2. A, effect of twice-weekly treatment with 2 µmol/L BRACO-19 or a vehicle control on the growth of DU145 cells. Treatment time (days) against the total number of cells (x105) or total number of population doublings. Initial seeding was 1 x 105 cells (n = 3). B, number of DU145 cells that stained positive for senescence-associated marker ß-galactosidase (ß-gal) activity (as a percentage of total cell number) following treatment with 2 µmol/L BRACO-19 or a vehicle control. Counts were repeated three times for each time point.

View larger version (47K): [in this window] [in a new window]   Figure 3. A, Western blots against p21 and p16 in DU154 cells treated with 2 µmol/L BRACO-19 or a vehicle control. Treatment time (days) above each corresponding band. Each lane contained 6 µg of total cellular protein. (Not enough cells were present after 28 days of treatment to obtain sufficient amounts of protein for a Western blot; n = 2.) Columns, normalized expression of protein plotted against treatment time. *, P < 0.05. B, western blots against p21 (B) and p16 (C) and in DU145 cells following 24, 48, or 72 hours of treatment with a vehicle control (VC) or 2 µmol/L BRACO-19 (B-19). Columns, expression of each protein in the vehicle control or BRACO-19-treated DU145 cells following normalization with a loading control (ß-tubulin). *, P < 0.05.

Exposure of mouse 3T3 fibroblast cells to the same concentration of BRACO-19 over a period of 28 days did not produce any significant effect on the growth characteristics of this cell line (Fig. 4).

View larger version (12K): [in this window] [in a new window]   Figure 4. Effect of twice-weekly treatment with 2 µmol/L BRACO-19 or a vehicle control on the growth of murine 3T3 cells, with the treatment time (days) plotted against the total number of cells (x105). Initial seeding was 1 x 105 cells (n = 3).

View larger version (125K): [in this window] [in a new window]   Figure 5. Metaphase spreads stained with Giemsa stain from DU145 cells treated twice weekly with a vehicle control. Spreads are for 1 (A) and 3 (B) weeks. Spreads for DU145 cells treated with 2 µmol/L BRACO-19 are for 1 (C) and 3 (D) weeks.

	Discussion

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1. Inhibition of the growth of DU145 tumor cells in culture by noncytotoxic mechanisms at concentrations employed that are far below the threshold for acute cytotoxicity by this agent. Affected cells rapidly enter replicative senescence as shown morphologically and by changes in the senescence-associated proteins p21 and p16^INK4a.
   
2. Induction of chromosomal end-to-end fusions in DU145 cells in the same time scale. These are distinct from random chromosomal abnormalities and visually show a maximum of two fusions that is consistent with BRACO-19 induction of telomere uncapping. The small number of fusions that were not exclusively end-to-end in control cells was unsurprising, as tumor cells generally display such fusions both as a cause and as a consequence of their genetic instability. However, the possibility of chromosome translocation cannot be disregarded due to the complexity of the metaphase configurations found in the treated cells. In the latter, individual chromosome banding patterns were not readily discernable due to the morphology of the long fused chromosomes. Ultimately, the points of fusion in the majority of these chromosomes were visually evaluated as being telomeric in origin.
   

The observed effects on cell growth have some parallels with those of conventional cytotoxic agents, such as Adriamycin (16, 17), where telomere dysfunction has also been implicated. However, by contrast with BRACO-19, these drugs produce such effects at cytotoxic concentrations with these agents and also produce other nonspecific effects that relate to DNA duplex binding.

The data presented here suggest that the ability of BRACO-19 to induce short-term growth arrest and replicative senescence is not dependent on changes in the average telomere length of this cell line, as growth inhibition and senescence were apparent after only 7 days of treatment, with growth ceasing completely after 21 days. DU145 cells have a mean telomere length of 2 kb. Their mean population doubling time is 53 hours (calculated from Fig. 1). Assuming that 100 to 200 bp of DNA are lost per round of replication, the onset of replicative senescence and thus growth arrest should start occurring after no >24 days if it is due solely to inhibition of the catalytic function of telomerase. The present observations are therefore consistent with a model in which BRACO-19 directly binds to the 3' single-stranded overhang and induces G-quadruplex formation. This four-stranded structure is incompatible with telomerase attachment to the 3' single-stranded overhang. We suggest that induction of the G-quadruplex complex is functionally equivalent to direct 3' end exposure (6, 7), which is known to rapidly produce destabilization of telomere maintenance and induction of DNA damage responses via telomere uncapping (18–20). The effects reported here may not depend on the initial shortness of telomeres in target cells; we have shown that equivalent effects are produced in cell lines such as SKOV-3, which has a much longer mean telomere length compared with DU145 cells (>5 kb).⁴ However, we cannot rule out the strong possibility of effects if there is a critical subpopulation of cells with especially short telomeres (21), for which telomerase inhibition would very rapidly lead to senescence. We also cannot exclude other telomere-associated mechanisms, including interference with telomere-associated proteins other than telomerase.

Telomerase inhibition and/or telomere uncapping have been shown previously to induce the activation of senescence pathways in tumor cells (6, 7) as well as end-to-end telomere fusions (20). The induction of senescence has been reported to be associated with increases in the expression of p21 and p16^INK4a. We find that there is a large (3-fold) increase in p21 expression in the initial 72-hour period after the start of BRACO-19 treatment, which continues until about day 14, when it starts to level off. By contrast, the level of p16^ink4a expression does not alter until approximately this time, when it also increases 3-fold. These changes seem to be largely independent of p53 status, because the DU145 cell line has been characterized as mutant p53, although p53-dependent apoptosis has been reported in this cell line (22). The data presented here suggest a model in which p21 is involved in detecting the uncapping and initiates the senescence pathway and then p16^INK4a maintains it in an activated state. This is in agreement with a recent report (23) demonstrating that telomere shortening triggers senescence through a p21, not a p16^INK4a, pathway.

The ability of G-quadruplex binding small molecules such as BRACO-19 that possess only low levels of cytotoxicity to rapidly induce growth arrest in tumor cells is in striking contrast to the long timeframe originally envisaged for the inhibition of telomerase catalytic activity to translate into senescence. It suggests that a therapeutic approach based on them would similarly produce significant anticancer responses without having the problem of an extended time scale for effects to be manifest. Thus, these data provide a further rationale for our intention to select a preclinical development candidate compound from this series of compounds for entering clinical trials. We are encouraged in this goal by the relative lack of toxicity of BRACO-19 in the normal IMR90 fibroblast cell line, with an IC₅₀ >5-fold lower than with tumor cell lines, and by the lack of effects on the growth characteristics of the murine fibroblast cell line 3T3 (Fig. 4). These cells have exceptionally long telomeres and are telomerase positive, suggesting that this combination, which compares with the telomeric characteristics of human germ line telomeres, results in insensitivity to BRACO-19. Interestingly, the ALT line GM847, which also has extended telomeres but is telomerase negative, is highly sensitive to this agent.⁵

	Footnotes

 
Grant support: Cancer Research UK grant C129/A406 (S. Neidle) and research studentship (C.M. Schultes), and Institute of Cancer Research studentship (C.M. Incles).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

⁴ C. Incles and H. Koehler, unpublished observations.

⁵ C. Gerner and S. Neidle, unpublished observations.

Received 4/ 2/04; revised 7/13/04; accepted 8/20/04.

	References

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