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Regarding reproducible results and continuity in research, these days scientists are facing difficulties in the genetically stabilizing living cells. Making culture and subculture are time consuming and easily lead to contamination or genetic drift as smaller portion of the population is selected. But we can stabilize the cells at cryogenic temperature at -1960 C or -770K. Stabilizing the cells at cryogenic temperature is known as cryopreservation. Certain small molecules are able to enter cells and prevent dehydration and formation of intracellular ice crystals which can lead to cell death and destroy the cell organelles during the freezing process. These small molecules are known as cryoprotective agents. Cryoprotective agents are of two types: penetrating and non-penetrating. Penetrating cryoprotectants enter into the cell and prevent dehydration and ice formation. Two common penetrating cryoprotectants are dimethyl sulfoxide and glycerol. Glycerol is generally used cryoprotective agent for red blood cells while dimethyl sulfoxide is primarily used for cryoprotection of most other cells and tissues. Non penetrating cryoprotective agents have ability to leak solute reversibly under osmotic stress.
Blood can provide classical field of application for cryopreservation. Cryopreservation method is useful for all different types of blood cells but in exception case for granulocytes no method has been developed yet. Frozen red blood cells are used for various diagnostic and clinical purposes. Cryopreservation protocols vary with different cell lines. The methods are depend on (1) cell concentration, (2) Protective solutions used, (3) temperature-time histories and (4) storage temperature. Sometimes some of the cryoprotectants are not suitable in all concentrations, for example dimethyl sulfoxide (Me2SO4) for platelets and glycerol for red blood cells.
Cryoprotective agent is selected on basis of cell type to be preserved. The most suitable cryoprotective agent for most of the cells is glycerol because of its lesser toxic effect in comparison with other cryoprotective agents. However, glycerol has some limitations, the major is its slow movement among the membrane of cells which are permeable for it.
Cryopreservation is widely use in clinical medicine, agriculture aquaculture and biomedical research but it is an inefficient technique that induces extensive cytoplasmic and genetic DNA damage. The main aim of cryopreservation is to preserve the genetic material- it is the main success of cryopreservation although DNA damage occurs in the form of double strand brakes (DSBs) or mutation which is kind of drawback of the technique.
The maintenance of genetic integrity and cell viability are essential for the proper function and survival of all the organisms. When human DNA battered by endogenous cellular metabolites and exogenous damaging agents, the normal cell cycle arrest by cell cycle checkpoints and DNA is repaired by sequential event.
The main regulators of checkpoint pathway in human DNA damage are ataxia telangiectasia, mutated (ATM), ATM and Rad3-related (ATR) and DNA- dependent protein kinase (DNA-PK), although the initial activation of these regulators are not fully understood.
The ATM and ATR protein kinases:
Mutated ATM is a product of ATM gene which is found in the patients with the genetic disorder ataxia telangiectasia. However, since non mammalian disease is associated with ATR but recently such deficiency is found to be embryonic lethal in mice.
ATM responds primarily to DSBs induce by IR. DSBs have been found as the major signal to active ATM although certain evidences also show that ATM can be activated by other signals also. (While) ATR is activated by UV or stalled replication fork. ATR also plays a supportive role in the DSBs response.
Mechanism for the activation of ATM/ATR:
By knowing the case of activation of ATM and ATR, it is well understood how they function as damage sensors. Treatment of DNA-cellulose matrix with restriction enzymes or IR can facilitate ATM binding to DNA ends while in the case of ATR, its affinity of binding gets stimulated with UV damaged DNA. ATM and ATR also interact with the foci, which is made of many proteins. These proteins are co localised at the site of DNA damage. Supporting the evidence of role of ATM and ATR in DNA repair system, they are mostly found at the foci after the few minutes of damage. However, recently Kozlow and co-workers found that ATM can be activated without DNA. According to them ATP can activate ATM by mechanism of autophosphorylation. In this process ATR and DNA-PK are not activated.
G1 checkpoint is the best understood mammalian checkpoint which prevents damaged DNA from being replicated. In their checkpoint accumulation and activation of p53 protein is controlled by the ATM and ATR kinases. In normal growing cells p53 interact with MDM2which maintain its low level and which associate p53 for nuclear export and proteosome mediated degradation in the cytoplasm. However in the DNA damage by IR S20 residue of p53 is phosphorylated by downstream kinase CHK2 which is activated by ATM by phosphorylation at position T68. As a result of this p53 is accumulated because the S20 which is phosphorylates p53, blocks the p53/MDM2 interaction. Although ATM directly phosphorylate the MDM2 on S395, because of this MDM2/p53 interaction become possible but it prevents p53 nuclear export to the cytoplasm. So degradation also can not take place. In the similar way role of ATR in the phosphorylation of p53 S20is less known but in in-vitro condition S20 phosphorylation take place by the ATR dependent kinase.
In addition of S20, S15 also has crucial role in p53 transcriptional transactivation activity. p53 up-regulates the number of target genes which are also involve in DNA damage response. This response is activated by S15 which is directly phosphorylated by ATM or ATR in response to IR, UV or stalls of DNA replication forks.
The S-phase checkpoint is the least understood mammalian repair pathway where cell cycle progression is monitored and the rate of DNA synthesis would be decreased following DNA damage. In radio resistant DNA synthesis (RDS) cell from cancer prone individuals affected with ataxia telangiectasia (AT) or Nijmegen breakage syndrome (NBS) fail to slow their rate of replication by IR exposure. This result brings in the related gene products that are ATM and NBS 1 respectively in the S phase checkpoint pathway. It is experimentally proved that S-phase checkpoint gets activated by IR damage via 2 parallel branches and both branches are controlled by ATM. IR damage in first branch provoke the phosphorylation of ChK2 kinase which then target CDC25A phosphatase and make it unable for its normal functioningof removing inhibitory phosphorylates from CdK2. The inactivated CdK2/cyclin E and CdK2/cyclin A complexes leads incompletion of DNA synthesis.
In the second branch of IR-induced S-phase checkpoint the activity of both ATM and NBS 1 is required, though it is independent of Cdc25A. ATM phosphorylates a number of downstream substrates like NBS1 at site S343, the product of the breast cancer susceptibility gene1 (BRCA1) at site S1387 and structural maintenance of chromosome protein 1 (SMC1) at site S957 and S966 after IR damage. S-phase checkpoint is activated by loss of any of these proteins or mutations.
The role of ATR in S-phase checkpoint is still unclear. ATR phosphorylates ChK1 at site S317 and S345 to start a slow IR-induced S-phase checkpoint response. ChK1 phosphorylates Cdc25A for degradation.
Delay of the cell cycle previous to chromosome segregation is an important event of G2 pathway. It is regulated by the cycline dependent kinase Cdc2. ATM and ATR regulate the phosphorylation on Cdc2 and lead to DNA damage. Both responses, to IR and to UV damage and replication blocks are governed by ATR.
DNA-PK is activated by DNA damage which is induced by IR or UV. DNA-PK is activated at primary DNA damage as a sensor rather than downstream effector of DNA damage signalling. DNA-PK is present in the nucleus at high level and maintains its activity throughout the cell cycle. DNA-PK may be activated by the interaction with DNA and other proteins. It is also projected that DNA-PKcs is offered to DNA by Ku which make DNA-PKcs ââ‚¬" DNA interaction and change in DNA-PKcs that gives catalytic charge to DNA-PK complex. It leads to that DNA-PK can be activated in the absence of Ku also. In a similar way DNA-PK is also activated by protein-protein interaction.
DNA-PK and p53: formation of close contact
DNA damage is also indicated by DNA-PK with the help of p53 and thus form a protein complex. This protein complex act as a sensor complex which binds to abnormal DNA structures, thus detects the breakage in DNA replication. Also in the DNA-PK mediated pathway, p53 act as effector.
DNA repair pathways
Base excision repair is a multistep process and it corrects the non bulky damage of bases that occurs because of natural alterations like oxidation, methylation, deamination or spontaneous loss of DNA base. Although these alterations are simple and natural, they are highly mutagenic and dangerous for genomic stability. BER possess two sub pathways: (1) short patch; (2) long patch. Both pathways are activated by cleavage of DNA glycosylase with N-glycosidic bond between the damaged base and the sugar phosphate backbone of DNA. After this cleavage pyrimidine/apurinic site is formed on the other hand.
Nucleotide excision repair pathway is based on the diversity of lesions on which it is going to act. Because of UV component from the sunlight, pyrimidine dimers are formed in the lesions.
DSB cause by variety of sources like exogenous agents for example ionizing radiation and sometimes genotoxic agents. Replication of single stranded DNA breaks also cause DSB. DSB becomes the most serious type of DNA damage because it creates the problems for the basic function like transcription replication and chromosome segregation. DSBs affect on both strands of DNA duplex therefore DNA can not use other strand for repair and it leads to deregulated gene expression and carcinogensis. There are two distinct pathways for DSB repair: (1) homologous recombination; (2) non-homologous end joining.
Homologous recombination repair system retrieves genetic information from homologous undamaged DNA pair and correct the damaged pair in error free manner. It usually takes place in S- and G2- phase. On the other hand, non homologous end joining does not use homologous undamaged pair so it can not do correction in error free manner.