Quantitative Analysis of ATM, ATR and γ-H2AX as DNA Damage Sensor Proteins through Western Blotting

 

Bhavna Dwivedi2, P. K. Mishra1, S. K. Mishra2

1Bhopal Memorial Hospital and Research Centre, Bhopal

2Department of Biotechnology, Govt. New Science College, Rewa 486491 (MP)

*Corresponding Author E-mail: bhavna18.bt@gmail.com

 

ABSTRACT:

Living cells sometimes encounter continuous or accidental exposure of genotoxic agents which misbalances the integrity of subcellular components. DNA damage by these genotoxic agents initiates various cellular responses. These cellular responses include sensors, transducers and effectors. Sensor protein act at the frontline of the DNA response mechanism. The transducer amplifies the intracellular signals to the down stream regulatory molecules. Then the effector molecules progresses towards the execution of cell cycle arrest, DNA repair or apoptosis. The most important sensor proteins are ATM and ATR. One of the most imperative substrate that is phosphorylated by these protein kinases is γ-H2AX which is a vital component for nuclear foci formation to execute DNA repair mechanisms. This complete process is under tight regulation and any imbalance in these processes may lead to hypersensitivity to cellular stress and susceptibility to DNA damage, genomic effects and resistance to apoptosis, thus initiating the cascade of carcinogenesis. There are various agents that modifies DNA molecule, for example; environmental isocyanates and their derivatives that have capability to cause toxicogenomic effects. Isocyanates are thus becoming of interest in the field of genetic toxicology as they may react with DNA to produce DNA damage.

 

KEYWORDS: DNA Damage, Genotoxic agent, ATM.

 


INTRODUCTION:

To ensure faithful duplication and inheritance of genetic material, the cell has evolved with the ability to detect and propagate the initial DNA damage signal to elicit cellular responses that include cell cycle arrest, DNA repair, transcriptional reprogramming, senescence and apoptosis, which collectively have been termed the DNA damage response. Dysregulation of components involved in these processes contributes to genomic instability, which in turn leads to tumorigenesis. This is supported by the fact that clinical mutations in proteins that play a role in the DNA damage response often predispose individuals to cancer development. Cellular response to genotoxic stress is an intricate process, and it usually starts with the “sensing” or “detection” of the DNA damage, followed by a series of events that includes signal transduction and activation of transcription factors (Yang et al., 2004).

 

Signalling pathways are rapidly activated after exposure to DNA damaging stresses and proteins are recalled in the active and functional form, in order to efficiently participate in DNA damage response (Jang & Lee, 2004).  The DNA-damage response is a conserved mechanism that enables cells to withstand endogenously and exogenously induced DNA lesions. In response to DNA damage, rapid recruitment of a host of proteins ensues, enabling the sensing, amplification and transduction of the DNA-damage signal to promote cell-cycle arrest, DNA repair, senescence or apoptosis. Accumulation of these proteins can be readily detected in vivo as nuclear dots or foci because these proteins surround double-strand breaks (DSBs) and themselves become markers for DSBs. This network of proteins has become increasingly complex because knowledge of this system has evolved from a linear kinase signalling cascade in which phosphorylation is the main signalling modification to the currently proposed intricate network involving protein ubiquitylation, phosphorylation, methylation and signal-amplification loops to regulate the many processes involved in DNA repair that are integral to the maintenance of genomic stability (Harper & Elledge, 2007).

The two main sensor molecules, Ataxia telangiectasia mutated (ATM) and ATM and Rad 3 related (ATR), act in parallel branches at the front line of the DNA damage response pathway. They respond primarily to different types of DNA damage (Gatei et al., 2001). ATM reacts mainly to the DNA-damaging agents that cause DSBs, such as Infra red (IR), and its downstream targets include Check point protein kinase 2 (CHK2), Breast cancer associated protein1 (BRCA1) and p53 (Matsuoka et al., 2000). ATR along with ATR-interacting protein (ATRIP), responds primarily to Ultra-Violet radiations (UV) and hydroxyurea (HU)-induced damage, which may potentially interfere with DNA replication (Cortez et al., 2001). ATR regulates CHK1 and BRCA1 (Chen, 2000), but phosphorylation of p53 is also possible (Tibbetts et al., 1999). Due to ATM and ATR, BRCA1 becomes phosphorylated not only at overlapping but also at distinct residues, depending on the type of the DNA lesion (Gatei et al., 2001). Moreover, the two pathways overlap and often cooperate with each other to ensure efficient repair without delay and to maintain genomic integrity (Liu et al., 2000). The real DNA damage sensors must have these properties to function effectively: First, a sensor must be able to detect a small number of DNA lesions within the genome of a cell. A single DNA DSB, for example, can be sufficient to cause apoptosis (Rich et al., 2000), or can directly inactivate key genes, lead to chromosomal translocations or generate unstable chromosomal abnormalities (Gent et al., 2001). Secondly, the sensor needs to trigger events that lead to an amplification of the initial signal so that global cellular changes can ensue. DNA damage sensors recognize the damage and initiate the subsequent events. Breaks in the DNA backbone are picked up by 'checkpoint' proteins, which sit at the top of complex signalling cascades that hail repair troops to the damage site. Two groups of proteins have been identified as checkpoint specific damage sensors: ATM and ATR (Durocher et al., 2001) and the RFC/PCNA (clamp loader/polymerase clamp) related Rad17-RFC/ Rad9-Rad1-Hus1 (9-1-1) complex. We attempt to evaluate the signalling pathways of different proteins ATM, ATR and their important substrate γ-H2AX involved in DNA damage responses in human kidney epithelial cells after exposed to isocynates to show its genotoxic effects. This hypothesis would enhance the understanding of the relevance of specific DNA repair pathways in counteracting the potentially harmful consequences of genetic insults. Therefore, it will provide the tools to investigate the effects of DNA repair disorders and decreased repair capacity on the toxicity and carcinogenic properties of genotoxins.

 

MATERIALS AND METHODS:

The HEK293 cells were seeded at the density of 1x105 cells/60 mm culture dishes in EMEM supplemented with 10% fetal calf serum and fetal bovine serum, 1% antibiotic– antimycotic (penicillin/streptomycin/amphotericin), and 2mM L-glutamine at 37°C in the humidified atmosphere of 5% CO2 in air as per ATCC catalogue instructions. After optimum confluency, the cells were transfected with an experimental agent, N-succinimidyl N-methylcarbamate. At the onset of the experiments, the cells were at an exponential and asynchronous phase of growth. The cells were treated with N-Succinimidyl N-Methylcarbamate and were incubated as per the experimental design as follows:

 

S. No.

Concentration (N-succinimidyl

N-methylcarbamate)

Incubation Time

1

Nil

Control

2

6μl

6hrs

3

6μl

12 hrs

4

6μl

24 hrs

5

6μl

48 hrs

6

6μl

72 hrs

7

6μl

96 hrs

Following cells were collected for various parameters

 

At first protein samples were prepared then western blotting done. For that the samples (proteins) were mixed with the 2X sample buffer and then along with markers (For SDS Profile) were loaded into the wells by the help of Hamilton syringe and were run for minimum 2 hours with a constant current. In our experiment we provided 150V for stacking gel and 200V in resolving gel. A piece of Nitrocellulose membrane was cut according to the size of gel and was wet firstly in distilled water for 5 minutes and then transferred in 1X pre cooled transfer buffer for 5 minutes. Hofaer semidry transfer unit accomplished electrophoretic transfer of the protein bands onto the nitrocellulose membrane. Transfer is done on basis of capillary action by providing current. Transfer unit rapidly transfer proteins from polyacrylamide gel onto a membrane by means of a low current and low voltage electrode transfer with minimal joule heating. Transfer is completed in 2 hour. When the transfer of proteins was completed, the nitrocellulose membrane was separated from the SDS-Polyacrylamide gel and soaked in a concentrated non antigenic protein solution (blocking solution; e.g., a solution of fat free milk). Blocking reagent blocked the non-specific sites of proteinsas well as it masked all the proteins on membrane. The nitrocellulose membrane was placed in an incubation vessel containing Blocking reagent (5%) and incubated for one hour at room temperature on the tilting shaker to block non specific binding sites.

 

Antibody treatment:

Primary antibody (A Rabbit Polyclonal IgG) that binded to the transferred protein was detected by using a secondary antibody (Anti-rabbit IgG/ Anti-mouse IgG) AP conjugated (Alkaline phosphatase) which binds to the primary antibody and was visualized by using an ALP (Alkaline phosphatase) conjugated substrate. Blocking reagent was discarded and washed with 1X PBST three times with an interval of 10 minutes at room temperature on the tilting shaker. The washing solution was removed and now added primary antibody (A Rabbit Polyclonal IgG Antibody in a 1:1000 dilutions) solution to the nitrocellulose membrane and incubated for 2-3 hours at room temperature or over night at 2-4 oC.  After incubation primary antibody solution was discarded and washed with1X PBST three times with an interval of 10 minutes at room temperature on the tilting shaker. PBST is strong tween-20 detergent it washed unbounded antibodies and avoids false binding. The washing solution was removed and secondary antibody (Anti-rabbit IgG AP conjugated diluted in a ratio of 1:2500) was added to the nitrocellulose membrane and incubated for 2-3 hours at room temperature. Secondary antibody solution was discarded and washed with 0.1% PBST three times with an interval of 10 minutes at room temperature on the tilting shaker. The washing solution was removed carefully so that the nitrocellulose membrane should not be completely dried. AP conjugated (Alkaline phosphatase) substrate was used and it was fluorescent so always prevented from light. Substrate reagent (5 ml) was added and incubated at room temperature on the tilting shaker in dark room for 5-10 minutes the bands were developed on the nitrocellulose membrane. The nitrocellulose membrane was washed with 0.1% PBST 2-3 times to remove unbound substrate and was photographed to make the record of the protein bands.

 

RESULT:

In the quantitative analysis of proteins through western blotting the expression of proteins were increased with time in treated cells as comparison to controls. The intensity of band is proportional to protein expressed. This data shows progressive expression of both proteins (ATM and ATR) which was maximum at 24 hrs. Here β-actin acts as a loading control which is a housekeeping gene. Loading controls are essential for proper interpretation of western blot. Beta- Actin is a relatively stable cytoskeletal protein generally present at a constant level in cells, regardless (in most cases) of experimental treatment or technical procedure. For this reason, measurement of beta-Actin is generally used as an internal control for experimental error.

 

 

Western Blott analysis of ATM and ATR proteins in different time interval with comparisons to β-actin load as control.

CONCLUSION:

Isocyanates are considered as highly reactive molecules because of their potential to modify biomolecules under physiological conditions. These compounds form covalent adducts with critical macromolecules such as nucleic acids resulting in a series of biotransformation events that initiate with the generation of the reactive intermediates (Shelby et al., 1987; Pearson et al., 1990; Slatter et al., 1991; Marczynski et al., 1992). DNA damage leading to cellular demise in mammalian cells upon treatment with isocyanates has been reported (Beyerbach et al., 2006). It has been also shown that ‘carbamate’ the reactive intermediate of isocyanates also induces the analogous upshot (Yoon et al., 2001). MIC, one of the most toxic isocyanates is known to exert immunological, mutagenic and genotoxic alterations (Deo et al., 1987; Saxena et al., 1988; Goswami et al., 1986 ) and since MIC is an important industrial byproduct with diverse applications. We evaluated the genotoxicity of MIC on cultured human kidney epithelial cell line. DNA damage responses in these cells were evaluated using N-Succinimidyl Nmethylcarbamate, a MIC substitute. Previous data have shown that MIC exposure can lead to a series of biotransformation reactions in mammals (Slatter et al., 1991), thereby exerting mutagenic and genotoxic alterations (Anderson et al., 1988). Extent of DNA damage following treatment of cultured human kidney epithelial cell line with N-Succinimidyl N-methylcarbamate showed a time course dependent response. Previous study have demonstrated that the qualitative study of the IMR-90( human lung fibroblast) cells treated with N-succinimidyl N-methylcarbamate showed an elevated expression of phospho-ATR, phospho-ATM and accumulated phospho-H2AX foci indicative of increased nuclear relocalization through aggregates and enhanced binding to the damaged sites (Mishra et al., 2009). Previous data have shown that western blot analysis of ATM, ATR and γH2AX after treatment with 5-azadC showed increased expression of these proteins. In our findings  Also quantitative evaluation of these proteins through western blot had been showed elevated expression of these proteins with time in bands pattern. ATM and ATR, protein kinases, act as central mediators in response to DNA DSBs. An important substrate for the ATM kinase cascade is H2AX. It is a variant isoform of the histone H2A protein family. It is demonstrated that histone H2AX becomes extensively phosphorylated on serine 139 residues at the site of DNA double strand breaks thereby forming large distinct nuclear γH2AX foci. In conclusion, the result  presented herein demonstrates that expression of proteins is maximum at 24hrs indicating the extent of DNA damage to be maximum at this point when cultured mammalian cells are treated with N-succinimidyl N-methylcarbamate. Isocynates induces DNA damage responses in these cell lines suggestive of causing immune alterations. We anticipate these data along with other studies reported in the literature would help to design better approaches in risk assessment of occupational and accidental exposure to isocyanates and also helpful to understand about isocyanate induced genomic alterations in relation to the development of carcinogenicity. How much an individual is exposed to these agents and how their cells respond to DNA damage are critical determinants of whether that individual will develop cancer (Kastan and Bartek, 2004). These cellular responses are important for determining toxicities and because these response pathways seem to be major protectors from cancer development, the study of these pathways could lead to effective and new approaches to the reduction of cancer development. For e.g. disruptions in the ATR pathway do cause genomic instability, and ATR is activated by most cancer chemotherapies. Furthermore, ATR signaling is a promising target for cancer drug development (Collins and Garrett, 2005; Kaelin, 2005).

 

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Received on 11.03.2014          Modified on 22.03.2014

Accepted on 18.04.2014      ©A&V Publications All right reserved

Research J.  Science and Tech. 6(2): April- June 2014; Page 91-94