**2.2 Studies with cultured human Peripheral Blood Mononuclear Cells (PBMC)**

Heparinized vacutainers (Griener, Astria) were used to draw 3-5 ml venous blood from healthy, non-smokers, non-alcoholic male donors (age 25-30 years). The blood was layered on the ficoll-histopaque column (Sigma Aldrich Chemicals, USA) and centrifuged at low speed at 26+2°C, the interface between plasma and histopaque comprising PBMCs was collected and washed three times with serum free RPMI-1640 (HiMedia, India). The washed cells were suspended @1×106 cells/ml in complete RPMI-1640 containing 10% fetal bovine serum, 100 units/ml penicillin sodium salt, 100 μg/ml streptomycin sulphate, 2 mg/ml sodium bicarbonate. Phytohemagglutinin (PHA, Difco, Hamburg, Germany) was added to stimulate the cells proliferation. The cultures were setup in 96 well flat-bottomed micro titer plates (Tarson, India) at 37°C, 5% CO2. Each well had 150 μl volume containing 1.5×105 cells. The 22-24 hour old cultured PBMCs were irradiated first with low dose of 60Co-γ-radiation (0.07 Gy, using Gamma Cell GC 220, Canada dose rate 0.0078 Gy/s) and then after suitable time interval with lethal dose of 60Co-γ-radiation (5.0 Gy, using Gamma Cell-5000, BRIT, India; dose rate 1.26 Gy/s).

The cell proliferation was quantified using Hoechst 33342. The cells were washed at least three times with saline in microtiter plate, freshly prepared Hoechst 33342 solution in serum free RPMI (10 μg/ml) medium was added and the suspension was incubated at 37°C for 30 min. (Blaheta et. al., 1991). Fluorescence was measured at ex 355 nm and em 460 nm in fluorescence spectrophotometer (Varion, Australia).

To score the micronuclei, cytochalasin B (Sigma Aldrich Chemicals, USA) was added at 44 hour after initiation of human PBMC culture and the cells were harvested at 72 hour. 1×106 cells were washed, the cell pellet was suspended in 200 µl carnoy solution (methanol: acetic acid, 3:1) and incubated at 4°C for 2 hour. This cell suspension was laid on the chilled slides, dried overnight at 26 2°C and stained with hoechst 33342 (10 μg/ml) at 26 2°C for 30 min. in dark. Micronuclei were counted at ex 355 nm and em 460nm as per criteria described (Fenech, 1993). At least 1000 cells per sample were scored at 1000× magnification under oil immersion.

### **2.3 Western blotting**

152 Gamma Radiation

(UVP Inc., U.K.). Real-time one-step rt-PCR kit with SYBR green as flourophore (Qiagen, Germany) was used as per manufacturer's protocol to perform quantitative rt-PCR using iCycler (Bio-RAD, US, software version 2.1). The fold changes were determined by

Fold change = 2-( Ct values of control- Ct value of irradiated sample)

Where Ct: threshold cycle

For microarray studies, the labeled cDNA was synthesized from total RNA by using CyScribeTM First-Strand cDNA Labeling Kit (Amersham Biosciences). Either Cy3-dUTP or Cy5-dUTP was incorporated into the cDNA of samples under comparison. The cDNAs were dried in a vacuum trap. Pre-printed DNA microarrays with complete set of 6400 Open Reading Frames (ORFs) of *S. cerevisiae* genome (Microarray Centre of the University Health Network, Toronto, Canada), were used in this study. The slides were first hybridized in prehybridization solution (6x SSC, 0.5% SDS, 1% bovine serum albumin) for 1 h and then hybridized overnight with labeled probe at 42 °C in a water bath. Before using as a hybridization probe, the labeled cDNA was re-suspended in 40 µl of hybridization solution (50% Formamide, 6x SSC, 5x Denhardt's, 0.5% SDS, 20 µg of poly(A) and salmon sperm, Invitrogen). For each test, cDNAs from the un-irradiated control and from the stress dose irradiated samples were together hybridized on to one chip. Further, for each test, two different hybridizations were performed by swapping the fluorochromes to cross check the transcriptional changes, if any, due to experimental procedures. At least two DNA microarrays were analyzed for each test condition. The chips were scanned at a resolution of 10 µm and data was analyzed using GenePix Pro 4.0 analysis software (Axon Instruments,

To study the DNA damage in individual chromosomes by pulsed field gel electrophoresis, the samples were prepared as described earlier (Bala & Jain 1996, Bala & Mathew, 2002). In brief, the cell suspension was washed with PB, centrifuged; pellet was treated with lyticase enzyme and then immobilized in low melting agarose plugs using the mould provided by BioRad USA. The plugs were first treated with LET buffer [0.5 *M* EDTA pH 8.0, 0.01 *M* Tris(hydroxymethyl)-aminomethane pH 7.0, 7.5% Mercaptoethanol] for 20 h at 37 oC. The LET buffer was removed, plugs were washed two times with NDS buffer [0.01 *M*  Tris(hydroxymethyl)-aminomethane pH 7.0,7.5% EDTA pH 8.0, 1% n-luaryl sarcosine] . The plugs were then treated with NDS buffer containing 2mg/ml Proteinase K for 20 h at 48 oC. Sufficient washings were given in EDTA (0.5 *M,* pH 8.0) thereafter. The plugs were stored at 4 oC before electrophoresis. The pulsed-field gel electrophoreses (PFGE) was for 20 h (60 sec

*XRS2* AGCAACAATACTGAGAAGG TGAAATTGGAAATACTCGGA *MRE11* GTCACTCTACCAAGTACTGA CCATATCACCATATCCAGGAA *RAD50* GGCTTTCATCTCTCAGGA ATTCCTGGGTGAGGGGAA *SSC1* GTCCCACAAATCGAAGTCAC GGCATTGTTGCCGTTGTTG *OXI3* GAAGTATCAGGAGGTGGTGAC TCCCACCACGTAGTAAGTATCG *OGG1* CAGGATGAAAGTGAGCTATGT CAGATCTATTTTTGCTTCTTTG

calculating the fold change in threshold cycle (Δ Ct').

Gene Forward primer Reverse primer

Table 2. Primers for genetic studies with *Saccharomyces cerevisiae* 

Union City, CA).

Protein extraction and Western blotting was as per procedures standardized in our laboratory (Bala & Goel, 2007). Briefly, 4x107 cells/ ml of *S. cerevisiae* were lysed and treated with 160 ml of 50% trichloroacetic acid (TCA), washed with 1.5 ml of chilled acetone, resuspended in 100 ml of extraction buffer (4% SDS; 0.16 M Tris-Cl, pH 6.8; 20% Glycerol; 0.38 M b-mercaptoethanol) and heated for 4 min at 95 oC. For extracting proteins from PBMCs, standard protocol was used. Briefly 5x106 cells were suspended in PB containing protease inhibitors for 1.5 hours at 4 oC. The cells were ruptured by sonication and soluble proteins were collected after centrifugation in cold. Total soluble proteins were quantified by using Bradford's reagent and resolved by one dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using Mini-PROTEAN II (BIO-RAD, US). Gels were stained with Coomassie brilliant blue R-250. Electro-blotting was on nitrocellulose membrane

Radiation Induced Radioresistance – Role of DNA Repair and Mitochondria 155

Fig. 1. Effect of different pre-irradiation doses (4,10, 20 Gy) and of dose rates of stress dose (20 Gy) on RIR in *Saccharomyces cerevisiae.* dT (h): duration in hours between pre-irradiation

There are reports to show that there is difference in the nature and quantum of DNA damage by stress doses delivered at different dose rates (Chaubey et al., 2006). The higher RIR (survival) by stress doses delivered at lower dose rate as compared to the same stress dose delivered at higher dose rate suggested that nature of damage generated by stress

**3.1.2 Alteration in gene expression after irradiation with low dose of 60Co-gamma-**

As many as 110 open reading frames (ORFs) displayed more than 2 fold increase in transcription at 4.5 h after the low dose irradiation (20 Gy) and some of the annotated once

The functional groups of the up-regulated genes were DNA damage, repair, synthesis, energy generation, metabolism and stress response. Besides this, many transcripts with

and lethal irradiation (400 Gy). The values are average + S.D. of three experiments.

dose, is an important determinant of induction of protective mechanisms.

(From Dwivedi et al., 2008).

**radiation - whole genome analysis** 

are listed in Table 3.

(Millipore) and treatment with primary and secondary antibody was as described earlier (Bala & Goel, 2007).

#### **2.4 Statistical analysis**

Each experiment based on CFUs assay, had three replicates and was repeated at least three times. The data was presented as the average of three experiments + S.D. For estimating differential gene expression, DNA damage and protein expression, the data was analyzed using paired t-test. For cell survival, mutagenesis and recombinogenesis the data was analyzed using two-way ANOVA. *P* < 0.05 was considered significant.

#### **3. Results and discussion**

#### **3.1 Studies with** *Saccharomyces cerevisiae*

#### **3.1.1 RIR inducing doses, survival and mutagenesis**

Systematic study with cultures grown to different phases (mid-log phase, late log phase and stationary phase) showed that the stationary phase cultures did not show any RIR. Mid-log phase cultures showed 25% increase, while late log phase cultures showed only 12% increase in survivors in comparison to the non-pre-irradiated cultures. This comparison was made at pre-irradiation dose 20 Gy (LD10), challenge dose 400 Gy (LD50) and the time interval between stress dose irradiation and challenge dose irradiation 4.5 h (Sharma & Bala, 2002). This was in agreement with earlier reports (Cai & Liu, 1990), where mitogen stimulated human lymphocyte cultures showed far better radio-adaptive response than the resting cells. The RIR, since was maximum with mid–log phase cells, further studies were planned with the mid-log phase cells. Pre-treatment with three different doses of 60Cogamma-ray viz. 4, 10 and 20 Gy (<LD10) showed that RIR increased with increase in the preirradiation dose. In comparison to non-pre-irradiated controls, the 4 Gy pre-irradiated samples showed maximum 13% increase in survivors, 10 Gy pre-irradiated samples showed maximum 27% increase in survivors and 20 Gy pre-irradiated samples showed maximum 32% increase in survivors after lethal irradiation (400 Gy). The time of maximal increase in survivors was delayed at higher stress doses and was approximately 10 h after irradiation at 20 Gy, 6 h after irradiation at 4 or 10 Gy (Figure 1). However, there was no linear correlation between the pre-irradiation 60Co-gamma-ray dose and increase in survival due to RIR. These studies suggested that priming of cells with small radiation doses may induce some signaling events which may lead to RIR. The pre-irradiation (stress) dose (20 Gy), thereafter, was delivered at two different dose rates i.e. 0.0078 Gy/s and 1.26 Gy/s to the mid-log phase cells. It was observed that in comparison to non-pre-irradiated cultures, the cultures pre-irradiated (20Gy) at lower dose rate (0.0078 Gy/s) and lethally irradiated with 400 Gy, showed a maximum of 32% increase in survivors while cultures pre-irradiated at higher dose rate (1.26 Gy/s) showed a maximum of 25% increase in survivors after lethal irradiation. Further, in comparison to non-pre-irradiated cultures, the pre-irradiated cultures showed decrease in gene convertants and revertants when irradiated with lethal dose (400 Gy, Dwivedi et al., 2001). The dose rate also impacted the mutations and gene conversion quantum and time kinetics. Pre-irradiation dose (20 Gy), delivered at lower dose rate decreased the gene conversions and mutations for a longer time period in comparison to the same dose delivered at higher dose rate (Figure 1).

(Millipore) and treatment with primary and secondary antibody was as described earlier

Each experiment based on CFUs assay, had three replicates and was repeated at least three times. The data was presented as the average of three experiments + S.D. For estimating differential gene expression, DNA damage and protein expression, the data was analyzed using paired t-test. For cell survival, mutagenesis and recombinogenesis the data was

Systematic study with cultures grown to different phases (mid-log phase, late log phase and stationary phase) showed that the stationary phase cultures did not show any RIR. Mid-log phase cultures showed 25% increase, while late log phase cultures showed only 12% increase in survivors in comparison to the non-pre-irradiated cultures. This comparison was made at pre-irradiation dose 20 Gy (LD10), challenge dose 400 Gy (LD50) and the time interval between stress dose irradiation and challenge dose irradiation 4.5 h (Sharma & Bala, 2002). This was in agreement with earlier reports (Cai & Liu, 1990), where mitogen stimulated human lymphocyte cultures showed far better radio-adaptive response than the resting cells. The RIR, since was maximum with mid–log phase cells, further studies were planned with the mid-log phase cells. Pre-treatment with three different doses of 60Cogamma-ray viz. 4, 10 and 20 Gy (<LD10) showed that RIR increased with increase in the preirradiation dose. In comparison to non-pre-irradiated controls, the 4 Gy pre-irradiated samples showed maximum 13% increase in survivors, 10 Gy pre-irradiated samples showed maximum 27% increase in survivors and 20 Gy pre-irradiated samples showed maximum 32% increase in survivors after lethal irradiation (400 Gy). The time of maximal increase in survivors was delayed at higher stress doses and was approximately 10 h after irradiation at 20 Gy, 6 h after irradiation at 4 or 10 Gy (Figure 1). However, there was no linear correlation between the pre-irradiation 60Co-gamma-ray dose and increase in survival due to RIR. These studies suggested that priming of cells with small radiation doses may induce some signaling events which may lead to RIR. The pre-irradiation (stress) dose (20 Gy), thereafter, was delivered at two different dose rates i.e. 0.0078 Gy/s and 1.26 Gy/s to the mid-log phase cells. It was observed that in comparison to non-pre-irradiated cultures, the cultures pre-irradiated (20Gy) at lower dose rate (0.0078 Gy/s) and lethally irradiated with 400 Gy, showed a maximum of 32% increase in survivors while cultures pre-irradiated at higher dose rate (1.26 Gy/s) showed a maximum of 25% increase in survivors after lethal irradiation. Further, in comparison to non-pre-irradiated cultures, the pre-irradiated cultures showed decrease in gene convertants and revertants when irradiated with lethal dose (400 Gy, Dwivedi et al., 2001). The dose rate also impacted the mutations and gene conversion quantum and time kinetics. Pre-irradiation dose (20 Gy), delivered at lower dose rate decreased the gene conversions and mutations for a longer time period in comparison

analyzed using two-way ANOVA. *P* < 0.05 was considered significant.

(Bala & Goel, 2007).

**2.4 Statistical analysis** 

**3. Results and discussion** 

**3.1 Studies with** *Saccharomyces cerevisiae*

**3.1.1 RIR inducing doses, survival and mutagenesis** 

to the same dose delivered at higher dose rate (Figure 1).

Fig. 1. Effect of different pre-irradiation doses (4,10, 20 Gy) and of dose rates of stress dose (20 Gy) on RIR in *Saccharomyces cerevisiae.* dT (h): duration in hours between pre-irradiation and lethal irradiation (400 Gy). The values are average + S.D. of three experiments. (From Dwivedi et al., 2008).

There are reports to show that there is difference in the nature and quantum of DNA damage by stress doses delivered at different dose rates (Chaubey et al., 2006). The higher RIR (survival) by stress doses delivered at lower dose rate as compared to the same stress dose delivered at higher dose rate suggested that nature of damage generated by stress dose, is an important determinant of induction of protective mechanisms.

#### **3.1.2 Alteration in gene expression after irradiation with low dose of 60Co-gammaradiation - whole genome analysis**

As many as 110 open reading frames (ORFs) displayed more than 2 fold increase in transcription at 4.5 h after the low dose irradiation (20 Gy) and some of the annotated once are listed in Table 3.

The functional groups of the up-regulated genes were DNA damage, repair, synthesis, energy generation, metabolism and stress response. Besides this, many transcripts with

Radiation Induced Radioresistance – Role of DNA Repair and Mitochondria 157

DNA dependent ATPase/DNA helicase B

Ferrichrome-type siderophore transporter

Table 4. Some important genes down regulated (>1.5 folds) after the 20 Gy irradiation

**3.1.3 Confirmation of stress dose induced time dependent changes in selected** 

Fig. 2. Effect of stress dose irradiation on relative time dependent changes in gene

the value of untreated control was assigned as one. RFU: relative fluorescence units

expression of MRE11, RAD50 and XRS2 as studied by Real Time- reverse transcription PCR;

**Rad50 Xrs**

0 h 3 h 4.5 h 6 h

Significant over expression of genes from the DNA damage, response, repair complex, prompted us to perform real time quantitative PCR for the *MRX* complex (*MRE11, RAD50*  and *XRS2)* of which *RAD50* is an essential gene. The β-actin gene, though considered as a house keeping gene, showed differences in the stress dose irradiated cultures in comparison to the non-pre irradiated cultures, suggesting that β-actin gene could not be used as a house keeping gene. The experimental data was, therefore, compared with reference to the unirradiated control at the corresponding time. The results obtained from the real time quantitative rt-PCR (Figure 2) confirmed the significant increase in *RAD50* transcripts at 4.5 h in stress dose irradiated cultures and supported the information obtained by microarray

Stress-responsive regulatory protein

Glycerol-3-phosphate dehydrogenase (NAD+),cytoplasm ic

Heat shock protein

Heat Shock protein Heat Shock protein Cytosolic HSP70 Secreted glycoproteins

Acid trehalase, vacuolar

Heat Shock Protein

**Gene Function** 

**transcripts as well as associated genes** 

**3.1.3.1 The MRX complex** 

(from Dwivedi et al., 2008*)* 

0

0.5

**Mre11**

**Fold change (RFU)** 

1

1.5

2

2.5

(Table 3).

*HSP26 GPD1 HSP12 HSP30 SSA1 YGP1 ECM32 ATH1 ARN1 DDR48 MSN2* 

unknown function (not listed in Table 3), were also up-regulated. Some genes such as IRE1, HSP12 were down-regulated 4.5 h after irradiation (20 Gy) (Table 4). Sahara et al., 2002 reported that Hsp12p might play a role in protein binding in yeast. The Ire1p and Hac1p participate in the "unfolded protein response" (UPR) pathway. It is predicted that in our study the UPR pathway was down regulated. Other genes that were down regulated were DDR48, MSN2. Further studies are planned to understand the role of these genes and the pathways in which they participate to induced RIR.


Table 3. Up regulated (> 2.0 folds) transcripts, 4.5 h after the irradiation (20 Gy). Categorization of ORFs into functional groups is based on SGD Library.


Table 4. Some important genes down regulated (>1.5 folds) after the 20 Gy irradiation

#### **3.1.3 Confirmation of stress dose induced time dependent changes in selected transcripts as well as associated genes**

#### **3.1.3.1 The MRX complex**

156 Gamma Radiation

unknown function (not listed in Table 3), were also up-regulated. Some genes such as IRE1, HSP12 were down-regulated 4.5 h after irradiation (20 Gy) (Table 4). Sahara et al., 2002 reported that Hsp12p might play a role in protein binding in yeast. The Ire1p and Hac1p participate in the "unfolded protein response" (UPR) pathway. It is predicted that in our study the UPR pathway was down regulated. Other genes that were down regulated were DDR48, MSN2. Further studies are planned to understand the role of these genes and the

Table 3. Up regulated (> 2.0 folds) transcripts, 4.5 h after the irradiation (20 Gy).

Categorization of ORFs into functional groups is based on SGD Library.

pathways in which they participate to induced RIR.

Significant over expression of genes from the DNA damage, response, repair complex, prompted us to perform real time quantitative PCR for the *MRX* complex (*MRE11, RAD50*  and *XRS2)* of which *RAD50* is an essential gene. The β-actin gene, though considered as a house keeping gene, showed differences in the stress dose irradiated cultures in comparison to the non-pre irradiated cultures, suggesting that β-actin gene could not be used as a house keeping gene. The experimental data was, therefore, compared with reference to the unirradiated control at the corresponding time. The results obtained from the real time quantitative rt-PCR (Figure 2) confirmed the significant increase in *RAD50* transcripts at 4.5 h in stress dose irradiated cultures and supported the information obtained by microarray (Table 3).

Fig. 2. Effect of stress dose irradiation on relative time dependent changes in gene expression of MRE11, RAD50 and XRS2 as studied by Real Time- reverse transcription PCR; the value of untreated control was assigned as one. RFU: relative fluorescence units (from Dwivedi et al., 2008*)* 

Radiation Induced Radioresistance – Role of DNA Repair and Mitochondria 159

*Saccharomyces cerevisiae* is an excellent eukaryotic model system to study DNA repair mechanisms because DNA repair pathways are highly conserved between human and yeast. Furthermore, yeast and human mitochondria resemble each other in structure and function. Mitochondria are the major sites of energy (ATP) production in the cell. Mitochondria also perform many other cellular functions, such as respiration and heme, lipid, amino acid and nucleotide biosynthesis. Mitochondria also maintain the intracellular homeostasis of inorganic ions and initiate programmed cell death. Mitochondria are the major source of endogenous reactive oxygen species (ROS) in cells as they contain the electron transport chain that reduces oxygen to water by addition of electrons during oxidative phosphorylation. The rt-PCR studies with the mitochondrial genes (*SSC1* gene coding for mtHsp70, *OXI3* gene coding for COX1 respiratory component of complex-IV and *OGG1*gene) showed that the expression of *OXI3* was more than unirradiated controls up to 6 h and that of *SSC1* only at 2 and 10 hours after irradiation (20 Gy, Figure 4a,b). The expression of OGG1 was increased up to 2 hour only, after irradiation [(20 Gy), data not shown]. The mitochondrial genome of eukaryotic cells is extremely susceptible to damage due to constant exposure to significant amounts of reactive oxygen species (ROS) produced endogenously by mitochondria as a by-product of oxidative phosphorylation (Gupta et al., 2005). It is known that inactivation of *OGG1* in yeast leads to spontaneous mutations in the mitochondrial genome. Our analysis revealed that irradiation with low doses of gamma radiation enhanced the expression of expression

3**.1.3.3 The mitochondrial genes** 

of OGG1 at 2 h only.

90

0

*Saccharomyces cerevisiae* (From Arya et al., 2006).

20Gy

0

20Gy

0

20Gy

Fig. 4a. Expression of *OXI3* gene after low dose pre-irradiation at different time intervals in

0

0h 2h 4h 6h 8h 10h

**Treatments**

20Gy

0

20Gy

0

20Gy

95

100

105

**Relative absorbance** 

110

115

The *RAD50/MRE11* complex possesses single-strand endonuclease activity and ATPdependent double-strand-specific exonuclease activity. Rad50 provides ATP-dependent control of mre11 by unwinding and/or repositioning DNA ends into the MRE11 active site. The rt-PCR studies showed that in non-irradiated controls, there was significant increase in *MRE11* transcript level from 0 h to 4.5 h. In comparison to the non-irradiated controls, the stress dose irradiated samples showed significantly higher level of *MRE11* transcripts at 3 h time interval after irradiation. Further, in comparison to non-irradiated controls, the stress dose irradiated samples showed significantly higher levels of *XRS2* up to 3 h and reduced levels at 4.5 h after irradiation. The Mre11 complex influences diverse functions in the DNA damage response. The complex comprises the globular DNA-binding domain and the Rad50 hook domain, which are linked by a long and extended Rad50 coiled-coil domain. Recently it is reported that functions of *MRE11* complex are integrated by the coiled coils of Rad50 (Hohl et al., 2011). *MRE11* is reportedly involved in DNA double-strand break repair and possesses single-strand endonuclease activity and double-strand-specific 3'-5' exonuclease activity. Its role in meiotic DSB processing is also reported (Smolka et al., 2007).

#### **3.1.3.2 The heat shock proteins**

The western blotting studies with members of *HSP70* family showed that in untreated controls, level of *Kar2 p* did not increase significantly from 0 h to 4.5 h. In comparison to non-irradiated controls, the stress dose irradiated samples showed significantly higher *Kar2 p* level at 3 and 4.5 h. The *Ssa1p* transcript level did not change in the untreated control from 0 h till 4.5 h. In stress dose irradiated samples, the *Ssa1p* level increased up to 3 h but decreased significantly at 4.5 h, in comparison to the non-irradiated control (Bala & Dwivedi 2005). By microarray technique also the *SSA1* level were found to be lower in the stress dose irradiated samples as compared to the untreated control at 4.5 h (Table 4). In stress dose irradiated cultures, the *Ssa2p* level was significantly higher than the non-irradiated control at 0h and 3 h (Figure 3).

Fig. 3. The effect of low dose irradiation (20 Gy) on expression of Kar2p, Ssa1p and Ssa2p. The top strip of membrane blot shows the protein expression in unirradiated controls.

#### 3**.1.3.3 The mitochondrial genes**

158 Gamma Radiation

The *RAD50/MRE11* complex possesses single-strand endonuclease activity and ATPdependent double-strand-specific exonuclease activity. Rad50 provides ATP-dependent control of mre11 by unwinding and/or repositioning DNA ends into the MRE11 active site. The rt-PCR studies showed that in non-irradiated controls, there was significant increase in *MRE11* transcript level from 0 h to 4.5 h. In comparison to the non-irradiated controls, the stress dose irradiated samples showed significantly higher level of *MRE11* transcripts at 3 h time interval after irradiation. Further, in comparison to non-irradiated controls, the stress dose irradiated samples showed significantly higher levels of *XRS2* up to 3 h and reduced levels at 4.5 h after irradiation. The Mre11 complex influences diverse functions in the DNA damage response. The complex comprises the globular DNA-binding domain and the Rad50 hook domain, which are linked by a long and extended Rad50 coiled-coil domain. Recently it is reported that functions of *MRE11* complex are integrated by the coiled coils of Rad50 (Hohl et al., 2011). *MRE11* is reportedly involved in DNA double-strand break repair and possesses single-strand endonuclease activity and double-strand-specific 3'-5' exonuclease activity. Its role in meiotic DSB processing is also reported (Smolka et al., 2007).

The western blotting studies with members of *HSP70* family showed that in untreated controls, level of *Kar2 p* did not increase significantly from 0 h to 4.5 h. In comparison to non-irradiated controls, the stress dose irradiated samples showed significantly higher *Kar2 p* level at 3 and 4.5 h. The *Ssa1p* transcript level did not change in the untreated control from 0 h till 4.5 h. In stress dose irradiated samples, the *Ssa1p* level increased up to 3 h but decreased significantly at 4.5 h, in comparison to the non-irradiated control (Bala & Dwivedi 2005). By microarray technique also the *SSA1* level were found to be lower in the stress dose irradiated samples as compared to the untreated control at 4.5 h (Table 4). In stress dose irradiated cultures, the *Ssa2p* level was significantly higher than the non-irradiated control

Fig. 3. The effect of low dose irradiation (20 Gy) on expression of Kar2p, Ssa1p and Ssa2p. The top strip of membrane blot shows the protein expression in unirradiated controls.

**3.1.3.2 The heat shock proteins** 

at 0h and 3 h (Figure 3).

*Saccharomyces cerevisiae* is an excellent eukaryotic model system to study DNA repair mechanisms because DNA repair pathways are highly conserved between human and yeast. Furthermore, yeast and human mitochondria resemble each other in structure and function. Mitochondria are the major sites of energy (ATP) production in the cell. Mitochondria also perform many other cellular functions, such as respiration and heme, lipid, amino acid and nucleotide biosynthesis. Mitochondria also maintain the intracellular homeostasis of inorganic ions and initiate programmed cell death. Mitochondria are the major source of endogenous reactive oxygen species (ROS) in cells as they contain the electron transport chain that reduces oxygen to water by addition of electrons during oxidative phosphorylation. The rt-PCR studies with the mitochondrial genes (*SSC1* gene coding for mtHsp70, *OXI3* gene coding for COX1 respiratory component of complex-IV and *OGG1*gene) showed that the expression of *OXI3* was more than unirradiated controls up to 6 h and that of *SSC1* only at 2 and 10 hours after irradiation (20 Gy, Figure 4a,b). The expression of OGG1 was increased up to 2 hour only, after irradiation [(20 Gy), data not shown]. The mitochondrial genome of eukaryotic cells is extremely susceptible to damage due to constant exposure to significant amounts of reactive oxygen species (ROS) produced endogenously by mitochondria as a by-product of oxidative phosphorylation (Gupta et al., 2005). It is known that inactivation of *OGG1* in yeast leads to spontaneous mutations in the mitochondrial genome. Our analysis revealed that irradiation with low doses of gamma radiation enhanced the expression of expression of OGG1 at 2 h only.

Fig. 4a. Expression of *OXI3* gene after low dose pre-irradiation at different time intervals in *Saccharomyces cerevisiae* (From Arya et al., 2006).

Radiation Induced Radioresistance – Role of DNA Repair and Mitochondria 161

these genes after low dose (20 Gy) irradiation (Figure 5a and 5b). This suggested that a

Fig. 5a. Sequencing of COX1 gene PCR product by forward primer (top chart) and reverse primer (bottom chart). Irradiation with low dose (20 Gy) did not induce changes in DNA

sequence.

RIR inducing doses were not inducing any mutations in the gene products.

Fig. 4b. Expression of *SSC1* (mt-HSP70) gene after low dose pre-irradiation at different time intervals in *Saccharomyces cerevisiae* (From Arya et al., 2006)

Maintenance of mitochondrial DNA (mtDNA) is essential for ensuring respiratory competence. *MGM101* was identified as a gene essential for mtDNA maintenance in *S. cerevisiae*. The MGM101p binds the DNA. The MGM101 function exclusively in the repair of DNA contained in the mitochondrial organelle, and is predicted to participate in base excision and/or nucleotide excision repair pathways. *Saccharomyces cerevisiae* contain 3 different Hsp70s i.e. *SSC1*, *SSQ1*, and *SSC3*. Amongst these, *SSC1* is the most abundant constitutively expressed multifunctional Hsp70 and is essential for the viability of yeast cells. It plays a critical role in protein translocation across the mitochondrial inner membrane and folding of almost all pre-proteins targeted to the mitochondrial matrix compartment, thus maintaining protein homeostasis in mitochondria. For proper translocation function, SSC1p moves to the translocation Tim23-channel as a core component of "import motor complex" via the peripheral membrane protein, Tim44. Besides translocation function, SSC1p plays a crucial role in folding of proteins that are imported into the mitochondrial matrix The mitochondrial genome of eukaryotic cells is extremely susceptible to damage due to constant exposure to significant amounts of ROSs produced endogenously by mitochondria as a by-product of oxidative phosphorylation. It is known that inactivation of OGG1 in yeast leads to spontaneous mutations in the mitochondrial genome. Our analysis revealed that irradiation with low doses of gamma radiation enhanced the expression of OGG1 after 2 h. The inactivation of human OGG1 is known to induce both the spontaneous and induced mutations in the mitochondrial genome.

#### **3.1.4 Sequencing of mitochondrial genes**

The amplified product of two of the mitochondrial genes *COX1* and *SSC1* were sequenced using commercial services. No significant change was observed in DNA sequence of both

0h 2h 4h 6h 8h 10h

80

0

20Gy

**3.1.4 Sequencing of mitochondrial genes** 

0

intervals in *Saccharomyces cerevisiae* (From Arya et al., 2006)

20Gy

0

20Gy

Fig. 4b. Expression of *SSC1* (mt-HSP70) gene after low dose pre-irradiation at different time

Maintenance of mitochondrial DNA (mtDNA) is essential for ensuring respiratory competence. *MGM101* was identified as a gene essential for mtDNA maintenance in *S. cerevisiae*. The MGM101p binds the DNA. The MGM101 function exclusively in the repair of DNA contained in the mitochondrial organelle, and is predicted to participate in base excision and/or nucleotide excision repair pathways. *Saccharomyces cerevisiae* contain 3 different Hsp70s i.e. *SSC1*, *SSQ1*, and *SSC3*. Amongst these, *SSC1* is the most abundant constitutively expressed multifunctional Hsp70 and is essential for the viability of yeast cells. It plays a critical role in protein translocation across the mitochondrial inner membrane and folding of almost all pre-proteins targeted to the mitochondrial matrix compartment, thus maintaining protein homeostasis in mitochondria. For proper translocation function, SSC1p moves to the translocation Tim23-channel as a core component of "import motor complex" via the peripheral membrane protein, Tim44. Besides translocation function, SSC1p plays a crucial role in folding of proteins that are imported into the mitochondrial matrix The mitochondrial genome of eukaryotic cells is extremely susceptible to damage due to constant exposure to significant amounts of ROSs produced endogenously by mitochondria as a by-product of oxidative phosphorylation. It is known that inactivation of OGG1 in yeast leads to spontaneous mutations in the mitochondrial genome. Our analysis revealed that irradiation with low doses of gamma radiation enhanced the expression of OGG1 after 2 h. The inactivation of human OGG1 is known to induce both the spontaneous and induced mutations in the mitochondrial

The amplified product of two of the mitochondrial genes *COX1* and *SSC1* were sequenced using commercial services. No significant change was observed in DNA sequence of both

0

**Treatments**

20Gy

0

20Gy

0

20Gy

100

**Relative absorbance**

genome.

120

140

these genes after low dose (20 Gy) irradiation (Figure 5a and 5b). This suggested that a RIR inducing doses were not inducing any mutations in the gene products.



Fig. 5a. Sequencing of COX1 gene PCR product by forward primer (top chart) and reverse primer (bottom chart). Irradiation with low dose (20 Gy) did not induce changes in DNA sequence.

Radiation Induced Radioresistance – Role of DNA Repair and Mitochondria 163

Lane 1). No significant change in the fluorescence intensity or mobility of bands could be recorded after low dose irradiation (20 Gy) (Lane 2). However, in comparison to untreated controls (lane1), in samples irradiated with 200 Gy, there was observable decrease in the fluorescence intensity of high molecular weight bands and increase in the smear intensity along the lanes (Figure 6, lane 3). This suggested that 20 Gy was too small a radiation dose to cause sufficiently large number of double strand breaks to be detected by PFGE. Although, presence of other types of DNA damage viz. base damage, DNA cross-links or single strand breaks as predicted by ionizing radiation at this dose could not be ruled out. The increase in the smear along the lanes in 200 Gy irradiated samples was due to settling down of broken DNA fragments along the lanes. This was similar to our earlier observations with X-irradiated (Bala & Jain, 1996) and 60Co-irradiated yeast cells (Bala & Mathew, 2002) and indicated that radiation dose 200 Gy could cause DNA double strand breaks immediately after irradiation. The samples pre-irradiated with 20 Gy, incubated in PBG for 2 h and then irradiated with 200 Gy showed greater DNA bands intensities in higher molecular weight region (Lane 4) as compared to non-pre-irradiated but 200 Gy irradiated samples (Lane 3). This suggested that cells which were pre-irradiated with 20 Gy and maintained in PBG for 2 h prior to lethal dose (200 Gy) irradiation, suffered considerably lower DNA damage as compared to the lethally-irradiated

Fig. 6. (Gel picture and corresponding densitometry): The pre-irradiation with low dose (20 Gy) reduces chromosomal DNA damage induced by lethal doses of 60Co--radiation (200

Gy) as studied by pulsed-field gel electrophoresis. Lane1: untreated control; Lane 2: 60Co--ray (20 Gy); Lane 3: 60Co--ray (200 Gy); Lane 4: 60Co--ray (20 Gy) +

incubation in PBG for 2 h + 60Co--ray (200 Gy).

1 2 3 4


Fig. 5b. Sequencing of SSC1 (mt HSP70) gene PCR product by forward primer (top chart) and reverse primer (bottom chrt). Irradiation with low dose (20 Gy) did not induce changes in DNA sequence.

#### **3.1.5 Chromosomal DNA damage and repair in** *S. cerevisiae* **cells showing RIR**

Study of chromosomal DNA damage was considered important because of its role in low dose induced responses (Bala & Dwivedi 2005; Collis et al., 2004)*. S. cerevisiae* has small genome divided into sixteen chromosomes of sizes ranging from 240 to 2200 kb, which can be easily resolved into discrete bands by pulsed-field gel electrophoresis (PFGE)**.** In our study plan, PFGE could resolve the genomic DNA into several bands (Figure 6 a,b,

Fig. 5b. Sequencing of SSC1 (mt HSP70) gene PCR product by forward primer (top chart) and reverse primer (bottom chrt). Irradiation with low dose (20 Gy) did not induce changes

**3.1.5 Chromosomal DNA damage and repair in** *S. cerevisiae* **cells showing RIR** 

Study of chromosomal DNA damage was considered important because of its role in low dose induced responses (Bala & Dwivedi 2005; Collis et al., 2004)*. S. cerevisiae* has small genome divided into sixteen chromosomes of sizes ranging from 240 to 2200 kb, which can be easily resolved into discrete bands by pulsed-field gel electrophoresis (PFGE)**.** In our study plan, PFGE could resolve the genomic DNA into several bands (Figure 6 a,b,

in DNA sequence.

Lane 1). No significant change in the fluorescence intensity or mobility of bands could be recorded after low dose irradiation (20 Gy) (Lane 2). However, in comparison to untreated controls (lane1), in samples irradiated with 200 Gy, there was observable decrease in the fluorescence intensity of high molecular weight bands and increase in the smear intensity along the lanes (Figure 6, lane 3). This suggested that 20 Gy was too small a radiation dose to cause sufficiently large number of double strand breaks to be detected by PFGE. Although, presence of other types of DNA damage viz. base damage, DNA cross-links or single strand breaks as predicted by ionizing radiation at this dose could not be ruled out. The increase in the smear along the lanes in 200 Gy irradiated samples was due to settling down of broken DNA fragments along the lanes. This was similar to our earlier observations with X-irradiated (Bala & Jain, 1996) and 60Co-irradiated yeast cells (Bala & Mathew, 2002) and indicated that radiation dose 200 Gy could cause DNA double strand breaks immediately after irradiation. The samples pre-irradiated with 20 Gy, incubated in PBG for 2 h and then irradiated with 200 Gy showed greater DNA bands intensities in higher molecular weight region (Lane 4) as compared to non-pre-irradiated but 200 Gy irradiated samples (Lane 3). This suggested that cells which were pre-irradiated with 20 Gy and maintained in PBG for 2 h prior to lethal dose (200 Gy) irradiation, suffered considerably lower DNA damage as compared to the lethally-irradiated

Fig. 6. (Gel picture and corresponding densitometry): The pre-irradiation with low dose (20 Gy) reduces chromosomal DNA damage induced by lethal doses of 60Co--radiation (200 Gy) as studied by pulsed-field gel electrophoresis. Lane1: untreated control; Lane 2: 60Co--ray (20 Gy); Lane 3: 60Co--ray (200 Gy); Lane 4: 60Co--ray (20 Gy) + incubation in PBG for 2 h + 60Co--ray (200 Gy).

Radiation Induced Radioresistance – Role of DNA Repair and Mitochondria 165

involved in double-strand break (DSB) repair, DNA recombination and cell cycle checkpoint control (Carson et al., 2003). The complex participates in single-strand endonuclease activity and double-strand-specific 3'-5' exonuclease activity. The protein expression of Mre11p in low dose irradiated cells was enhanced (about 1.5 times) as compared to non pre-irradiated cell after 5.0 hours of irradiation. This was similar to the enhanced the expression of *MRE11* in *S. cerevisiae* (Table 3, Figure 2). *NBS1* or *p95* is another component of the *MRN* complex, which has a role in the recruitment of the *MRN* complex to double strand break sites for DNA repair. *NBS1* plays a critical role in the cellular response to DNA damage and the maintenance of chromosome integrity. *NBS1* modulate the DNA damage signal sensing by recruiting PI3/PI4-kinase family members ATM, ATR, and probably DNA-PKcs to the DNA damage sites and activating their functions (Frappart 2005; Stiff et al., 2005). It can also recruit *MRE11* and *RAD50* to the proximity of DSBs by an interaction with the histone H2AX. *NBS1* also functions in telomere length maintenance by generating the 3' overhang which serves as a primer for telomerase dependent telomere elongation. The Nbs1p levels in low dose irradiated cells were significantly reduced (nearly 2 times) as compared to non-pre-irradiated cell after 5.0 hours of irradiation. It is not clear why the protein levels were reduced. *NBS1,* since, is inducible gene, time dependent studies are now planned to understand the role of *NBS1* in RIR. After 5 hour of low dose exposure Rad50p level was similar as in unirradiated cells (Figure 7). *RAD50* is required to bind DNA ends and hold them in close proximity This could facilitate searches for short or long regions of sequence homology in the recombining DNA templates, and may also stimulate the activity of DNA ligases and/or restrict the nuclease activity of *MRE11A* to prevent nucleolytic degradation past a given point (Jager et al., 2001, Waltes et al., 2009). In our study, the levels of RAD50p did not

alter 5 hour after low dose irradiation in comparison to the untreated controls.

Fig. 7. Change in expression of Mre11p, Nbs1p and Rad50p in human PBMCs 5 hour after

irradiation with low dose (0.07 Gy) of 60Co--rays. Results were mean ±SD.

cells (lane 3). During analysis of pulsed-field gels throughout this study, the intensity changes in the individual bands in the lower molecular weight region were not given much importance because their intensities were influenced by the intensities of DNA fragments settling down as smears in the lower molecular weight regions and this has been shown to create errors in data analysis in our earlier studies (Bala & Jain 1996, Bala & Mathew 2002). **I**nduction of gene transcription or protein expression has been reported after low dose irradiation (Franco et al., 2005). Our studies showed that low dose radiation enhanced DNA repair ability and produced protective proteins to minimize the indirect damaging effects of subsequent high dose radiation.
