**2. What is telomere instability? Telomere loss, dysfunction and erosion**

Telomere instability refers to the chromosomal instability caused either by the loss of the chromosome ends (one or both) or the dysfunction of telomeres [26, 27]. These phenomena can take place in the short (at first cell división after the induction of chromosome damage by a given mutagen) or in the long term (in the progeny of the exposed cells). Once telomere instability arises, the involved chromosomes tend to associate or fuse with each other [26, 27].

Chromosomal instability is usually defined as a delayed increase in the production of new aberrations or a delayed appearance of excessive levels of aberrations in the clonal surviving cells after exposure to a given mutagen [28]. However, whenever a chromosome lacks one or both of its ends or suffers telomere dysfunction it becomes unstable, so we can refer to chromosome -or more properly, telomere- instability to the one that arises since the first cell division after the chromosome damage event at the telomeric region occurs.

Telomeric instability may arise when a chromosome, due to a break in one or both of its ends, loses one or both telomeres (ie, becomes an "incomplete chromosome") and, therefore, the chromosome end is exposed to enzymatic degradation or fuses with another chromosome end or enters to what is called a breakage-fusion-bridge (BFB) cycle (see below for details). We refer to this type of instability as telomere instability by telomere or chromosome end loss [26, 27, 29]. Alternatively, if one or both telomeres of the chromosome are excessively shortened, the chromosome tends to associate or fuse with other damaged chromosomes. This shortening can affect any of the four telomeres of a (metaphase) chromosome, resulting in duplication or loss of telomeric signals (after Fluorescence In Situ Hybridization or FISH, each FISH signal representing a block or specific set of telomeric sequences) [26, 29]. It may also be the case that any of the telomeric proteins (the shelterin complex) or telomeric RNA be altered or lost. In all these cases, we refer to telomeric instability by telomere dysfunction because the telomere has lost its protective function, either by excessive shortening (termed "telomere erosion or attrition") or by loss or alteration of the proteins or RNA associated with the telomeric DNA [26]. This phenomenon can be studied at the cytogenetic level by telomere FISH and is visible through telomeric chromosomal aberrations such as telomeric fusions, associations or loss or duplication of telomeric signal [26, 29, 30].

#### **2.1. Telomere or chromosome end loss**

As previously noted, true telomere loss is due to chromosome breakage at one or both ends of the chromosome, and can generate chromosome instability, both by allowing degradation of the ends of chromosomes and promoting chromosome fusions. Fusion can occur between sister chromatids, or between different chromosomes if telomeres are lost in more than one of the chromosomes of a given cell. Chromosome fusion results in chromosome instability through the abovementioned BFB cycles [26, 27, 29], when chromosomes, after telomere loss, repeatedly fuse and break for many cell generations. BFB cycles can continue for multiple cell generations, leading to extensive chromosomal rearrangements, and terminate when the unstable chro‐ mosome eventually acquires a new telomere and so becomes stable [26, 27, 29]. BFB cycles involving sister chromatid fusions result in several types of chromosome rearrangements, including terminal deletions, inverted duplications, DNA amplification, duplicative and nonreciprocal translocations, and dicentric chromosomes, all of which have been associated with human cancer. Chromosomes lacking one telomere remain unstable until they are capped, and lost telomeres after a BFB cycle can be acquired by several mechanisms, including nonreciprocal translocation, duplication/translocation, subtelomeric duplication, or direct telomere addition [26, 27, 29]. For a detailed description of BFB cycles see [26, 27].

#### **2.2. Telomere dysfunction and erosion**

As previously stated, dysfunctional telomeres arise when they lose their end-capping function or become critically short (a phenomenon called telomere erosion or attrition), which causes chromosomal termini to behave like a DSB [9, 31]. In effect, dysfunctional (uncapped or shorten) telomeres are sensed as true DSB, according to the presence of DNA damage response proteins at telomeres in senescent cells or shelterin deficient cells [32]. Therefore, dysfunctional telomeres act as DSB, interfering with the correct rejoining of broken ends. Both telomeres and DSB are DNA ends, and as such, both recruit many of the same proteins. As previously mentioned, proteins governing the DNA damage response are intimately involved in the regulation of telomeres, which undergo processing and structural changes that elicit a transient DNA damage response [19]. Chromosomes with dysfunctional telomeres tend to fuse with one another, producing dicentrics, which can give rise to the abovementioned BFB cycles [26, 27]. It must be taken into account that telomere shortening does not always mean telomere dysfunction. Only when telomeric repeats loss gives rise to a defective telomere structure a dysfunctional telomere appears. Thus, telomere erosion refers to a dysfunctional telomere which became critically short, so it cannot function properly.

Telomere dysfunction at the chromosomal level is commonly assessed applying the telomere FISH technique to metaphase chromosomes [26, 29, 30]. A normal metaphase chromosome exhibits four telomeric signals, two at each end (one per chromatid). When the telomere becomes dysfunctional, one or more of these telomeric signals are lost or duplicated [26, 29, 30]. The presence of chromosome ends with undetectable telomeric hybridization signals has been shown to be a good indicator of critically short and probably dysfunctional telomeres in mammalian cells [33-36]. It is important to mention that not all telomere involving chromo‐ somal aberrations imply telomere dysfunction, but only those ones directly involving terminal telomeric repeats (see [26, 29] for details).

Telomeres suppress the DNA damaging response, so dysfunctional telomeres activate DNA damage checkpoints [19, 32]. In addition, dysfunctional telomeres induce metabolic and mitochondrial compromise [37], promote carcinogenesis [38, 39], induce chromosome instability [27], and triggers cellular senescence [40].
