DNA repair during regeneration in Ambystoma mexicanum

Abstract

The remarkable regenerative capabilities of the salamander Ambystoma mexicanum have turned it into one of the principal models to study limb regeneration. During this process, a mass of low differentiated and highly proliferative cells, called blastema, propagates to reestablish the lost tissue in an accelerated way. Such a process implies the replication of a huge genome, 10 times larger than humans, with about 65.6% of repetitive sequences. These features make the axolotl genome inherently difficult to replicate and prone to bear mutations. In this context, the role of DNA repair mechanisms acquires great relevance to maintain genomic stability, especially if we consider the necessity of ensuring the correct replication and integrity of such a large genome in the blastema cells, which are key for tissue regeneration. On the contrary, DNA damage accumulation in these cells may result in senescence, apoptosis and premature differentiation, all of them are mechanisms employed to avoid DNA damage perpetuation but with the potential to affect the limb regeneration process. Here we review and discuss the current knowledge on the implications of DNA damage responses during salamander regeneration.

1 INTRODUCTION

The capacity to repair damaged tissue in adult mammals is an essential achievement, however in most of the cases this phenomenon results in the formation of scars, a tissue type that while helping to maintain the homeostasis of the organism does not restore the functions of the originally-damaged tissue.¹ Though, some organisms have evolved the impressive skill of regenerating damaged or lost tissues and structures, such as limbs and tail, without scarring and restoring the original function. It has been shown that some of these regeneration processes resemble embryonic development.²

Ambystoma mexicanum, also known as axolotl, is one out of more than 30 salamander species from the Ambystoma genus.³ The axolotl is one of the most used organisms to study epimorphic regeneration, a complex procedure that relies on the formation of a specialized structure known as blastema.⁴ A. mexicanum possesses the remarkable ability to regenerate a great variety of tissues from heart, brain, gills and jaw to the tail and legs, these latter being the most widely used to study regeneration for hundreds of years.^{3, 5}

In the axolotl, successful regeneration is achieved through the interplay of multiple processes, some of them more understood than others. Such processes involve diverse cell reprogramming events and a tight balance between cell proliferation and differentiation. The correct inheritance of genetic information of new cells produced to form the new tissue, and their correct differentiation, are essential for complete regeneration and function restoration. In this context, the role of DNA repair mechanism acquires great relevance during tissue regeneration, especially if we consider the necessity of ensure the correct replication and integrity of a large genome (32 000 Mb),⁶ which as all the DNA sequences are prone to suffer double strand breaks (DSBs) and other insults in the course of replication.⁷ Throughout evolution, cells have developed a plethora of mechanisms to face and overcome DNA damage. In specific, there exist two main pathways involved in DSBs resolution, non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ is a non-completely faithful mechanism based in recovery DNA integrity, but it does not necessarily conserve sequence fidelity, since several nucleotides can be added or deleted around the damaged sites. On the other hand, HR restores structure and also sequence, since it is based in homology searching and use of the sister chromatid, therefore it is considered error-free.⁸ The correct deployment of these mechanisms is crucial to maintain genomic stability and, in consequence, cellular homeostasis, which is indispensable to reach proper tissue regeneration. Hence, in this review we will analyze and discuss the potential involvement of DNA repair machinery in A. mexicanum epimorphic regeneration, with special emphasis in the maintenance of genomic stability in the proliferative cells that populate the blastema.

2 CELLULAR CONTRIBUTIONS TO BLASTEMA FORMATION

After axolotl limb or tail amputation, the first step to reach homeostasis is wound healing, in this event the stump is covered by a thin layer of epidermal cells formed by migrating and proliferate cells that finally compose the structure known as wound epidermis, which covers the underlying tissues after the cut.⁹ Afterwards, underneath this epidermal layer, differentiated cells reprogram to a more potent state and behave as progenitor cells which start to proliferate and contribute the major cell population that forms the blastema. Although in less extent, resident quiescent stem cells also proliferate and contribute to populate the blastema, the fundamental structure of epimorphic regeneration. Later, a fraction of blastema cells will eventually start differentiating to form the tissues of the new limb. It is important to mention that although blastema cells appear to be of a single cell type, as they look phenotypically identical, it has recently been determined that most of them conserve the identity of the type of tissue they come from, making them lineage-restricted.¹⁰ As briefly mentioned above, blastema progenitor cells are originated by two main procedures: the re-entry to cell cycle of quiescent stem cells that reside in the adult tissue, and cell dedifferentiation of mature cells that constitute remaining limb tissues. The latter ones must undergo reprogramming to acquire a less differentiated state, while keeping molecular traits of their lineage of origin.^{9, 11-13} The contribution of these two mechanisms have started to be elucidated, and recent studies have demonstrated that blastema cell population in salamanders relies on both proliferation of resident quiescent stem cells and cell dedifferentiation at different levels. The prevalence of one mechanism over the other depends on the species, tissue type and developmental stage of the organism. For instance, it has recently been described that in the axolotl, after limb amputation, the completely differentiated and heterogeneous population of connective tissue (CT) cells undergo dedifferentiation to constitute the majority of the homogeneous group of blastema progenitor, multipotent, cells which behave as embryonic limb bud cells and are in charge of guiding the regeneration of the lost tissues.¹³ In contrast, the regeneration of muscular tissue of the axolotl's limb relies only on the contribution of Pax7 positive cells, a specific marker of muscle stem cells, and participation of muscle cells dedifferentiation was not observed, suggesting that for this tissue the major source of muscle regenerated cells could be the mitotic activation of resident muscle quiescent stem cells. A different situation occurs in the newt Notophthalmus viridescens, another amphibian with high regeneration capacity, who reaches muscle regeneration through the usage of Pax7-negative cells, hence depending on dedifferentiation of mature cells to populate the blastema.¹⁴ Equally important, after axolotl tail amputation the spinal cord regeneration is maintained by neural stem cells which translate to a highly proliferative status and recapitulate and embryonic-like gene expression pattern to later produce the different cell types required to achieve fully regeneration.¹⁵

Regardless of the origin of these cellular progenitors, it is imperative to consider that in the axolotl's blastema, cells accelerate their cycle rates in order to build new tissues. This constant mitotic activity implies the replication of a genome 10 times larger than that of humans (3200 Mb), in a short time, each round of division.^{6, 16} The need to constantly duplicate such a large genome, increases also the mutational rate inherent to the replication process, activated metabolism and the fine-tuned regulation of factors that modulate proliferation.¹⁷ As a sum of all these factors one interrogation emerges, how in A. mexicanum the fidelity in the mitotically inherited genetic information and genomic stability are maintained in blastema cells during regeneration?

3 MAINTENANCE OF GENOMIC STABILITY IN BLASTEMA CELLS

Limb epimorphic regeneration has been studied in urodeles (axolotl), anurans (Xenopus spp) and teleosts (zebrafish). Independently of their cellular origin, blastema cell's nature has been described either as stem cells^18-20 or multipotent progenitor cells.¹³

In diverse developmental contexts, including limb morphogenesis and limb regeneration, both proliferative stem cells and/or multipotent progenitor cells divide asymmetrically to self-renew and generate daughter cells, which may also suffer several rounds of division and eventually will differentiate to populate a specific, or replace lost tissues. In the case of limb regeneration, blastema cells proliferate to reestablish tissue homeostasis, additionally they are capable of maintaining the pool of progenitor cells to continue with their duty in the newly formed tissue.^{15, 21} In contrast with fully differentiated cells, the constant rounds of mitotic events of multipotent progenitors and stem cells require a specialized and robust DNA Damage Response (DDR) with the intention of preventing the accumulation of DNA insults, repair them and avoid the propagation of mutations which, on the contrary, could affect the functionality of the resulting tissue, organs, or viability of the organism.²² Despite their ability to face and repair a vast amount of DNA damage, the high proliferation rates in these cells may also result counterproductive and be responsible for other types of genomic injury. For example, most of the time stem cells remain in a reversible non-proliferating state best known as quiescence, in phase G0 of cell cycle. Although in its quiescent state, stem cells maintain the capability to activate themselves in response to external stimuli and proliferate, the quiescence condition is also considered a way to reduce metabolic activity and therefore preserve their viability in the long-term, however, it can also occur that these cells accumulate mutations, since most of the DNA repair machinery and checkpoints are cell cycle-dependent.^{23, 24} Interestingly, if a quiescent stem cell suffers DNA damage independently of DNA replication while remains in G0 phase and later is called to re-enter into the cell cycle, this cell must go first through the G1 stage where the main DNA repair pathway is the unreliable NHEJ.²⁵ Accordingly, the passage through G1 after injury accumulation, mainly double-strand breaks, may lead to the generation of mutations which are inherited to the offspring. This may be not the case for the vast majority of proliferative axolotl blastema cells, which proliferative lifespan is limited and most of them will differentiate in the diverse tissues of the new limb, and only a few of them may become new satellite and quiescent stem cells.

In addition, it has to be considered that fully differentiated cells, which are called to dedifferentiate in the regeneration process, bear a genomic history as a result of their development, differentiation, metabolism, and exposition to external agents, accordingly their DNA material could not be intact and present somatic mutations as an accumulated product of all of these features.²⁶ Therefore, the implementation of somatic cells to dedifferentiate and later reestablish the lost tissues implies other risks to genomic stability, since mutations already present in dedifferentiated cells will be added to those acquired through proliferation and re-differentiation of blastema cells, which²⁷ could be perpetuated in the progeny, if they are not properly repaired. For example, several studies are underscoring the concern about genomic stability in fully reprogrammed cells, namely induced pluripotent stem cells (iPSC) which are comparable to dedifferentiated cells regarding its provenance from somatic cells but with lineage restrictions. In this context, an increase in de novo mutations in iPSC, variations in the copy number,²⁸ and propensity to DSBs have been shown.²⁹ Even mutations in mitochondrial DNA originated from parental blood or skin cells have been observed,³⁰ however these parameters have not been evaluated yet in dedifferentiated cells during regeneration.

Back to axolotl regeneration, the potential accumulation of mutations in the blastema cells could provoke genomic aberrations which may affect the proper function of differentiated daughter cells and, on the other hand, the perpetuations of these mutations in the cells which will become stem cells in the new limb, could affect their stemness capacity under homeostatic conditions and in future regeneration rounds. Actually, it has been observed in A. mexicanum that after several rounds of amputation-regeneration, the capability to restore a complete right size limb is reduced, producing remarkably smaller limbs compared to the previous ones,³¹ besides multiple studies have determined that regeneration capacity diminishes as the axolotl ages (reviewed in Viera et al.³²). However, it has not been determined yet whether this altered regeneration is due to satellite stem cell pool exhaustion where DNA damage might be involved or as a product of the continuous accumulation of genomic alterations in CT cells which need to efficiently reprogram into multipotent progenitor blastema cells for repetitive regeneration rounds. These observations strongly suggest that A. mexicanum must rely on a broad repertoire of mechanisms to deal with DNA damage and yet conserve blastema cells' genomic health when tissue regeneration is in progress. In this regard, the transcriptional expression of some molecular factors involved in DNA repair throughout regeneration process has been measured using microarray³³ and RNAseq³⁴ approaches. These studies showed that, in average, by day 14th post-amputation (dpa), there is an increase in the transcript levels of genes from several of the major pathways involved in DNA repairing such as base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end-joining. Accordingly, it was recently shown that γ-H2AX, a phosphorylated version of the histone H2AX directly associated with DSBs, is accumulated when regeneration is active (~14 dpa), as well as an increase in the corresponding gene expression (h2afx) was observed. Since the γ-H₂AX/H2AX ratio is decreased during this process, such observation indicates that H2AX is being phosphorylated to correct the replication mistakes thus avoiding potential genotoxic stress that could disturb cell cycle process. Remarkably, the level of γ-H₂AX does not correlate with those of comet assay, a test used to determine DNA strand breaks, at 14 dpa.³⁵ However it has to be considered that critics are emerging on using only the comet assay as the technique to evaluate DNA damage, due to its lack of reproducibility, as evidenced in several publications.^36-41 Therefore the opposite observations in Sousounis et al.,³⁵ study suggest that further research, using diverse techniques besides comet to asses DNA damage, are needed to fully evidence which DNA insults are occurring in blastema cells and if they are being efficiently repaired in some cells, while in other they accumulate perhaps leading to cell senescence.

4 DNA DAMAGE IN PROLIFERATIVE CELLS

Multipotent progenitor blastema cells, similarly to stem cells, possess a remarkable feature that may increase their susceptibility to genomic insults, their highly proliferative status²², in fact, mutations associated with replication are more common in highly proliferative cells because they replicate more frequently than somatic cells.⁴² During cell division, prior to mitosis, DNA content in the cells must be replicated in a way that genomic stability is preserved, this means that the genome is replicated only once per cycle with the adequate speed and accuracy and, at the same time, cells must deal with a plethora of situations that could affect the DNA replication process; because when replication is compromised the DNA becomes more sensitive to suffer breaks, which are one of the most deleterious damages.^{7, 8} In the replication context, the hindrances that may halt DNA duplication can be divided into two categories: i) those related with directly DNA damage, mainly caused by external elements such as UV light, ionizing radiation and alkylating agents,⁴³ and ii) those that are product of the natural cell metabolism like reactive oxygen species (ROS) and hydrolysis which may provoke DNA depurination, base oxidation and interstrand cross-links (ICL) among others^{44, 45} (see Figure 1). Additionally there are other risk elements related to the natural topology of the DNA, which can also end in DNA damage, like late replication zones, DNA-binding proteins and the encounter with transcriptional units, which are capable to affect the progress of the replication machinery and even stop it (see Figure 1). Moreover, the DNA sequence itself can make DNA replication difficult due to secondary structures, among these are: H; Z and S-DNA forms; cruciform; hairpins, slipped-strand DNA and G-quadruplexes. Normally, these patterns are formed in sequences containing a considerable number of repeats like dinucleotides, trinucleotides, inverted and direct tandem repeats, as well as mini and microsatellites, pseudogenes, transposable elements (TE) and long terminal repeats (LTRs). The diverse types of repeats may induce unstable chromosome areas identified as “rare” fragile sites.^46-48

FIGURE 1

DNA replication hindrances. Several natural impediments may affect the progression of the replication machinery, provoking stalling and later DNA breaks. The encounter with DNA-protein or interstrand cross-links, adducts, and transcriptional units can affect replication due to their volume or difficulty to open the strands, on the other hand, the DNA sequence located in repeats and late replication zones may generate secondary structures prone to break when are replicated

Interestingly, after the sequencing and ensemble of the axolotl large genome, it was noticed that the difference of size in comparison with another smaller vertebrate genomes is mainly due to the number of repetitive sequences present, which constitute about 65.6% of the genome, represented by long interspersed nuclear elements (LINEs) and LTRs.⁶ In this context, it has been observed that the RT (retrotransposon) element LINE-1 is re-activated during limb regeneration in the axolotl, and its copy number increases in consecutive amputation and regeneration rounds.⁴⁹ Of note, the expression and mobilization of LINE-1 has been linked to harmful genomic rearrangements like duplications, inversions, and deletions⁵⁰ and although the role of this TE has not been elucidated yet in the regenerative environment, it is possible that its deregulation could lead to aberrations mostly after repetitive regeneration as it has been observed in previous studies.³¹

The abundance of repeats suggests that the A. mexicanum genome is not easy to replicate since it would be prone to collide with impassable secondary structures and stall, leading to the generation of DSBs, which may provoke loss of information or chromosomal rearrangements, depending on whether they are repaired by NHEJ or HR respectively.^{7, 51} Indeed, it has been shown that even the more reliable HR pathway may fail in the search of homology when the damaged site is at repeated sequences.⁵² Besides, as was previously mentioned, during spinal cord regeneration in axolotl tail a cell cycle acceleration has been observed in neural stem cells, revealed by a shortening of S and G1 stages, and such high and fast proliferation is one of the major drivers to achieve tissue replenish in this context.^{15, 16, 53} In summary, the previous facts emphasize the imperative task of replicative cells to duplicate a huge genome, full of repetitive sequences, in a shorter period when regeneration is ongoing. Such scenario highlights the relevance of DNA repair to maintain the genomic stability in blastema cells.

5 THE FATE OF DAMAGED BLASTEMA CELLS

The proper function of the cells that proliferate to populate the blastema, and those that conform a mature blastema, is fundamental for tissue regeneration in A. mexicanum. Therefore, DNA genome integrity must be conserved in highly proliferative cells, such as the previously described, to accomplish correct tissue de novo formation. However, when this type of cell population is compromised there are different paths that cells can take, that would result in the preservation of their integrity at the expense of the number or vice versa. In brief, four possible mechanisms have been described by which progenitor and stem cells respond to an excess of irreversible DNA damage, and in the context of regeneration they could also be applied to the other cell populations of the blastema. These mechanisms are senescence, apoptosis, differentiation, and cancer^{51, 54} (see Figure 2).

FIGURE 2

Possible outcomes for progenitor and stem cells with damaged DNA. A healthy stem cell can be affected in their DNA integrity due to external and internal sources, once this cell has accumulated an irreparable or unbearable DNA damage it could follow different pathways to deal with it such as, turn it-self into a senescent cell, undergo apoptosis, differentiates or develop a tumorigenesis process, all of them with the potential of exhausting the cells pool and therefore affect the regeneration

6 SENESCENCE

Cellular senescence is normally recognized as a regulated process where the cells are permanently taken out of the cell cycle as a response to different harmful stimuli and to avoid the propagation of damaged cells which could ultimately affect tissue homeostasis, consequently senescence can act as an alternative to apoptosis. In this regard, the foremost senescence incentives are telomere erosion, oxidative damage, oncogene activation and DNA damage (reviewed in Yun et al.,⁵⁵). Recent studies have found that senescent cells can be generated in other cellular processes different from those related to genomic stress. At present, senescence occurrence can be classified into three distinct types: replicative, programmed, and premature stress-induced senescence. Replicative senescence is mainly associated with telomere erosion and it is detected as DNA damage by the cells, thus activating the DDR, alternatively, programmed senescence occurs during development and therefore might not be linked to DNA damage. Finally, stress-induced senescence is a response to different situations where DDR is activated and remarkably it might or not be accompanied with DNA damage since other types of stressors can induce a response that from time to time overlap with DDR.⁵⁶ The role of senescent cells during development has been evaluated in amphibia, in specific, these cells were observed during axolotl development in a structure known as pronephros, which constitutes the embryonic kidney, in the olfactory epithelium of nerve fascicles, lateral organs, and gums. On the other hand, during Xenopus laevis development, the presence of senescent cells was determined in cement gland, pronephros, anterior cartilage, midbrain, and hindbrain, therefore in this developmental context senescent cells could be contributing to tissue degeneration and remodeling of larval structures.^{57, 58}

Interestingly, it has been observed in newts and axolotls that there exists an increase in the occurrence of senescent cells in the regenerating limb, where these cells reach their maximum point at mid blastema stage, and after this period the number of senescent cells diminishes gradually until the limb is completely regenerated, when they become almost undetectable.⁵⁹ In contrast, when the developing limb in the same organism was assayed, the senescence occurrence did not take place. These results suggest that this kind of senescence may be a specific feature of regeneration⁵⁹ as this singularity has also been observed during zebrafish fin regeneration.⁶⁰ However, the presence of senescent cells in actively regenerating limbs but not in those suffering re-differentiation, suggests that in less differentiated and highly proliferative cells in early blastema stages, more insults to DNA may be occurring and causing that certain cells, where DNA damage is severe and cannot be properly repaired, undergo senescence. Perhaps in the transition from a mature blastema to redifferentiation the rate on division is diminishing, thus the number of senescent cells is also reduced. Nonetheless, to prove these hypotheses it is necessary to determine, with diverse techniques besides comet, whether early and mid-blastema accumulate more DNA damage and if these is causing the occurrence of the reported senescent cells in salamander blastema stages, compared to what occurs in a developing limb stage.

In the framework of regeneration, ROS are involved in cellular signaling that mediate the proliferation of progenitor cells in organisms capable of regenerating.^61-64 The important role of these molecules has been observed in axolotl tail and spinal cord regeneration.⁶⁵ Nevertheless, it is well known that ROS are able to induce a plethora of insults inside the cell, in specific they are capable to affect the DNA molecule and activate the DDR resulting in stress-induced senescence. Interestingly, Saxena et al.⁶⁶ determined that fibroblasts from mammalian species (Acomys cahirinus and Oryctolagus cuniculus) with remarkable regeneration capabilities can resist H₂O₂ treatments preventing senescence and the associated DNA damage, as well as avoiding oxidative stress induced-mitochondrial dysfunction, this oxidative resilience is accomplished through the implementation of enhanced detoxification methods. Although it has not been proven yet whether the axolotl's cells present similar protective features against ROS, it is undoubtedly an interesting topic to tackle in the near future.

7 APOPTOSIS

Apoptosis is a form of programmed cell death, deployed when the cells are exposed to different stimuli such as DNA damage, persistent cellular stress, hypoxia, viral infection, increased levels of reactive oxygen species, accumulation of misfolded proteins, and cytoskeletal damage among others.⁶⁷

As the cells that contribute to the blastema are originated from different types of cells, the role of apoptosis in these two key populations could be different in the context of tissue regeneration. Going back to our previous comparison of dedifferentiated cells and iPSCs, it has been observed in the latter a reluctance to undergo apoptosis even when the induced cells have suffered intentional DNA damage, therefore these cells accumulate mutations that may trigger tumorigenesis.⁶⁸ On the other hand, the relationship between apoptosis and highly proliferative cells becomes an interesting topic to explore due to the necessity to eradicate cells that bear a compromised genome with the objective of maintaining either the progenitor or stem cells pool functional and secure genomic stability in the progeny. Nonetheless, if the apoptotic signal is strong enough it may provoke that cells move into a process of elimination that can compromise the whole cell pool. On the opposite side, if the cells do not respond to DNA insults, they will accumulate mutations over the time, which will eventually generate altered gene expression, loss of phenotype and hence disruption of stem cells homeostasis.⁶⁹ It is important to mention that in the case of stem cells, not all of them have the same sensitivity to go through apoptosis after DNA damage, for instance, human colonic stem cells are relatively more hardy to undergo apoptosis and as they are not dying are consequently more prone to accumulate mutations in comparison with stem cells of the small intestine.^{24, 70} Interestingly, not only a decrease in stem cells number could affect tissue regeneration, but also their quality, since an increase of human hematopoietic stem cells has been observed with age, however the functional capacity of the pool is generally decreased (reviewed in Sperka et al.,⁵⁴).

Apoptosis induction has been studied in regenerating tissues in some model organisms. In general, these studies suggest that apoptosis has a role during the first stages of limb regeneration since there is a slight increase in TUNEL positive cells, an assay designed to detect fragmented DNA produced in the finals steps of apoptosis.^{71, 72} Furthermore, results from proteomic studies reported a few apoptotic cells in the first steps of regeneration and later a raise of anti-apoptotic genes as well as a decrease in some genes with pro-apoptotic activity,⁷³ thus supporting the hypothesis generated by TUNEL assays that apoptotic cells are only present at the beginning of regeneration and after that, the cells are secured from apoptosis in regenerating salamander limbs. These data are also consistent with the observation that p53 activity, a major regulator of DDR and a molecule involved in preventing the proliferation of cells with genomic aberrations,⁷⁴ is reduced during early stages of regeneration thus enabling progression through the cell cycle checkpoints,⁷⁵ of note, apoptotic cells incidence is minimal in posterior stages of regeneration being the exception of retinal regeneration, since apoptotic cells persist most time of the process.⁷⁶ The presence of apoptotic cells during stages of dedifferentiation and blastema formation during regeneration, suggest that death cell could be implicated in the restructuration of the tissue and elimination of cellular debris generated by the injury, indispensable steps prior blastema conformation.^{71, 72} It would be interesting to determine whether population of apoptosis-resistant blastema cells carry some kind of DNA damage that is somehow “ignored”, thus compromising the genomic stability of daughter cells.

8 DIFFERENTIATION

Interestingly, there is a wide body of evidence suggesting that once progenitor cells accumulate DNA damage, different pathways are activated to promote their premature differentiation, for instance, human melanocyte stem cells (MSC) lose stemness after ionizing radiation pushing cells toward premature differentiation. Noteworthy is the fact that when the protein kinase ataxia-telangiectasia mutated (ATM), implicated in assembling and regulating the response to DSBs, is absent, the cells are more sensitive to suffer this transformation, suggesting the involvement of ATM in genomic stability of MSC.⁷⁷ A similar outcome has been observed in other cell types, like embryonic stem cells,⁷⁸ hematopoietic stem cells,⁷⁹ and neural stem cells from humans, where after radiation-induced DNA damage astrocytic differentiation in vivo is promoted.⁸⁰ However, so far there is no evidence of premature differentiation of blastema progenitor cells as a consequence of DNA damage.

9 CANCER

Tissue regeneration is a process characterized by high-speed proliferation of blastema cells, and it is not difficult to conceive it as a perfect environment to cell transformation. Since malignant tumors may be derived from an affected or incomplete regeneration, it has been suggested that regeneration is able to contribute to the foundation of malignant transformation and also be a way to avoid or amend growth abnormalities.⁸¹

Among the cellular categories that contribute to blastema formation, the stem cells are the only tissue units that possess the lifespan and proliferative capacity to accumulate a significant amount of DNA damage, an assertion that turns them more susceptible to malignant transformation causing the appearance of cancer-prone stem cells.^{69, 82} In fact, there are several similarities between normal and malignant stem cells, like the ability to divide asymmetrically to self-renew and produce daughters with diverse proliferation status, therefore it has been hypothesized that malignant stem cells might be originated from normal tissue stem cells.⁵¹ On the other side, the similarity between blastema dedifferentiated cells and cancer cells is undeniable, thus earlier results interestingly have shown how an adult tissue is capable of returning to a less differentiated stage, gaining migratory skills and high proliferation without developing abnormal cell growth.⁸¹

Tumor generation is rarely observed in axolotl and other urodele amphibians. Over the years, several investigations have tried to induce tumorigenesis in urodele through the usage of strong carcinogenic substances such as dibenzanthracene, methylcholanthrene, methylnitronitrosoguanidine, benzo(a)pyrene, 4-Nitroquinoline, and others. When applied to tissue in regeneration the main affectations observed were retardation in the regeneration time, abnormal polarity, morphological abnormalities, accessory limb structures, and arrest of the regeneration, however, tumorigenesis was unsuccessful in these tissues, conversely, in non-regenerating tissues, some of the chemical employed were able to induced tumor formation.^83-87 In this context, it is important to clarify that the absence of tumorigenesis during regeneration does not mean a lack of mutagenic activity since it was determined that re-amputation of a 4NQO-induced regenerate distal to the site where the carcinogen was implanted resulted in and abnormal limb that replicates the original affectation, in contrast, re-amputation proximal to implantation site will produce completely normal limbs.⁸⁸ These data strongly suggest the occurrence of carcinogenic-induced genomic mutations that are perpetuated in the blastema cells and inherited to their progeny, thus repeating the original phenotype but not developing tumors. Under different conditions, a reversion to normal cells phenotype has been observed after inoculation of tumor cells prior to amputation, a situation that although contradictory, places regeneration as a possible corrective process in this context.^{89, 90} Additionally, it has been found that p53 from axolotl, presents multiple amino acids changes usually found in tumors.⁹¹ These observations only underscore the importance of understanding the mechanism for “tumor resistance” in axolotl and other urodele amphibians during regeneration. Remarkably there are other organisms with incredible resistance to cell transformation, such as the case of the naked mole-rat (NMR) Heterocephalus glaber, an animal with the longest lifespan among rodents, around 30 years, and well-studied cancer resistance (reviewed in Shepard et al.,⁹²). NMR possess a stable genome characterized by a low number of transposable elements, only 25%,⁹³ in comparison with the more of 60% of the axolotl.⁶ In addition, it has been argued that NMR deploy an enhanced DNA damage response since several genes involved are upregulated in this organism, also an absence of replicative senescence has been declared. These and other features, such as resistance to oxidative damage and constitutive telomere maintenance, may be responsible for the lack of signs of aging and cancer resistance (reviewed in Shepard et al.,⁹²). It is important to note that the axolotl shares some of these peculiarities with the NMR, however more studies are needed to understand how the regenerating, and non-regenerative, tissues avoid tumorigenesis when the cells are threatened with carcinogenic compounds. In this regard, specific experiments addressing how DNA repair mechanisms are modulated, as well as the proliferation of DNA-damaged cells without tumor formation.

10 CONCLUSIVE REMARKS

The preservation of genomic integrity and genetic information fidelity is crucial in all cell types, but in highly proliferative stem/progenitor cells it becomes a high priority due to their fundamental role in morphogenesis, tissue regeneration and stem cell pool maintenance. The high proliferative capacity of blastema cells implies a possible inheritance of DNA alteration with the potential to affect the homeostasis of the regenerated tissue. In the axolotls regeneration context, cell proliferation is increased in amount and speed which supposes the replication of an immense genome with more than 60% of repetitive sequences in a shorter time, therefore, the DNA repair mechanisms must be efficient enough to counteract all of the DNA insults that could compromise cell viability, induce senescence, and even affect complete and functional regeneration, this is supported by recent findings in limb regeneration where a variety of genes involved in DDR have been identified up-regulated, including the histone H2AX and its phosphorylation.³⁵ These results correlate with the observation of senescent cells in proliferative blastema cells of axolotls and newts.⁵⁹ Although these observations strongly suggest an important participation of DNA damage response pathways during tissue regeneration, the contribution of the diverse DNA repair mechanisms to achieve successful regeneration has not been elucidated yet. Without a doubt, the study of how axolotl and other urodele amphibians maintain genomic stability will shed light on the tight regulation of these pathways and the interplay with other components that are necessary for appropriate limb regeneration.

ACKNOWLEDGMENTS

We acknowledge to the Consejo Nacional de Ciencia y Tecnología (CONACYT) for its contribution with the scholarship N° 589716 to the PhD student Ulises Omar García Lepe.

AUTHOR CONTRIBUTIONS

Ulises García-Lepe: Conceptualization; investigation; visualization; writing-original draft; writing-review and editing. Alfredo Cruz-Ramírez: Investigation; supervision; validation; visualization; writing-review and editing. Rosa María Bermúdez-Cruz: Investigation; supervision; validation; visualization; writing-review and editing.

CONFLICT OF INTEREST

The authors declare no conflict of interest.