**Telomere Instability Induced by Anticancer Drugs in Mammalian Cells**

Alejandro D. Bolzán

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64928

#### **Abstract**

Telomere instability results from chromosome end loss (due to chromosome breakage at one or both ends) or, more frequently, telomere dysfunction. Dysfunctional telomeres arise when they lose their end-capping function or become critically short, which causes chromosomal termini to behave like a DNA double-strand break. At the chromosomal level, this phenomenon is visualized by using Fluorescence In Situ Hybridization (FISH), as chromosomal aberrations directly involving terminal telomeric repeats: loss or dupli‐ cation of telomeric signals, association or fusion of telomeres of different chromosomes, telomere sister chromatid exchanges, translocation or amplification of telomeric sequen‐ ces, and extrachromosomal telomeric signals. At the molecular level, telomere instability arises due to the loss or modification of any of the components of the telomere (telomere DNA, telomere-associated proteins or telomere RNA). Since telomeres play a fundamen‐ tal role in maintaining genomic stability, the study of telomere instability in cells exposed to anticancer drugs is of great importance to understand the genomic instability associat‐ ed with chemotherapy regimens. In this chapter, we will summarize our current knowl‐ edge about telomere instability induced by anticancer drugs on mammalian cells.

**Keywords:** Telomere, telomere instability, telomere loss, telomere dysfunction, telomere erosion, chemotherapy, anticancer drugs

### **1. Introduction**

#### **1.1. What are telomeres?**

Classically defined as the chromosome ends, telomeres (from the Greek, *telo =* end, and *mere =* part) [1] are nowadays defined as specialized nucleoprotein complexes localized at the physical ends of linear eukaryotic chromosomes, that maintain their stability and integrity [2, 3]. They protect chromosomes from degradation, recombination or fusion, by preventing the

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ends of linear chromosomes from being recognized as DNA double-strand breaks (DSB) by the DNA repair machinery, i.e., they distinguish natural DNA ends from DNA ends resulting from breakage events [2, 3].

In all vertebrates, telomeres are composed of tandem arrays of short, repetitive G-rich sequences (TTAGGG)n, oriented 5′ → 3′ towards the end of the chromosome, ending in an essential 3′ single-stranded overhang that ranges in length from ~50 to 400 nt [4-6], bound by a specialised multiprotein complex known as "shelterin" or telosome [7-9]. The length of the double-stranded telomeric repeat varies greatly among species [2]. In normal human cells, the DNA at each chromosome terminus spans 5-20 kb in length [6, 10, 11], terminating in a 3" single-stranded overhang 100-400 nt in length [10], whereas in human tumor cells, telomere length varies from 1 to 20 kb [12-14].

The telosome is constituted by 6 proteins (POT1, TPP1. TIN2, TRF1, TRF2 and RAP1) and is charged with protecting chromosome ends from activating a DNA damage response, inhibit‐ ing inappropriate repair mechanisms, and maintaining telomeric length and structure [7-9]. Besides telomeric repeats and shelterin, telomeres also comprise (UUAGGG)n-containing RNA molecules (telomeric repeat-containing RNA or TERRA), a novel class of RNA for which several functions have been suggested [15-18]. TERRA transcription occurs at most or all chromosome ends and it is regulated by RNA surveillance factors and in response to changes in telomere length. Therefore, telomeres are composed of DNA, proteins, and RNA. In addition to the shelterin complex, many proteins involved in DNA repair are also associated with telomeres [19].

Telomere length is maintained by a dynamic process of telomere shortening and lengthening. Usually, telomere shortening occurs due to nucleolytic degradation and incomplete DNA replication, due to the inability of lagging strand synthesis to completely replicate chromoso‐ mal ends (i.e., the so-called "end replication problem") [9]. Telomere shortening is usually prevented by telomerase, a reverse transcriptase-like enzyme containing an RNA subunit (TERC, "Telomere RNA Component") and a catalytic protein subunit called "Telomerase Reverse Transcriptase" (TERT, "Telomere Reverse Transcriptase") which works via an RNA template -using exclusively single-strand 3" telomeric overhangs as primers- [9], by adding telomeric repeats to the chromosome ends. Although repressed in the majority of normal somatic cells (with the exception of a transient S phase activity thought to maintain the singlestranded overhang]), telomerase is present in immortal cell lines, germline cells, stem cells, activated lymphocytes, and most of the tumor cells analyzed so far [20, 21]. Telomerase activity favors 3" overhangs over blunt DNA ends for an addition of telomere sequence, at least *in vitro* [22, 23]. Loss of telomerase enzymatic function leads to progressive telomere shortening over time, eventually resulting in the disappearance of detectable telomeric DNA and the formation of end-to-end chromosome fusions, followed by growth arrest or cell death [24].

It has been suggested that TERRA may be involved in the regulation of telomerase activity and that the accumulation of TERRA at telomeres can interfere with telomere replication, leading to telomere loss [15-18]. Telomere elongation can also occur in the absence of telomer‐ ase through the so-called ALT (for 'Alternative Lengthening of Telomeres') mechanism, which involves homologous recombination between telomeres and has been described in several tumor cells and immortalized cell lines [25].

ends of linear chromosomes from being recognized as DNA double-strand breaks (DSB) by the DNA repair machinery, i.e., they distinguish natural DNA ends from DNA ends resulting

In all vertebrates, telomeres are composed of tandem arrays of short, repetitive G-rich sequences (TTAGGG)n, oriented 5′ → 3′ towards the end of the chromosome, ending in an essential 3′ single-stranded overhang that ranges in length from ~50 to 400 nt [4-6], bound by a specialised multiprotein complex known as "shelterin" or telosome [7-9]. The length of the double-stranded telomeric repeat varies greatly among species [2]. In normal human cells, the DNA at each chromosome terminus spans 5-20 kb in length [6, 10, 11], terminating in a 3" single-stranded overhang 100-400 nt in length [10], whereas in human tumor cells, telomere

The telosome is constituted by 6 proteins (POT1, TPP1. TIN2, TRF1, TRF2 and RAP1) and is charged with protecting chromosome ends from activating a DNA damage response, inhibit‐ ing inappropriate repair mechanisms, and maintaining telomeric length and structure [7-9]. Besides telomeric repeats and shelterin, telomeres also comprise (UUAGGG)n-containing RNA molecules (telomeric repeat-containing RNA or TERRA), a novel class of RNA for which several functions have been suggested [15-18]. TERRA transcription occurs at most or all chromosome ends and it is regulated by RNA surveillance factors and in response to changes in telomere length. Therefore, telomeres are composed of DNA, proteins, and RNA. In addition to the shelterin complex, many proteins involved in DNA repair are also associated with

Telomere length is maintained by a dynamic process of telomere shortening and lengthening. Usually, telomere shortening occurs due to nucleolytic degradation and incomplete DNA replication, due to the inability of lagging strand synthesis to completely replicate chromoso‐ mal ends (i.e., the so-called "end replication problem") [9]. Telomere shortening is usually prevented by telomerase, a reverse transcriptase-like enzyme containing an RNA subunit (TERC, "Telomere RNA Component") and a catalytic protein subunit called "Telomerase Reverse Transcriptase" (TERT, "Telomere Reverse Transcriptase") which works via an RNA template -using exclusively single-strand 3" telomeric overhangs as primers- [9], by adding telomeric repeats to the chromosome ends. Although repressed in the majority of normal somatic cells (with the exception of a transient S phase activity thought to maintain the singlestranded overhang]), telomerase is present in immortal cell lines, germline cells, stem cells, activated lymphocytes, and most of the tumor cells analyzed so far [20, 21]. Telomerase activity favors 3" overhangs over blunt DNA ends for an addition of telomere sequence, at least *in vitro* [22, 23]. Loss of telomerase enzymatic function leads to progressive telomere shortening over time, eventually resulting in the disappearance of detectable telomeric DNA and the formation of end-to-end chromosome fusions, followed by growth arrest or cell death [24].

It has been suggested that TERRA may be involved in the regulation of telomerase activity and that the accumulation of TERRA at telomeres can interfere with telomere replication, leading to telomere loss [15-18]. Telomere elongation can also occur in the absence of telomer‐ ase through the so-called ALT (for 'Alternative Lengthening of Telomeres') mechanism, which

from breakage events [2, 3].

120 Telomere - A Complex End of a Chromosome

length varies from 1 to 20 kb [12-14].

telomeres [19].

The analysis of the short- and long-term chromosomal instability produced by anticancer drugs is of great importance to understand the genomic instability associated with chemo‐ therapy regimens. Since telomeres play a fundamental role in maintaining chromosomal/ genomic stability, the study of telomere instability induced by antineoplastic drugs is of clinical interest. Therefore, in this chapter, we will consider in detail the phenomenon of telomere instability and summarize our current knowledge concerning the main data available about telomere instability induced by anticancer drugs on mammalian cells. In the next section, we will see what is telomere instability and how it can be generated in the cells.
