Zinc protoporphyrin binding to telomerase complexes and inhibition of telomerase activity

Abstract Zinc protoporphyrin (ZnPP), a naturally occurring metalloprotoporphyrin (MPP), is currently under development as a chemotherapeutic agent although its mechanism is unclear. When tested against other MPPs, ZnPP was the most effective DNA synthesis and cellular proliferation inhibitor while promoting apoptosis in telomerase positive but not telomerase negative cells. Concurrently, ZnPP down‐regulated telomerase expression and was the best overall inhibitor of telomerase activity in intact cells and cellular extracts with IC50 and EC50 values of ca 2.5 and 6 µM, respectively. The natural fluorescence properties of ZnPP enabled direct imaging in cellular fractions using non‐denaturing agarose gel electrophoresis, western blots, and confocal fluorescence microscopy. ZnPP localized to large cellular complexes (>600 kD) that contained telomerase and dysskerin as confirmed with immunocomplex mobility shift, immunoprecipitation, and immunoblot analyses. Confocal fluorescence studies showed that ZnPP co‐localized with telomerase reverse transcriptase (TERT) and telomeres in the nucleus of synchronized S‐phase cells. ZnPP also co‐localized with TERT in the perinuclear regions of log phase cells but did not co‐localize with telomeres on the ends of metaphase chromosomes, a site known to be devoid of telomerase complexes. Overall, these results suggest that ZnPP does not bind to telomeric sequences per se, but alternatively, interacts with other structural components of the telomerase complex to inhibit telomerase activity. In conclusion, ZnPP actively interferes with telomerase activity in neoplastic cells, thus promoting pro‐apoptotic and anti‐proliferative properties. These data support further development of natural or synthetic protoporphyrins for use as chemotherapeutic agents to augment current treatment protocols for neoplastic disease.

The natural fluorescence properties of ZnPP enabled direct imaging in cellular fractions using non-denaturing agarose gel electrophoresis, western blots, and confocal fluorescence microscopy. ZnPP localized to large cellular complexes (>600 kD) that contained telomerase and dysskerin as confirmed with immunocomplex mobility shift, immunoprecipitation, and immunoblot analyses. Confocal fluorescence studies showed that ZnPP co-localized with telomerase reverse transcriptase (TERT) and telomeres in the nucleus of synchronized S-phase cells. Zn PP also co-localized with TERT in the perinuclear regions of log phase cells but did not co-localize with telomeres on the ends of metaphase chromosomes, a site known to be devoid of telomerase complexes. Overall, these results suggest that ZnPP does not bind to telomeric sequences per se, but alternatively, interacts with other structural components of the telomerase complex to inhibit telomerase activity. In conclusion, ZnPP actively interferes with telomerase activity in neoplastic cells, thus promoting pro-apoptotic and anti-proliferative properties. These data support further development of natural or synthetic protoporphyrins for use as chemotherapeutic agents to augment current treatment protocols for neoplastic disease.

| INTRODUC TI ON
Telomerase is a cellular reverse transcriptase that is reactivated in about 85% of all cancers. 1 The enzyme maintains adequate lengths of chromosomal 3' DNA telomeric strand ends, which are continuous sequences of -(TTAGGG)n--that progressively shorten with each replication cycle because of the DNA polymerase 3' end replication problem. Using an RNA template, telomerase adds complementary DNA bases to the 3' telomere end which prevents chromosomal end damage and enables prolonged cellular proliferation, the hallmark of cancer cells. In rapidly dividing malignant cells, telomeres need constant repair to enable high replication rates. 2 This activity is so crucial to malignancy that even in the 15% of neoplastic cells that do not express telomerase, telomeric ends are maintained by an alternative recombination process. 3 Considerable evidence supports the feasibility of telomerase inhibitors as chemotherapeutic agents 4-6 for a number of neoplastic diseases. As a class, planar, positively charged polyaromatic compounds such as porphyrins have been shown to have anti-telomerase activity. 7 Porphyrins are known to bind and stabilize single-stranded telomeric DNA sequences at guanine secondary structures known as quadruplexes (G4) 8 and impact telomerase presumably through substrate inhibition. 7,9,10 Metalloprotoporphyrins (MPP) such as FePP and ZnPP, represent a subclass of important naturally occurring porphyrins that are also known to bind G4 structures in general 11 and some telomeric sequences specifically. 12,13 Additionally, ZnPP and conjugated derivatives such as ZnPP-polyethylene glycol have been widely studied in experimental rodent systems for use as chemotherapeutic agents. 14,15 Considering the widespread interest in porphyrins as telomerase inhibitors as well as work with ZnPP as a chemotherapeutic agent, it is surprising that no studies have addressed potential interactions of ZnPP and other MPPs with telomerase.
The aim of the present study was to determine whether common MPPs impact telomerase expression and enzymatic activity in established hepatoma cells. We show that ZnPP abruptly halts DNA synthesis and promotes apoptosis, while concomitantly depressing the expression of telomerase as well as other proliferative proteins such as cyclin D1 and β-catenin. Furthermore, ZnPP effectively inhibits telomerase activity in intact cells, crude cellular lysates, and immunoprecipitates (IP) while localizing to large protein complexes that contain telomerase. The effects of ZnPP on apoptosis and toxicity were observed primarily in cells that contain telomerase, thus directly implicating a role for telomerase inhibition in the chemotherapeutic activities of ZnPP. These data indicate that ZnPP and perhaps other natural or synthetic MPPs should be studied further for use as chemotherapeutic agents in a wide range of telomerase positive neoplasms.
Electrophoresis supplies were purchased from Bio-Rad (CA).

| Cell lines and cell culture
Huh7, Hek293, and the HCV permissive clonal line Huh7.5 cells were maintained in routine cultures as described. 16 The human hepatoma cell line (Huh5.15) with replicating sub-genomic HCV RNA (genotype 1b) (Huh5.15NS) 17 was cultivated as described. 18 Wild type Hek293 cells were obtained from University of Iowa Tissue Culture stocks and passed routinely in minimal essential medium containing 10% fetal bovine serum. Telomerase negative U2OS osteosarcoma cells were obtained from American Type Culture Collection and passed using recommended media conditions. U2OS extracts were routinely tested by immunoblot analysis to ensure TERT negativity.

| Cell proliferation
MPPs were tested for effects on cell proliferation and viability using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye conversion assay (Cell Titer 96, Promega) as we described 22 with some modifications. Cells were plated into 96 well plates and allowed to attach overnight. MPPs were added to the cultures and then incubated for the times indicated. At assay time, MTT reagent was added and absorbance measured at 570 nm. Controls included buffer blanks containing MPP because of background absorbances reported for MPPs by us 22 and others. 23 The formula used for determination of viable cells relative to controls was: Cell viability was also directly determined with trypan blue staining as previously described and closely correlated with MTT assay. 24

| Apoptosis assay
For detection of MPP apoptotic effects, cells were treated with MPPs (0, 2.5, 5 and 10 µM) for 48 h and assayed using PE Annexin V Apoptosis Detection Kit (Cat. No. 559763, BD Biosciences) as recommended by the manufacturer.

| Quantification of telomerase activity by realtime quantitative PCR and TRAP assay
Telomerase reverse transcriptase (TERT) enzymatic activity was determined using Telomeric Repeat Amplification Protocol (TRAP) with Real-time quantification as described previously, 25 or measured directly using α-32 P-dGTP incorporation as described 26  telomerase activity was derived from a standard curve of reference samples and data were analyzed using relative fluorescence units as compared to controls. In some cases, telomerase reaction products were visualized and quantified with TRAPeze system (EMD/Millipore).
After separation of the DNA products using 10% non-denaturing PAGE and SYBR fluorescence labeling [1x SYBR safe DNA gel stain (Invitrogen)], gel bands were imaged with iBright 1500 (Invitrogen).
The intensity of the sample's TRAP ladder and internal control was first measured using GelAnalyzer [GelAnalyzer 19.1 (www.gelan alyzer. com)] as recommended. Then, the relative telomerase activity was determined by the ratio of the intensity of the sample's TRAP ladder (telomerase products, TP) to that of the internal control (IC) band.

| Direct telomerase activity assay
For direct telomerase activity assay, a modified procedure of

| TERT overexpression and Immunoprecipitation
Plasmid pCl neo-hEST2 together with TERC (pBS U3-hTR-500) were transfected into log phase Hek293 cells and non-denaturing cellular lysates were prepared 48 h later in lysis buffer (Cell Signaling Technology, Beverly, MA). Immunoprecipitation was performed as described previously. 21 Briefly, transfected cells were harvested, Normal rabbit or mouse IgG was always used as a control (Santa Cruz, CA). Aliquots of the IP were also assayed in triplicate by TRAP assay and quantified using realtime PCR as described above. In some cases, aliquots were electrophoresed on non-denaturing agarose gels after treatment with MPP and/or nucleases.

| SDS-Polyacrylamide gel electrophoresis (PAGE) and Western blot assays
For SDS -PAGE, cellular lysates, and protein preparations such as IP were dissolved in Laemmli buffer, boiled for 1 min and separated on denaturing SDS gels as described. 28 After electrophoretic transfer of separated proteins to nitrocellulose sheets, western blot immunoassays employed enhanced chemiluminescence for signal detection (ECL TM Prime, Amersham). 28

| Cellular fluorescence labeling
Cells were grown to semi-confluency while attached to coverslips, then (green) and Alexa Fluor 568 (red) filters were used to visualize TERT and ZnPP respectively. antibodies for immunofluorescence or labeled with ZnPP or telomere probe as described above.

| S-phase synchronized cells
To synchronize cells in S phase, we performed double thymidine block essentially as described 29 with modifications. Briefly, Huh7 cells were seeded onto coverslips and then treated with 2 mM thymidine (Sigma, T9250) for 18 h, released for 9 h, again treated with 2 mM thymidine for 18 h, then released 2 h before use. To determine the optimal timepoint for collecting cells in S phase, we assayed cultures with flow cytometry and Western blot for cyclin A expression ( Figure S4). The cells were fixed with 4% paraformaldehyde and washed with PBS. Telomere FISH and ZnPP staining was then conducted as described below.

| Fluorescence in situ hybridization (FISH)
We performed telomere FISH 16 using a peptide nucleic acid (PNA) probe specific to telomeres, and labeled with TelC-Alexa488 (PNA Bio, Newbury Park, CA,) as per the manufacturer's instructions with modifications. Briefly, 0.2 µl of PNA probe was added to 20 µl of hybridization solution which was used to cover cells attached to slides.
Hybridization was at 80°C for 10 min, then 37ºC overnight. After washing, slides were incubated with 10 μM ZnPP 2 h at room temperature, washed twice in PBS, and mounted with VECTASHIELD.
Confocal fluorescence microscopy used a Zeiss LSM710 confocal fluorescence microscope using 63x oil objective.

| Statistical determinations
All mean values for enzymatic and proliferation assays were deter-

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guide topha rmaco logy.  with their minimal effects on DNA synthesis and apoptosis (Figure 1).
The effects of ZnPP on TERT expression were apparent in different Huh 7 constructs and, interestingly, in a NS 5.15 HCV replicon, ZnPP promoted disappearance of both 120 kDa telomerase monomer as well as the 45 kDa C-terminal TERT fragment that we previously reported to be specific for HCV infected cells ( Figure 2B). 21 We next evaluated the effects of MPPs on telomerase activity in cultured cells ( Figure 3A) as well as non-denatured cell lysates ( Figure 3B-E). In cultured cells incubated with ZnPP, telomerase activity was reduced in a dose-dependent fashion, (EC 50 = 5.6-5.8 µM, upper and middle panels respectively) while SnPP or FePP, had none to mild effects (EC 50 > 10 µM, either cell line) ( Figure 3A). The loss of telomerase activity with time of ZnPP treatment in the NS 5.15 HCV replicon ( Figure 3A, lower panel) reflected the disappearance of TERT seen in the WB ( Figure 2B).
The possibility that MPPs can directly inhibit telomerase activity in cellular extracts, similar to porphyrin quadruplex ligands, 6 was addressed next. Because of concerns that some quadruplex ligands inhibit Taq DNA polymerase in addition to telomerase, we assayed MPP inhibition at both steps of the TRAP procedure with a strategy similar to that of others. 36 Using equivalent extracts but separate assays, MPP was either included in the telomerase RT extension step or the extension step was conducted without MPP and then MPP added only for the amplification steps with Taq DNA polymerase.
To avoid further potential errors introduced by Real-time quantification, TRAP products were labeled with SYBR green, visualized on denaturing gels, and each lane was quantified by density measurements as described in the Methods. The latter step also ruled out the possibility that decreases in activity were artifactual due to fluorescence signal quenching by some MPPs. 37 ZnPP was significantly more active (IC 50 = 2.5 µM) than FePP and SnPP (both IC 50 > 10.0 μM), ( Figure 3B-D respectively). All three MPPs had minimal effects on Taq polymerase during telomerase product extension and the slight inhibition of Taq polymerase seen for ZnPP was not directly concentration-dependent. However, at least one MPP, CoPP, clearly inhibited Taq polymerase and could not be reliably assayed via TRAP assay (see Figure S2).
To confirm that ZnPP specifically inhibited telomerase as seen in the TRAP assays, direct telomere extension assays were conducted in the presence of α-32 P-dGTP. An IC 50 of 3.8 μM for ZnPP obtained by direct extension assay ( Figure 3E) was quite similar to the IC 50 obtained with TRAP assay (2.5 μM) ( Figure 3B Table 2. ZnPP, SnPP, and non-metal, "free" Lewis base protoporphyrins exhibit autofluorescence 37 ; a property that has proven useful to study intracellular activities of MPPs such as nuclear localization and DNA or cellular adduct binding. 13 fluorescence emission. We investigated ZnPP binding to native, nondenatured telomerase-containing complexes after separation on large pore, (0.8%), agarose gels. 27,40 Initially, non-denaturing acrylamide gels were considered for these studies, however, we discovered that ZnPP labeled complexes would not enter the largest pore size possible, a result also noted by others 23,39 (see Figure S3).
Initially, increasing amounts of cellular extracts were incubated with varying amounts of ZnPP, then electrophoresed on large pore agarose gels. ZnPP was then visualized fluoroscopically using visible red wavelengths [608-632 nm Ex and 675-720 nm EM] or wide band UV ( Figure 4A upper and lower panels respectively). ZnPP bound to high molecular weight complexes in a concentration dependent manner and the complexes electrophoresed with a mobility just above thyroglobulin (670 kD), quite similar to sizes noted by us and others for TERT ribonuclear protein particles separated by glycerol gradient centrifugation 21,41 and large pore agarose/acrylamide gels. 40 Under these conditions, free ZnPP migrated slightly cathodal. While ZnPP binding was easily identified in cellular extracts, no binding was detectable in bulk protein incubations of BSA or IgG ( Figure 4B). ZnPP also labeled complexes in intact cells

F I G U R E 3 MPP inhibition of telomerase enzymatic activity. (A) Telomerase inhibition by MPP in cultured cells. Log phase Huh7, Hek293
and Huh5.15 HCV replicon cells, (upper, middle, and lower panels, respectively), were incubated with MPPs (ZnPP, SnPP, or FePP) for 48 h, then telomerase activity was determined by TRAP assay in enzymatic lysate. Points represent mean ± SD, n = 6, ANOVA, #p < .01, *p < .001, **p < .01 paired t-test of ZnPP versus other MPPs or vehicle only controls. (B-D) Telomerase inhibition by MPP in enzymatic extracts. Enzymatic extracts were prepared from semi-confluent phase Huh7 cells and aliquots were assayed in triplicate using TRAP assay. MPPs were added to RT-PCR reactions either before or after the RT telomere elongation step to test whether MPPs inhibit Taq polymerase. Visualization of amplified telomeric products was on 10% non-denaturing polyacrylamide gels using SYBR fluorescence labeling (upper panels) followed by quantification with densitometry and plotted (lower panel). IC = Internal Taq polymerase control. Plotted points represent the mean ± SD with n = 3. (E) MPP inhibition of telomerase activity as determined with direct α-32 P-dGTP extension assays. Aliquots of IP eluates from 10 7 Hek293 cell pellet lysates overexpressing hTERT, TERC, and dyskerin were incorporated into direct telomere extension assays using biotin-linked DNA substrate. Reactions were incubated at 37ºC for two hours, then biotin labeled products purified with strepavidin linked agarose beads and electrophoresed on denaturing acrylamide gels. Bands were visualized radiographically (upper panel) and relative activity was quantified with densitometry (lower panel). Points represent mean ± SD, n = 3, ANOVA *p < .01 paired t test of ZnPP versus other MPPs  Figure S3).

Telomerase inhibition by MPP in intact cells
ZnPP labeled complexes from Huh7 cells were next blotted onto nitrocellulose by capillary diffusion (conditions determined empirically, see Figure S4) and probed with specific anti-TERT or anti-dyskerin antibodies, the latter a positive control for telomerase holoenzyme ( Figure 4E). Both TERT and dyskerin were easily identified in the high molecular weight complexes binding ZnPP ( Figure 4E).
To investigate whether nucleic acids are components of the ZnPP labeled complexes, IP or crude lysates, were digested with DNase I or RNase A prior to labeling with ZnPP ( Figure 5C telomerase holoenzyme only associates with telomeres at DNA replication during S phase. 45 Consequently, we compared ZnPP localization in synchronized S-phase cells as compared to metaphase chromosomes which contain prominent telomere ends without telomerase. The percentages of cells in S phase were determined temporally with flow cytometry after double thymidine block and extracts were monitored on immunoblots with Cyclin A2 staining to determine optimal times for study, (Figure S5). Telomere sites were labeled with telomere sequence-specific fluorescent probe (PNA TeIC-Alexa488) and TERT was localized with specific antibodies ( Figure 6).
First, we looked at ZnPP co-localization with the telomere probe. While ZnPP clearly localized with telomeres in S phase cells ( Figure 6A left panels), it did not label the prominent telomeres on metaphase chromosome tips, ( Figure 6A, right panels) which are devoid of holoenzyme. Next, we investigated whether ZnPP would co-localize with TERT in S phase as compared to unsynchronized Huh7 cells. In S phase cells, TERT co-localized with ZnPP in the nucleus and at some cytoplasmic sites ( Figure 6B left panels). As we reported previously, TERT is sparsely present in the nucleus of unsynchronized, log phase Huh7 cells, but is chiefly found at perinuclear sites which co-localize with mitochondria. 21 Interestingly, even perinuclear TERT, likely lacking telomeric DNA, showed avid TERT-ZnPP co-localization ( Figure 6B, right panels).
Collectively, these data indicate that ZnPP can bind to telomerase complexes and/or associated components. While telomeric DNA does not appear to be a primary binding site of ZnPP per se, at least at prominent telomeres on metaphase chromosomes, the specific sites of interaction in the telomerase holoenzyme remain to be determined.

| DISCUSS ION
The development of telomerase inhibitors for use in chemotherapy began shortly after the discovery of telomerase over three decades  were shown to cause cell growth arrest and apoptosis in neoplastic cells which demonstrated that G4 telomeric binding sites are useful targets for chemotherapy. 10,48 In contrast to porphyrins, protoporphyrins have not been investigated for anti-telomerase behavior. Nevertheless, common MPPs such as FePP and ZnPP are known to bind selected oligomeric G-4 sequences in vitro, 13,49 thus suggesting that they are capable of behaving like porphyrins for telomerase inhibition. Biologically, ZnPP and longacting conjugates such as ZnPP-polyethylene glycol have been studied as chemotherapeutic agents in vivo in rodent models for some time 14 and promote tumor regression, both alone as well as synergistically with drugs such as cisplatin. 15 Consequently, investigation of ZnPP effects and sites of action on telomerase activity is timely and important.
We first evaluated the effects of common MPPs on telomerase activity, cellular proliferation, DNA synthesis, and apoptosis. ZnPP  behavior. Downregulation of TERT was also accompanied by a reduction in cyclin D1 and β-catenin which are not only important markers of cellular proliferation and apoptosis per se, 51 but they have known positive signaling relationships with telomerase. 34,35 The data shown in Figure 1 also support earlier work that documented the specific anti-proliferative actions of ZnPP. 23 ZnPP directly inhibited telomerase enzymatic activity in cellular lysates, IP, and intact cells in culture. This was verified by both TRAP and direct α-32 P-dGTP extension assays with similar IC 50 (Table 1).
To date, this is the first demonstration that ZnPP has direct antitelomerase activity and these findings support consideration of  (Table 2) is also close to the micromolar ranges of IC 50 reported for other synthetic experimental porphyrins. 6,46 The major components of the telomerase holoenzyme complex include TERT, dyskerin, p23, Hsp90, TERC and telomerase-associated protein. 53 TERT is absent from telomeres until it is assembled into telomerase holoenzyme and then recruited to selective chromosomal telomeric sites at the start of S phase. 45,54 Telomere addition is known to be coupled to DNA synthesis and the processes likely occur sequentially in neoplastic cells. 45 While the latter interaction may very well account for the profound depression of DNA synthesis in neoplastic cells, we cannot rule out the possibility that ZnPP also has a direct effect on DNA polymerases.
With the structural and functional characteristics of telomerase in mind, we investigated whether ZnPP can bind at or near Confocal immunohistochemical experiments demonstrated that ZnPP binds at or near telomere/telomerase complexes in S phase cells, which is the only time in the cell cycle when telomerase is found at telomeres. 45 Surprisingly, telomere rich sites on metaphase chromosomal ends did not overtly bind ZnPP ( Figure 6A). Consequently, ZnPP interactions with telomerase complexes appear more complex than just telomere G4 binding and may depend on S phase chromatin structure, site accessibility, and composition of the complexes. Furthermore, these data suggest there may be a primary interaction of ZnPP with TERT or closely associated proteins since there was co-localization of TERT and ZnPP in cytoplasmic as well as nuclear locations ( Figure 6B). Interestingly, earlier work showed that ZnPP and Aside from telomerase, other cellular sites, both nuclear and cytoplasmic, have been proposed to be responsible for the pro-apoptotic qualities of ZnPP. Since ZnPP is both a transcriptional inducer for heme oxygenase-1 (HO-1) 57 and a competitive HO-1 inhibitor, 58 earlier studies attributed many ZnPP mechanisms to HO-1 antagonism.
Presently it is not known whether ZnPP actions on the telomerase system are impacted by HO-1 antagonism and this concept requires further study. ZnPP also inhibited transcriptional promoter sites for cyclin D1 23 and attenuated Wnt/β-catenin expression leading to increased apoptosis. 59 Our proliferation studies support these findings, yet it is not yet clear whether telomerase inhibition may impact these other pathways.

| CON CLUS IONS
In summary, our data indicate that ZnPP interacts with the telomerase enzyme system at two major regulatory points: (1) downregulation of TERT and (2) direct inhibition of telomerase enzymatic activity.
Concomitantly, ZnPP attenuates DNA synthesis and cellular proliferation while promoting apoptosis. Structurally, ZnPP binds to TERT containing ribonuclear protein complexes and co-localizes with a subset of nuclear telomeres that likely contain holoenzyme complexes. Our findings support the use of ZnPP and potentially the development of other synthetic or natural protoporphyrins for use as chemotherapeutic agents in the treatment of neoplastic disease. Ongoing further experiments aim to characterize the binding and inhibition sites of ZnPP in the telomerase complexes.

ACK N OWLED G EM ENT
None.

CO N FLI C T O F I NTE R E S T
The authors declare there are no conflicts of interests.

E TH I C S S TATEM ENT
This paper is the original and authentic work of the authors. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data underlying the results are freely available as part of the article and no additional source data are required.