Genomic instability

Genomic instability is a condition that can lead to cancer development (Hanahan & Weinberg, 2011) and is referred to the fact that cancer cells are defective in the quality of DNA replication and the subsequent DNA partition to daughter cells during cell division. This may occur at the level of single nucleotides (leading to point mutations) or in entire chromosome (leading to chromosome aberrations and anomalies in the karyotype) (Lengauer et al., 1998; Sieber et al., 2005; Negrini et al., 2010). The preservation of the genomic stability is fundamental for cellular integrity to prevent errors from DNA replication or exposure to endogenous or exogenous genotoxins (Yao & Dai, 2014).

The genomic integrity is monitored by several surveillance mechanisms: DNA damage checkpoint, DNA repair machinery and mitotic checkpoint; a defect in the regulation of one of these mechanisms can lead to genomic instability, which may facilitate cells malignant transformation (Yao & Dai, 2014).

DNA damage checkpoint: p53 (tumor protein P53, the name derives from its apparent molecular mass) is a tumor suppressor gene that has an important role in DNA repair, cellular senescence, apoptosis and regulates cell cycle progression. It is the most frequently mutated gene in human cancers: p53 loss of function mutations are found in more than 50% of cancers (Ellenson et al., 2015; Baugh et al., 2018). P53 recognizes different forms of cellular stress, principally DNA damage, and, in response to them, p53 blocks cell proliferation, arresting cell cycle in phase G1 to inhibit the propagation of DNA damage (Levine, 1997; Brown et al., 2007; Ellenson et al., 2015]. P53 can also induce apoptosis (programmed cell death) or senescence when the damage is too severe, to avoid the possible malignant transformation (Efeyan et al., 2006; Zhang et al., 2011). P53 also responds to oncogenic stress: this occurs through the upstream p19ARF (alternative reading frame tumor suppressor) and MDM2 (mouse double minute 2 homolog) pathway. ARF is induced by elevated proliferation signals caused, for example, by the overexpression of MYC and oncogenic RAS. ARF then antagonizes MDM2 activity to block p53 function, leading to cell cycle arrest or apoptosis (Palmero et al., 1998; Zindy et al., 1998).

DNA Repair Pathway: there are different repair pathways, such as the nucleotide excision repair (NER) and the mismatch repair system (MMR). NER acts through several steps: recognition of the lesion site, incision of the damaged DNA strand, DNA synthesis and link of the uncouple flanks by ligase enzymes. Each component of the NER pathway is fundamental in the correct repair of the damaged sites (Mitchell et al., 2003). The mismatch repair system corrects the base-base mismatches caused by insertion/deletions or replication errors (Kunkel & Erie, 2005).

Mitotic Checkpoint: its function is to control the correct attachment of the spindle microtubules to all condensed chromosomes before the nuclear division during mitosis (Yao & Dai, 2014). There are multiple components that mediate the mitotic checkpoint functions during mitosis. For example, Aurora B is responsible for the phosphorylation of histone H3 serine 10 (H3S10) during mitosis (Carmena & Earnshaw, 2003): it is thought that this mechanism plays a role in super-condensation and super compaction of chromosomes during mitosis. Another protein involved is the Protein phosphatase 2A (PP2A), a serine/threonine phosphatase that dephosphorylates different substrates (Shi, 2009) and has an essential role for the maintenance of centromeric cohesion of sister chromatids before the entry in anaphase (Riedel et al., 2006; Tang et al., 2006).

Genomic instability is principally characterized by:

Microsatellite instability (MSI): microsatellites are short repetitive DNA sequences that are distributed in the entire genome. When there are DNA replication errors, these are repaired by nuclear enzymes of the DNA mismatch repair system. When one of the repair enzymes is not functioning, some replication errors are not corrected; these events may accumulate and facilitate tumor development (Esteller et al., 1999; Kanaya et al., 2005). MSI is caused by the accumulation of insertions and/or deletions in short DNA repeats or microsatellites leading to different lengths of repeat. The accumulation of these mutations affects genes with important regulatory functions, for example tumor suppressor genes (Tashiro & Katabuchi, 2014).

Chromosome instability: it consists of an increased rate of chromosomal incorrect aggregation during mitosis, resulting in an incorrect chromosome number and/or in an abnormal chromosome structure (Rao et al., 2009). Failure of the correct chromosome segregation leads to cell death or to malignant transformation (Yao & Dai, 2014). Genes that, when altered, may lead to chromosome instability include genes involved in chromosome condensation, sister-chromatid cohesion, function and centrosome/ microtubule formation and dynamics (Murray, 1995; Elledge, 1996; Paulovich et al., 1997).

Increased frequencies of base pair mutation: it has been found in hereditary cancers that the loss of function of DNA repair genes may cause increased frequency of base pair mutation. This event and MSI in hereditary DNA damage repair gene cases have indicated that the loss of genetic integrity maintenance may be a cause of genomic instability and the initiation of cancer: it has been proposed that the increased mutations in genes involved in the maintenance of genetic stability may be an early step in tumor progression (Yao & Dai, 2014).

Genomic instability

There are several methods to detect genetic instability in cancers: to assess instability is important to repeat measurements in the cell populations throughout the tumor development. For example:

The analysis of the karyotype of a single cell permits the identification of chromosome number abnormalities, called aneuploidy, and the identification of rearrangements in the entire genome, such as inversions and translocations (Beheshti et al., 2001; Wan & Ma, 2012). Fluorescence in situ hybridization (FISH), referred to as spectral karyotyping (SKY), is a method that has facilitated the assessment of chromosomal instability: this technique stains each chromosome with different colored fluorophore readily enabling the presence of rearrangements or modifications in chromosomes (Bayani & Squire, 2001; Wan & Ma., 2012).

PCR (polymerase chain reaction) is a multicellular approach, used to detect MSI: it amplifies known microsatellite regions, and the lengths of the PCR products (short tandem repeats) are compared in the tumoral and normal DNA to determine the state of MSI (Janavicius et al., 2010; Meyerson et al., 2010; Vilar & Gruber, 2010).

Flow cytometry is another multicellular approach that, through the measurement of cells in suspension while they are passing through a laser beam, scatter light and emit fluorescence, estimates cell ploidy measuring DNA content (that is proportional to the intensity of the fluorescence) and the stage of the cells in cell cycle. The subsequent comparison between the estimated ploidy in the G0/G1 normal and malignant cell may estimate genomic instability in cancer cells (D’Urso et al., 2010; Darzynkiewicz et al., 2017).

Accumulation of mutations

OECD (2016a) Organisation for economic co-operation and development. Test guideline 476: in vitro mammalian cell gene mutation tests using the hprt and xprt gene.

OECD (2016b) Organisation for economic co-operation and development. Test guideline 490: in vitro mammalian cell gene mutation tests using the thymidine kinase gene.

In vitro cell transformation

OECD (2016e) Organisation for economic co-operation and development. Guidance document 231 on the in vitro BHAS 42 Cell Transformation Assay.

This GD provides an in vitro procedure using the Bhas 42 CTA, which can be used for hazard identification of potential carcinogenicity of chemicals with initiating and/or promoting activity. The test method described is based upon the protocols of previous reports and that published by the EURL ECVAM.

Genetic errors that increase mutation rates are quite common in cancers: they accelerate the acquisition of several mutations that are necessary for the transformation and subsequent tumor progression (Ellenson et al., 2015).

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