**3.2.1 RIR in human PBMCs - cell proliferation, micronuclei formation**

Hoechst binds with DNA and an increase in fluorescence is observed with the increase in cell number. This assay was used to determine the effects of low doses of 60Co-gammaradiation on proliferation of PBMCs. The cells pre-irradiated with low dose of 60Co-gammarays (0.07 Gy) and 4-5 hours later irradiated with high dose 60Co-gamma-rays (5 Gy, LD 50) showed significantly higher cell proliferation (RIR), in comparison to non-pre-irradiated but lethally irradiated (5 Gy) cells. The RIR, however, was much less before 4h and after 5h of time interval between low dose and high dose exposure (Bala et al., 2002). The maximum increase was observed if 60Co-gamma-rays (5 Gy, LD 50) were given 5 h after the low dose (0.7 Gy) irradiation. In comparison to the non-pre-irradiated controls, the pre-irradiated cells showed decrease in micronuclei frequency and the decrease varied between the donors from 23.4% - 31.8% (Table 5, Bala et al., 2002).


Table 5. Micronucei (MN) in human PBMCs stained with hoechst 33342 and total 1000 cells were scored per sample at 400 magnification in fluorescence microscope. % decrease in MN = (MNlow dose+high dose -MNlow dose) X 100/MNhigh dose; \* indicates significant (p0.05) change with respect to 5.0 Gy irradiated controls (from Bala et al., 2002).

#### **3.2.2 Effect of RIR on MRN complex proteins in human PBMCs**

The *MRE11/RAD50/NBN* (*MRN*) complex in humans comprises genes, which are homologous/analogous the to *MRX* complex in *S. cerevisiae.* The *MRN* complex is

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

The phenomenon of radioadaptive response has been reported in human lymphocytes in various studies (Table 1). The advantages of the lymphocytes as a model to understand the low dose ionizing radiation response is due to their radiosensitivity. More over, the lymphocytes are found in circulating peripheral blood and therefore, can be easily obtained from peripheral venous blood. These cells involved in cell mediated immunity as well as humoral immunity and cell proliferation and their radiosensitivity is similar to that of

Hoechst binds with DNA and an increase in fluorescence is observed with the increase in cell number. This assay was used to determine the effects of low doses of 60Co-gammaradiation on proliferation of PBMCs. The cells pre-irradiated with low dose of 60Co-gammarays (0.07 Gy) and 4-5 hours later irradiated with high dose 60Co-gamma-rays (5 Gy, LD 50) showed significantly higher cell proliferation (RIR), in comparison to non-pre-irradiated but lethally irradiated (5 Gy) cells. The RIR, however, was much less before 4h and after 5h of time interval between low dose and high dose exposure (Bala et al., 2002). The maximum increase was observed if 60Co-gamma-rays (5 Gy, LD 50) were given 5 h after the low dose (0.7 Gy) irradiation. In comparison to the non-pre-irradiated controls, the pre-irradiated cells showed decrease in micronuclei frequency and the decrease varied between the donors

Donors Micronuclei per 1000 binucleated cells at doses (Gy) % decrease

I 13±0.33 15±1.02 245±11.08 167±8.92\* 31.8±2.29 II 18±0.89 16±1.26 166±9.67 89±6.65\* 23.4±2.14 X 12±0.65 14±0.88 298±15.38 206±8.77\* 30.8±2.47 Table 5. Micronucei (MN) in human PBMCs stained with hoechst 33342 and total 1000 cells were scored per sample at 400 magnification in fluorescence microscope. % decrease in MN = (MNlow dose+high dose -MNlow dose) X 100/MNhigh dose; \* indicates significant (p0.05)

The *MRE11/RAD50/NBN* (*MRN*) complex in humans comprises genes, which are homologous/analogous the to *MRX* complex in *S. cerevisiae.* The *MRN* complex is

**3.2.1 RIR in human PBMCs - cell proliferation, micronuclei formation** 

0 0.07 5.0 0.07+5.0

change with respect to 5.0 Gy irradiated controls (from Bala et al., 2002).

**3.2.2 Effect of RIR on MRN complex proteins in human PBMCs** 

subsequent high dose radiation.

**3.2 Studies with human PBMCs** 

proliferating cells of the hematopoetic tissue.

from 23.4% - 31.8% (Table 5, Bala et al., 2002).

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.

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