**4. Telomere instability produced by anticancer drugs in mammalian cells**

In the next sections, we will consider the main data available concerning the short- and longterm telomere instability induced by anticancer drugs in mammalian cells. Firstly, we will refer to those drugs whose effects on telomeres have been intensively investigated: bleomy‐ cin, streptonigrin, streptozotocin, paclitaxel, cisplatin, doxorubicin, etoposide and 5-azacyti‐ dine. Afterwards, we will shortly refer to other anticancer drugs whose effects on telomeres are barely known, such as gemcitabine, C-1027, ICRF-193, melphalan and 5-fluorouracil. It is important to bear in mind that, in this chapter, when we refer to telomeres, we refer to the very end of the chromosomes (which, at the molecular level is constituted by TTAGGG repeats and the associated RNA and proteins), not the subtelomeric region of them (constitut‐ ed by telomere-specific sequences located near the telomere). Therefore, we will not refer to those studies involving the effects of anticancer drugs on the subtelomeric regions of the chromosomes.

### **4.1. Bleomycin (BLM), Streptonigrin (SN) and Streptozotocin (STZ)**

Several years ago, we carried out in our laboratory a series of experiments to determine the short-term effects of three antibiotics with anticancer properties, bleomycin (BLM), streptoni‐ grin (SN) and streptozotocin (STZ) on mammalian telomeres and telomeric sequences (see [26] for review).

BLM (CAS No. 11056-06-7) is a chemotherapeutic drug isolated from *Streptomyces verticillus* which is commonly used to treat testicular cancer, lymphoma, lung cancer, cervical cancer and cancers of the head and neck [50]. This antibiotic is an S-independent clastogen and a radio‐ mimetic agent that generates free radicals and induces single- and double-strand breaks in DNA [50, 51]. SN (CAS No. 3930-19-6) is an aminoquinone antitumor antibiotic isolated from cultures of *Streptomyces flocculus*, which shows antitumor activity against a broad range of tumors, including breast, lung, head and neck cancer, lymphoma and melanoma [52], although its use in cancer therapy is very limited because it induces severe and prolonged bone marrow depression [52]. Despite of being considered a radiomimetic compound, SN is capable of producing chromosome damage both by S-independent and S-dependent mechanisms [52]. Moreover, SN causes inhibition of topoisomerase II by stabilizing the transesterification intermediate of the enzyme (called cleavable complex) [52]. STZ (CAS No. 18883-66-4) is is an antibiotic isolated from *Streptomyces achromogenes* [53, 54], usually used to experimentally induce diabetes mellitus in laboratory animals, and it has been considered a potential com‐ pound for the clinical treatment of some malignant diseases, including advanced pancreatic neuroendocrine tumors and colon cancer. STZ is a potent alkylating agent that directly methylates DNA, giving rise to chromosome and DNA damage [53, 54]. STZ exerts its clastogenic effect mainly in an S-dependent manner, inducing both chromatid- and chromo‐ some-type aberrations [53, 54].

The abovementioned studies, perfomed using FISH with a Peptide Nucleic Acid telomere probe (telomere PNA-FISH) in Chinese hamster cells (CHE cell line), showed that all the above drugs can induce the formation of incomplete chromosomes and terminal fragments [55-58]. These observations were made on metaphase cells obtained 18 h after treatment (i.e., in cells in their first mitosis after treatment) and indicated that, despite of the fact that BLM, SN, and STZ act on chromosomes in a different way, these drugs can induce short-term telomere instability by chromosome end loss in mammalian cells. The induction of short-term telomere instability by BLM was also demonstrated by Benkhaled et al. [59] in human lymphocytes. More recently, our studies were focused on the effects of these antibiotics on the progeny of the exposed cells, to determine if telomeres play some role in the long-term chromosomal instability induced by these drugs and if telomere instability can persist in the exposed cells for several generations after treatment. To accomplish our goal, we exposed rat cells (ADIPO-P2 cell line) to a single pulse of BLM, SN or STZ, and determined the type and frequency of chromosomal aberrations at 18 h (first mitosis after exposure), 10 days and 15 days after treatment by using PNA-FISH with a telomeric probe.

**4. Telomere instability produced by anticancer drugs in mammalian cells**

In the next sections, we will consider the main data available concerning the short- and longterm telomere instability induced by anticancer drugs in mammalian cells. Firstly, we will refer to those drugs whose effects on telomeres have been intensively investigated: bleomy‐ cin, streptonigrin, streptozotocin, paclitaxel, cisplatin, doxorubicin, etoposide and 5-azacyti‐ dine. Afterwards, we will shortly refer to other anticancer drugs whose effects on telomeres are barely known, such as gemcitabine, C-1027, ICRF-193, melphalan and 5-fluorouracil. It is important to bear in mind that, in this chapter, when we refer to telomeres, we refer to the very end of the chromosomes (which, at the molecular level is constituted by TTAGGG repeats and the associated RNA and proteins), not the subtelomeric region of them (constitut‐ ed by telomere-specific sequences located near the telomere). Therefore, we will not refer to those studies involving the effects of anticancer drugs on the subtelomeric regions of the

Several years ago, we carried out in our laboratory a series of experiments to determine the short-term effects of three antibiotics with anticancer properties, bleomycin (BLM), streptoni‐ grin (SN) and streptozotocin (STZ) on mammalian telomeres and telomeric sequences (see [26]

BLM (CAS No. 11056-06-7) is a chemotherapeutic drug isolated from *Streptomyces verticillus* which is commonly used to treat testicular cancer, lymphoma, lung cancer, cervical cancer and cancers of the head and neck [50]. This antibiotic is an S-independent clastogen and a radio‐ mimetic agent that generates free radicals and induces single- and double-strand breaks in DNA [50, 51]. SN (CAS No. 3930-19-6) is an aminoquinone antitumor antibiotic isolated from cultures of *Streptomyces flocculus*, which shows antitumor activity against a broad range of tumors, including breast, lung, head and neck cancer, lymphoma and melanoma [52], although its use in cancer therapy is very limited because it induces severe and prolonged bone marrow depression [52]. Despite of being considered a radiomimetic compound, SN is capable of producing chromosome damage both by S-independent and S-dependent mechanisms [52]. Moreover, SN causes inhibition of topoisomerase II by stabilizing the transesterification intermediate of the enzyme (called cleavable complex) [52]. STZ (CAS No. 18883-66-4) is is an antibiotic isolated from *Streptomyces achromogenes* [53, 54], usually used to experimentally induce diabetes mellitus in laboratory animals, and it has been considered a potential com‐ pound for the clinical treatment of some malignant diseases, including advanced pancreatic neuroendocrine tumors and colon cancer. STZ is a potent alkylating agent that directly methylates DNA, giving rise to chromosome and DNA damage [53, 54]. STZ exerts its clastogenic effect mainly in an S-dependent manner, inducing both chromatid- and chromo‐

The abovementioned studies, perfomed using FISH with a Peptide Nucleic Acid telomere probe (telomere PNA-FISH) in Chinese hamster cells (CHE cell line), showed that all the above drugs can induce the formation of incomplete chromosomes and terminal fragments [55-58].

**4.1. Bleomycin (BLM), Streptonigrin (SN) and Streptozotocin (STZ)**

chromosomes.

124 Telomere - A Complex End of a Chromosome

for review).

some-type aberrations [53, 54].

We found that BLM induces persistent telomere instability in mammalian cells, cytogenetically manifested as incomplete chromosome elements (i.e., chromosome end loss) and telomere FISH signal loss and duplication (i.e., telomere dysfunction) ([60] and Paviolo, unpublished data). In addition, our results suggested that BLM can induce delayed telomere instability in the form of telomere (end-to-end) fusions. Therefore, we concluded that BLM induces telomere instability at the chromosome level both by chromosome end loss and telomere dysfunction [60]. The delayed appearance of dicentric chromosomes and telomere fusions (which produces dicentric chromosomes without accompanying fragment) that we observed in ADIPO-P2 cells exposed to BLM suggests that the BFB cycles [27] might play a significant role in the mainte‐ nance of the long-term telomere instability induced by this compound. In effect, by inducing breakage at terminal regions of chromosomes, resulting in incomplete chromosomes, BLM could promote genome instability through BFB cycles, which can continue for multiple cell generations, leading to extensive chromosomal rearrangements in the progeny of the cells exposed to this compound. According to our data, the persistent telomere instability induced by BLM in rat cells is neither related to telomerase activity nor telomere length variations ([60] and Paviolo, unpublished data).

In the case of SN, we found that this drug induces persistent (i.e., up to 15 days after treatment) telomere dysfunction in ADIPO-P2 cells in the form of additional telomeric FISH signals, extrachromosomal telomeric FISH signals, and telomere FISH signal loss and duplications [61]. Several studies have provided a large body of evidence indicating that SN directly interacts with DNA, binding covalently but not interacting into the double helix (see [52] for review). Therefore, the persistence of the clastogenic action of SN in terms of telomere-associated aberrations could be due to the formation of a stable complex between SN and the DNA molecule, which may induce chromosome damage through a persistent cyclic redox process and the resulting generation of active oxygen species [52]. Moreover, we found that SN causes persistent inhibition of telomerase activity in rat cells [61]. A decreased telomerase activity could promote extensive telomere shortening, cytogenetically detected as telomere FISH signal loss. However, using the Flow-FISH technique, we were able to determine that SN does not have a persistent effect on telomere length in rat cells, since we observed a transient telomere lengthening in these cells only at 10 days after treatment (Paviolo, unpublished data). There‐ fore, the precise relationship between telomerase activity, telomere length and telomere instability in SN-exposed cells remains to be determined.

It is interesting to note that despite of the fact that both BLM and SN are radiomimetic compounds, they exhibit some important differences in their long-term effects on telomeres [60, 61]. First, BLM induces persistent chromosome end loss and telomere dysfunction, whereas SN induces persistent telomere dysfunction but not persistent chromosome end loss. Second, BLM induces telomere fusions, whereas SN not. Finally, BLM induces delayed increase of telomerase activity in mammalian cells, while SN decreases telomerase activity. Although these discrepancies could be due to differences between SN and BLM in their mode of action [50-52], further studies will be needed to confirm this assumption.

Finally, we found that also STZ induces persistent telomere dysfunction in rat cells, cytoge‐ netically detected mainly as telomere FISH signal loss and duplications, most of them being chromatid-type aberrations [62]. We observed that STZ induces significantly more signal loss than duplications, telomere loss thus being the most significant effect of STZ on telomere function at the chromosome level in ADIPO-P2 cells. As previously mentioned, telomere FISH signal loss and duplication were also observed in BLM- [60] and SN-exposed ADIPO-P2 cells [61]. Therefore, these types of aberrations seem to be the predominant chromosome aberrations directly related to telomere dysfunction induced by anticancer drugs. In addition, our experiments with rat cells exposed to STZ showed that this compound also induces long-term telomere instability in the form of incomplete chromosome elements, as previously observed in the short-term in Chinese hamster cells [57]. We found that STZ induces a transient increase in telomere length in ADIPO-P2 cells at 10 days after treatment, a delayed effect not related with telomerase activity, which remained unchanged in both treated and untreated cells [62]. Therefore, the persistence of chromosomal aberrations related to telomere dysfunction in rat cells exposed to STZ seems to be unrelated to telomerase activity or telomere length.

Besides the abovementioned studies, other researchers reported additional data on the effect of BLM on telomeres. No further studies have been made concerning the effects of SN and STZ on telomeres so far. The most important finding with regard to BLM was that human telomeric DNA sequences are a major target for this anticancer drug [63, 64]. In effect, these authors examined the DNA sequence specificity of BLM in a target DNA sequence containing 17 repeats of the human telomeric sequence and other primary sites of BLM cleavage and found that BLM cleaved primarily at 5'-GT in the telomeric sequence 5'-GGGTTA [63, 64]. The telomeric region constituted 57% of the 30 most intense BLM damage sites in the DNA sequence examined, these data indicating that telomeric DNA sequences are a major target for BLM damage. Previously, by using the Comet-FISH technique (i.e., single cell gel electropho‐ resis or Comet assay in combination with Fluorescent in situ hybridization), with a telomerespecific PNA probe, Arutyunyan et al. [65] found that BLM and Mitomycin C (MMC, CAS No. 50-07-7) induce breaks in telomere-associated DNA in human lymphocytes. The breakage frequency for telomeric DNA was found to be proportional to that of the total DNA, which suggests random induction of DNA breaks by these drugs. A year later, these authors using the same technique showed that, in human lymphocytes, BLM and also the anticancer drug cisplatin induce telomere DNA damage [66]. The action of cisplatin on telomeres will be considered in detail later in this chapter.

The induction of telomere DNA damage by BLM (and also MMC) in mammalian cells was confirmed by Hovhannisyan et al. [67], who analyzed the effect of these drugs in normal human leukocytes and three transformed cell lines (HT1080, CCRF-CEM and CHO) using the Comet-

FISH assay. They found significant differences between these cells with respect to quantita‐ tive head/tail distribution of telomeric signals after BLM exposure, which indicates that the extent of the telomere DNA damage induced by this compound depends on the cell type. Recently, Liu et al., studied the effect of BLM and other anticancer drugs on telomeres of a mouse spermatogonial cell line and found that BLM damages telomeric DNA (as seen by the colocalization of telomere and gamma-H2AX signals after FISH and immunofluorescence) [68].

#### **4.2. Paclitaxel**

It is interesting to note that despite of the fact that both BLM and SN are radiomimetic compounds, they exhibit some important differences in their long-term effects on telomeres [60, 61]. First, BLM induces persistent chromosome end loss and telomere dysfunction, whereas SN induces persistent telomere dysfunction but not persistent chromosome end loss. Second, BLM induces telomere fusions, whereas SN not. Finally, BLM induces delayed increase of telomerase activity in mammalian cells, while SN decreases telomerase activity. Although these discrepancies could be due to differences between SN and BLM in their mode

Finally, we found that also STZ induces persistent telomere dysfunction in rat cells, cytoge‐ netically detected mainly as telomere FISH signal loss and duplications, most of them being chromatid-type aberrations [62]. We observed that STZ induces significantly more signal loss than duplications, telomere loss thus being the most significant effect of STZ on telomere function at the chromosome level in ADIPO-P2 cells. As previously mentioned, telomere FISH signal loss and duplication were also observed in BLM- [60] and SN-exposed ADIPO-P2 cells [61]. Therefore, these types of aberrations seem to be the predominant chromosome aberrations directly related to telomere dysfunction induced by anticancer drugs. In addition, our experiments with rat cells exposed to STZ showed that this compound also induces long-term telomere instability in the form of incomplete chromosome elements, as previously observed in the short-term in Chinese hamster cells [57]. We found that STZ induces a transient increase in telomere length in ADIPO-P2 cells at 10 days after treatment, a delayed effect not related with telomerase activity, which remained unchanged in both treated and untreated cells [62]. Therefore, the persistence of chromosomal aberrations related to telomere dysfunction in rat

cells exposed to STZ seems to be unrelated to telomerase activity or telomere length.

considered in detail later in this chapter.

Besides the abovementioned studies, other researchers reported additional data on the effect of BLM on telomeres. No further studies have been made concerning the effects of SN and STZ on telomeres so far. The most important finding with regard to BLM was that human telomeric DNA sequences are a major target for this anticancer drug [63, 64]. In effect, these authors examined the DNA sequence specificity of BLM in a target DNA sequence containing 17 repeats of the human telomeric sequence and other primary sites of BLM cleavage and found that BLM cleaved primarily at 5'-GT in the telomeric sequence 5'-GGGTTA [63, 64]. The telomeric region constituted 57% of the 30 most intense BLM damage sites in the DNA sequence examined, these data indicating that telomeric DNA sequences are a major target for BLM damage. Previously, by using the Comet-FISH technique (i.e., single cell gel electropho‐ resis or Comet assay in combination with Fluorescent in situ hybridization), with a telomerespecific PNA probe, Arutyunyan et al. [65] found that BLM and Mitomycin C (MMC, CAS No. 50-07-7) induce breaks in telomere-associated DNA in human lymphocytes. The breakage frequency for telomeric DNA was found to be proportional to that of the total DNA, which suggests random induction of DNA breaks by these drugs. A year later, these authors using the same technique showed that, in human lymphocytes, BLM and also the anticancer drug cisplatin induce telomere DNA damage [66]. The action of cisplatin on telomeres will be

The induction of telomere DNA damage by BLM (and also MMC) in mammalian cells was confirmed by Hovhannisyan et al. [67], who analyzed the effect of these drugs in normal human leukocytes and three transformed cell lines (HT1080, CCRF-CEM and CHO) using the Comet-

of action [50-52], further studies will be needed to confirm this assumption.

126 Telomere - A Complex End of a Chromosome

Paclitaxel or Taxol (CAS No. 33069-62-4) is an anticancer drug, isolated from the bark of the Pacific yew *Taxus brevifolia*, that has been shown to be clinically effective against a wide range of human cancers, including ovarian, breast, lung and pancreatic cancers [69]. The anticancer effect of paclitaxel is attributable principally to irreversible promotion of microtubule stabili‐ zation and is hampered upon development of chemoresistance by tumor cells [70, 71].

It has been shown that paclitaxel and water-soluble poly (L-glutamic acid)-paclitaxel induce telomeric associations in a murine metastatic melanoma cell line (K1735, clone X-21), being the effect of the water-soluble form of paclitaxel more pronounced than the effect of paclitaxel alone [72]. Two years later, these authors analyzed the effects of the above compounds and two other water-soluble forms of paclitaxel (sodium-pentetic acid-paclitaxel and polyethylene glycol-paclitaxel) in the same murine cell line and found that these drugs induce the formation of telomeric associations [73]. In addition, they found that paclitaxel and its water-soluble conjugates induce extensive telomere erosion (visualized as reduced telomeric signal intensity after telomere FISH) but do not change telomerase activity [73]. Therefore, these drugs induce telomere dysfunction in mammalian cells by producing telomeric associations and telomere erosion (which means loss of telomeric repeats). Telomeric associations and reduction of telomeric signal intensity were also observed in Tax-18 and Tax-2-4, two paclitaxel-requiring mutant Chinese hamster ovary (CHO) cell lines [74]. Moreover, in these cells, cell death was driven by the loss of telomeric DNA repeats, as shown by the analysis of terminal telomeric restriction fragments [74]. Telomere erosion induced by paclitaxel can be enhanced by telomerase inhibitors, such as 3'-azido-3'-deoxythymidine (AZT) [75, 76]. More recently, using telomerase-deficient cells derived from mTERC−/− (mouse telomerase RNA componentminus) mice, Park et al. demonstrated that, upon telomere erosion, paclitaxel stimulates chromosomal fusion and instability in cells with dysfunctional telomeres [77]. Chromosomal fusions promoted by paclitaxel involve both q- and p-chromosome arms, being the q-arm fusions both unstable and lethal [77]. These chromosomal fusions occur in response to microtubule disruption induced by paclitaxel in cells with dysfunctional telomeres [77]. Thus, telomere dysfunction, rather than telomerase inhibition seems to be essential to sensitize transformed cells to paclitaxel.

#### **4.3. Cisplatin**

Cisplatin (CAS No. 15663-27-1), another well-known anticancer drug, was also found to interact with telomeric DNA sequences. Ishibashi and Lippard [78] showed that cisplatin can bind to telomeric repeats: Duplex DNA containing five telomeric repeats treated with cisplatin at formal platinum/strand ratios of 5 or 10 in water was platinated with efficiencies of 91.0% and 76.4%, respectively. More recently, Paul and Murray, using an automated capillary DNA sequencer investigated the interaction of cisplatin with purified telomeric DNA sequences and found that cisplatin strongly formed adducts with telomeric DNA sequences [79]. A similar result was obtained by Murray and Kandasamy, using a plasmid clone containing seven telomeric repeats and a sequence of ten consecutive guanine bases [80]. Although cisplatin preferentially damaged the guanine sequence, the telomeric DNA was also a major site of cisplatin adduct formation [80]. Furthermore, Nguyen et al. analyzed the DNA sequence specificity of cisplatin in a long telomeric tandem repeat (a human telomeric DNA sequence containing 17 tandem repeats) and found that the 3'-end of the G-rich strand of the telomeric repeat was preferentially damaged by this compound [81].

Even though several studies showed that cisplatin inhibits telomerase activity in a specific and concentration-dependent manner in several types of cancer cells (see [82, 83] for example), little is known about whether this compound induces telomere instability. Ishibashi and Lippard [78] by using Analysis of Terminal Restriction Fragment (TRF) Length (by Southern blot) showed that cisplatin induces telomere loss (shortening) and degradation in HeLa cells. A recent study from Liu et al. in a mouse spermatogonial cell line, showed that the alkylating compounds cisplatin and 4-hydroperoxycyclophosphamide (4OOH-CPA, a preactivated analog of cyclophosphamide, CAS No. 50-18-0) induce telomere dysfunction in mouse cells [68]. These authors found that these compounds decrease telomerase activity and shorten telomere length, thus causing telomere dysfunction [68]. Thus, cisplatin and 4OOH-CPA could induce long-term telomeric loss in mammalian cells, resulting from the inhibition of the enzyme telomerase.

#### **4.4. Doxorubicin and etoposide**

Doxorubicin (also called Adriamycin, CAS No. 23214-92-8) and etoposide (CAS No. 33419-42-0) are both topoisomerase II inhibitors with anticancer properties. As is the case with BLM and cisplatin, doxorubicin can also bind to human telomeric repeats. In effect, it was demonstrated that this drug binds to the human telomeric sequence 5'-d[GGG(TTAGGG) (3)]-3' (21-mer), assuming a G-quadruplex structure in the presence of K(+) [84]. It has been found that doxorubicin inhibits telomerase activity and shortens mean telomere length in human hepatoma cells [85]. Thus, it has been proposed that telomerase inhibition and telomere shortening by doxorubicin may contribute to its efficiency in the treatment of hepatocellular carcinoma. However, doxorubicin showed no effect on telomerase or telomere length in human ovarian cancer cells [86], induces telomere dysfunction (as determined by the presence of end-to-end chromosome fusions and end breaks by conventional staining with Giemsa, not telomere FISH) and decreases telomerase activity but has no effect on telomere length in breast tumor cells [87], and decreases telomerase activity in several human breast and stomach cancer cell lines [88]. Thus, the effect of doxorubicin on telomeres depends on the cell type. Moreover, it has been shown that doxorubicin induces senescence or apoptosis in rat neonatal cardio‐ myocites by regulating the expression levels of the telomere binding factors 1 and 2: High-dose doxorubicin strongly reduces TRF2 expression while enhancing TRF1 expression, and it determines early apoptosis, whereas low-dose doxorubicin induces downregulation of both TRF2 and TRF1 [89]. The exposed cells maintain telomere dysfunction and a senescent phenotype over time and undergo late death. Therefore, this study suggests that doxorubicin induces telomere dysfunction at the molecular level by regulating the expression levels of TRF1 and TRF2, both of them being part of the shelterin complex. A few years ago, it was reported that doxorubicin and etoposide induce progressive telomere shortening (assessed by flowfluorescence in situ hybridization and Southern blotting) in human mesenchymal stem cells (MSCs), obtained from bone marrow (BM) cells from normal adults and grown in the presence of platelet lysates [90]. A year later, Li et al. reported that the treatment of normal human T lymphocytes and fibroblasts with doxorubicin or etoposide led to significant shortening of telomeres, down-regulation of telomerase activity, diminished expression of telomerase reverse transcriptase (hTERT) and the telomere binding proteins TPP1 and POT1 and telomere dysfunction in these cells [91]. Therefore, both topoisomerase II poisons doxorubicin and etoposide, induce telomere dysfunction. However, recent data reported by Liu et al. showed that etoposide alone does not specifically affects telomeres of a mouse spermatogonial cell line and that this drug did not induce telomere dysfunction in these cells [68], but in combination with BLM and cisplatinum, etoposide produces telomere shortening in rat male germ cells [92]. In addition, it was demonstrated that etoposide did not affect telomere length in the neuro‐ blastoma cell line SHSY5Y, with very short telomeres and the acute lymphoblastic T cell line 1301, which displays extremely long telomeres [93]. Thus, the effect of etoposide on telomeres depends on the cell type.

#### **4.5. 5-azacytidine (5-AZA)**

at formal platinum/strand ratios of 5 or 10 in water was platinated with efficiencies of 91.0% and 76.4%, respectively. More recently, Paul and Murray, using an automated capillary DNA sequencer investigated the interaction of cisplatin with purified telomeric DNA sequences and found that cisplatin strongly formed adducts with telomeric DNA sequences [79]. A similar result was obtained by Murray and Kandasamy, using a plasmid clone containing seven telomeric repeats and a sequence of ten consecutive guanine bases [80]. Although cisplatin preferentially damaged the guanine sequence, the telomeric DNA was also a major site of cisplatin adduct formation [80]. Furthermore, Nguyen et al. analyzed the DNA sequence specificity of cisplatin in a long telomeric tandem repeat (a human telomeric DNA sequence containing 17 tandem repeats) and found that the 3'-end of the G-rich strand of the telomeric

Even though several studies showed that cisplatin inhibits telomerase activity in a specific and concentration-dependent manner in several types of cancer cells (see [82, 83] for example), little is known about whether this compound induces telomere instability. Ishibashi and Lippard [78] by using Analysis of Terminal Restriction Fragment (TRF) Length (by Southern blot) showed that cisplatin induces telomere loss (shortening) and degradation in HeLa cells. A recent study from Liu et al. in a mouse spermatogonial cell line, showed that the alkylating compounds cisplatin and 4-hydroperoxycyclophosphamide (4OOH-CPA, a preactivated analog of cyclophosphamide, CAS No. 50-18-0) induce telomere dysfunction in mouse cells [68]. These authors found that these compounds decrease telomerase activity and shorten telomere length, thus causing telomere dysfunction [68]. Thus, cisplatin and 4OOH-CPA could induce long-term telomeric loss in mammalian cells, resulting from the inhibition of the

Doxorubicin (also called Adriamycin, CAS No. 23214-92-8) and etoposide (CAS No. 33419-42-0) are both topoisomerase II inhibitors with anticancer properties. As is the case with BLM and cisplatin, doxorubicin can also bind to human telomeric repeats. In effect, it was demonstrated that this drug binds to the human telomeric sequence 5'-d[GGG(TTAGGG) (3)]-3' (21-mer), assuming a G-quadruplex structure in the presence of K(+) [84]. It has been found that doxorubicin inhibits telomerase activity and shortens mean telomere length in human hepatoma cells [85]. Thus, it has been proposed that telomerase inhibition and telomere shortening by doxorubicin may contribute to its efficiency in the treatment of hepatocellular carcinoma. However, doxorubicin showed no effect on telomerase or telomere length in human ovarian cancer cells [86], induces telomere dysfunction (as determined by the presence of end-to-end chromosome fusions and end breaks by conventional staining with Giemsa, not telomere FISH) and decreases telomerase activity but has no effect on telomere length in breast tumor cells [87], and decreases telomerase activity in several human breast and stomach cancer cell lines [88]. Thus, the effect of doxorubicin on telomeres depends on the cell type. Moreover, it has been shown that doxorubicin induces senescence or apoptosis in rat neonatal cardio‐ myocites by regulating the expression levels of the telomere binding factors 1 and 2: High-dose doxorubicin strongly reduces TRF2 expression while enhancing TRF1 expression, and it

repeat was preferentially damaged by this compound [81].

enzyme telomerase.

**4.4. Doxorubicin and etoposide**

128 Telomere - A Complex End of a Chromosome

5-azacytidine (5-AZA, Ladakamycin, CAS No. 320-67-2) and its deoxy derivative 5-aza-2'de‐ oxycytidine (Decitabine, CAS No. 2353-33-5) are demethylating compounds (inhibit DNA methyltransferases) with anticancer properties, usually employed against myelodysplastic syndrome and acute myeloid leukemia [94]. It has been shown that 5-aza-2'deoxycytidine, either alone or in combination with trichostatin A, induces up-regulation of shelterin genes, which leads to telomere elongation in breast cancer cell lines [95]. In addition, 5-AZA was found to induce DNA damage at telomeres and telomere dysfunction in acute myeloid leukemia cell lines [96]. Telomere dysfunction was coupled with telomere shortening, dimin‐ ished TERT expression and apoptosis in the exposed cells [96]. Thus, these authors suggested that another mechanism (besides DNA demethylation) by which 5-AZA exerts its anticancer activity is telomere dysfunction [96]. On the contrary, Choudhury et al., using the glioblastoma cell line SF-767, found that 5-AZA caused significant changes in DNA methylation of subte‐ lomeric regions of chromosomes but did not modify the telomere length in these cells [97]. Thus, further studies will be needed to clarify the effect of this compound on telomeres.

#### **4.6. Gemcitabine**

It has been recently reported that the cytidine analog gemcitabine (2′, 2′-diflurodeoxycytidine) (CAS No. 95058-81-4), an effective anticancer drug against several types of solid tumors, including colorectal, breast, pancreatic, renal and lung cancer [98], causes telomere attrition or shortening in Hela cells, by increasing the level and stability of TRF2 [99], that is required for the Xeroderma pigmentosum group F protein (XPF)-dependent telomere loss or degradation. By increasing TRF2 expression, gemcitabine enhances XPF activity, and because XPF is a nuclease, binding of the nuclease to telomeres may lead to inappropriate excision of telomeric DNA. The anticancer effect of gemcitabine is due to the incorporation of the active derivative compound dFdCTP into DNA in proliferating cells, leading to inhibition of DNA synthesis and repair. Thus, the above findings by Su et al. [99] suggest that the promotion of telomere attrition by induction of TRF2 is a new mechanism of action of gemcitabine against cancer. This effect of gemcitabine seems to be independent of telomerase, since this drug had no effect on telomerase activity in Hela cells 3 days after treatment. No further studies have been made to analyze the effect of gemcitabine on mammalian telomeres.

#### **4.7. C-1027**

The enediyne antibiotic C-1027 or Lidamycin (CAS No. 120177-69-7) is a new kind of macro‐ molecular antitumor antibiotics, produced by *Streptomyces globisporus* in soil, consisting of a noncovalently bound apoprotein and a labile chromophore which is responsible for most of the biological activities [100-102]. This drug is a potent anticancer drug with radiomimetic properties, which is being currently evaluated in Phase II clinical trials [103]. Several years ago, it was demonstrated in cultured human colon carcinoma HCT116 cells exposed to C-1027 that this drug induces telomere fusions (i.e., chromosomes joined end to end at their telomeres or fused together after complete loss of telomere sequences) in these cells [104]. Therefore, C-1027 induces short-term telomere dysfunction in human cells. No further studies on the effects of C-1027 on telomere stability have been performed so far.

#### **4.8. ICRF-193**

ICRF-193 ([meso-2, 3-bis (2, 6-dioxopiperazin-4-yl) butane], CAS No. 21416-68-2) is a topoiso‐ merase II catalytic inhibitor [105]. Two recent publications deal with the effect of ICRF-193 on telomeres [106, 107]. These studies show that this drug induces DNA damage at telomeres (as assessed by colocalization of telomere PNA-FISH signals and immunofluorescence of 53BP1 foci) [106] and telomere dysfunction in the HT1080 fibrosarcoma cell lines [106] and telomere shortening in mice cells [107]. In particular, it was found that ICRF-193 induces damage at telomeres properly capped by TRF2 but not by POT1 [106]. Moreover, ICRF-193 treatment blocks ALT-associated phenotypes in vitro and inhibits ALT cell proliferation in mice [107], which suggests that this drug could be used to prevent cell proliferation in cancer cells with an ALT mechanism of telomere elongation. No further studies on the effects of ICRF-193 on telomere stability have been performed so far.

#### **4.9. Melphalan**

Melphalan, L-phenylalanine mustard, L-PAM, Alkeran or L-Sarcolysine (CAS No. 148-82-3) is a chemotherapeutic drug belonging to the class of nitrogen mustard alkylating agents [108]. It has been reported that melphalan has no effect on telomerase activity in human testicular cancer cells [82]. More recently, by studying the induction and persistence of chromosome aberrations in bone marrow and spleen cells of p53+/- (and wild type) mice exposed for 4, 13, or 26 weeks to 2 mg/kg melphalan (MLP), Sgura et al. [109] were able to demonstrate that this drug induces telomere shortening in bone marrow cells of wild-type mice, while in p53+/- mice the exposure to this compound induces telomere elongation. No further studies on the effect of melphalan on telomeres have been reported so far.

### **4.10. 5-fluorouracil (5-FU)**

the Xeroderma pigmentosum group F protein (XPF)-dependent telomere loss or degradation. By increasing TRF2 expression, gemcitabine enhances XPF activity, and because XPF is a nuclease, binding of the nuclease to telomeres may lead to inappropriate excision of telomeric DNA. The anticancer effect of gemcitabine is due to the incorporation of the active derivative compound dFdCTP into DNA in proliferating cells, leading to inhibition of DNA synthesis and repair. Thus, the above findings by Su et al. [99] suggest that the promotion of telomere attrition by induction of TRF2 is a new mechanism of action of gemcitabine against cancer. This effect of gemcitabine seems to be independent of telomerase, since this drug had no effect on telomerase activity in Hela cells 3 days after treatment. No further studies have been made

The enediyne antibiotic C-1027 or Lidamycin (CAS No. 120177-69-7) is a new kind of macro‐ molecular antitumor antibiotics, produced by *Streptomyces globisporus* in soil, consisting of a noncovalently bound apoprotein and a labile chromophore which is responsible for most of the biological activities [100-102]. This drug is a potent anticancer drug with radiomimetic properties, which is being currently evaluated in Phase II clinical trials [103]. Several years ago, it was demonstrated in cultured human colon carcinoma HCT116 cells exposed to C-1027 that this drug induces telomere fusions (i.e., chromosomes joined end to end at their telomeres or fused together after complete loss of telomere sequences) in these cells [104]. Therefore, C-1027 induces short-term telomere dysfunction in human cells. No further studies on the effects of

ICRF-193 ([meso-2, 3-bis (2, 6-dioxopiperazin-4-yl) butane], CAS No. 21416-68-2) is a topoiso‐ merase II catalytic inhibitor [105]. Two recent publications deal with the effect of ICRF-193 on telomeres [106, 107]. These studies show that this drug induces DNA damage at telomeres (as assessed by colocalization of telomere PNA-FISH signals and immunofluorescence of 53BP1 foci) [106] and telomere dysfunction in the HT1080 fibrosarcoma cell lines [106] and telomere shortening in mice cells [107]. In particular, it was found that ICRF-193 induces damage at telomeres properly capped by TRF2 but not by POT1 [106]. Moreover, ICRF-193 treatment blocks ALT-associated phenotypes in vitro and inhibits ALT cell proliferation in mice [107], which suggests that this drug could be used to prevent cell proliferation in cancer cells with an ALT mechanism of telomere elongation. No further studies on the effects of ICRF-193 on

Melphalan, L-phenylalanine mustard, L-PAM, Alkeran or L-Sarcolysine (CAS No. 148-82-3) is a chemotherapeutic drug belonging to the class of nitrogen mustard alkylating agents [108]. It has been reported that melphalan has no effect on telomerase activity in human testicular cancer cells [82]. More recently, by studying the induction and persistence of chromosome aberrations in bone marrow and spleen cells of p53+/- (and wild type) mice exposed for 4, 13, or 26 weeks to 2 mg/kg melphalan (MLP), Sgura et al. [109] were able to demonstrate that this

to analyze the effect of gemcitabine on mammalian telomeres.

C-1027 on telomere stability have been performed so far.

telomere stability have been performed so far.

**4.7. C-1027**

130 Telomere - A Complex End of a Chromosome

**4.8. ICRF-193**

**4.9. Melphalan**

In the case of 5-fluorouracil (CAS No. 51-21-8), a thymine analog with anticancer properties commonly used against several types of solid tumors, including breast, colorectal, pancreatic, skin and cervical carcinoma [110], there is no information about its effects on telomeres as a single drug, since this compound is usually employed in chemotherapy in combination with several drugs. Thus, for example, it has been reported that, combined with cisplatin, 5-FU increases telomerase activity and causes long-term telomere elongation in colorectal carcinoma cells (LoVo and DLD-1 cell lines) [111]. It is interesting to mention that it has been recently demonstrated that overexpression of a human telomerase reverse transcriptase polypeptide (hTERT)C27, which induces telomere dysfunction by promoting end-to-end chromosome fusions, sensitizes HeLa cells and nasopharyngeal carcinoma cells to 5-FU cytotoxic effects [112, 113]. Thus, despite the fact that 5-FU does not induce telomere dysfunction, these recent studies suggest that combinational therapy of this drug with hTERTC27 may provide a novel approach to treat cancer.
