SPontaneous Additional Synthesis of DNA and formation OF chromosome aberrations

H.A.Hakimov a, G.F.Mahmudovaa , R.A.Yakubova a , E.A.Latypova a , A.P.Akifyev b

aInstitute of Genetics and Experimental Biology of Plants, Uzbekistan

bInstitute of General Genetics, Russia

Anumberofrecentresearchespointoutthenonrandomcharacterofallocation of chromosome aberrations (CA) along the length of a chromosome.Besides, thereareclearexperimentaldatawhichallowa suggestion thatexchangethemainreasonofexchange chromosomeaberrations(ECA) formation is not a distortion of mutagen induced DNA repair but some a regular process in cellular nucleus which initiates recombination processes resulted in ECA formation [1].

Sucha processwaspredictedabout 30 yearsagobyagroupofRussiangeneticists by the name of Spontaneous Additional Synthesis (SAS) of DNA [2]. Anumberofexperimentaldatainfavorofthishypothesiswasobtainedafterthat, howevera SAS is still a hypothetic process, the main features of which are not well studied yet.

Wehavefoundinourownpreliminary researchesonanimal (humanlymphocytes) andplant (Crepis capillaris and wheat) cells that incorporation of 5-bromo-2’-deoxyuridine (BrdU) at DNA in G0, G1 and the border of G1 and S-phases as well as in S-phase itself results to similar radiosensitizing effect on the CA criteria [3]. It should be noted that we used the BrdU as a marker in our experiments, because it incorporates only at DNA molecule. Thuswehavefacedwithafactthatincorporation of BrdU at minor fractions of DNA, for example, in G0 and G1-phases leaded to sharply expressed radiosensitizing effect analogous to the same in S-phase.

Materials and Methods

In the present work we conducted cytological analysis of a character of incorporation of BrdU at human lymphocytes DNA as well as the cytogenetic research of the peculiarities of modifying effect of the Etoposide - an inhibitor of Topo II, on a character of a radiosensitizing effect of BrdUin PHA–stimulated human lymphocytes. To do this we studied a result of combined action of BrdU andEtoposide in GO, G1, S and G2-phases of a cellular cycle. Duration of each treatment of cells was equal to 4 hours: 0-4 hrs in G0, 20-24 hrs in G1, 32-36 hrs in S and 44-48 hrs in G2- phase. At the end of each treatment cells were irradiated with gamma-rays of Co60 (4 Gy). A character of BrdU incorporation in G1 (10-24 hrs) and S-G2 -phase (34-48 hrs) has been studied by means of differential chromosome staining technique [4]. We have also conducted cytogenetic analysis of radiosensitizing effect of combined action of BrdU and Etoposide on human lymphocytes.

Results and Discussion

Theanalysisofdifferentiallystainedchromosomesshowsthat BrdU incorporation at DNA in different phases of cellular cycle has a specific character. InG0, G1 andG2 - phasesBrdU incorporatesatseparatelocusesof chromosomes and the staining occurs on the R-type. On the other hand in S-phase incorporation of BrdU takes place in accordance with the course of DNA replication process and chromosomes themselves were stained on harlequin type (Fig 1).

а) b)

Fig 1. DifferentiallystainedchromosomesofhumanlymphocytesaftertheirincubationwithBrdUin G1 (а) and S-G2-phase (b).

The cytogenetic analysis of combined action of BrdU and Etoposide has showna specific character of the modifying influence of Etoposide on radiosensitizing effect of BrdU depending on the phase of a cell cycle (Table 1). It turned out that DNA sequences which included in BrdU in G2-phasewere much more sensitive to combined action of BrdU and Etoposide on all four studied criteria, whereas G0- and G1- cells have expressed practically a similar character

Table 1. Cytogenetic analysis of combined action of BrdU and Etoposide on human lymphocytes

Phase/Treatment conditions / Aberrant cells (in %) / CA per one cell / ExchangeCA per one cell / DSBs per one cell
G0 / BrdU+irradiation / 54,0 / 1,2+/-0.2 / 0.3 +/- 0.1 / 0.8 +/- 0.1
Thymidine+irradiation / 29,5 / 0.4+/-0.1 / 0.10 +/- 0.04 / 0.3 +/- 0.1
BrdU+Et / 23,0 / 0,6/-0,1 / 0.10 +/- 0.04 / 0.4 +/- 0.1
BrdU+Et+irraditation / 58,8 / 1,2+/-0,1 / 0,24+/-0,07 / 0,8+/-0,1
Thymidine+Et+irradiation / 40,7 / 0,8+/-0,1 / 0, 2+/-0,06 / 0,5+/-0,1
G1 / BrdU+irradiation / 61,0 / 1,5+/-0,2 / 0,4+/-0,1 / 0,9+/-0,1
Thymidine+iiradiation / 29,5 / 0,6+/- 0.1 / 0,13 +/- 0,04 / 0,4+/- 0.1
BrdU+Et / 42,1 / 1,4+/-0,4 / 0,3+/-0,1 / 0,9+/-0,2
BrdU+Et+irraditation / 58,8 / 1,1+/-0.2 / 0.3+/-0.1 / 1,1+/- 0.2
Thymidine+Et+irradiation / 36,7 / 1,0+/-0,3 / 0,2+/-0,1 / 1,0+/-0,3
G2 / BrdU+irradiation / 38,0 / 0.6+/-0.2 / 0.1 +/- 0.04 / 0.3 +/- 0.1
Thymidine+iiradiation / 36,0 / 0.5+/-0.1 / 0.06 +/- 0.02 / 0.3 +/- 0.1
BrdU+Et / 42,3 / 0.8+/-0.2 / 0.10 +/- 0.06 / 0.5 +/- 0.1
BrdU+Et+irraditation / 88,9 / 4,5+/-0,7 / 0,44+/-0,16 / 2,0+/-0,4
Thymidine+Et+irradiation / 66,7 / 1,3+/-0,3 / 0,2+/-0,1 / 0,8+/-0,2

of their sensitivity to radiosensitizing action of BrdU, doesn’t matter whether this action was combined with Etoposide or not. On the contrary an incubation of lymphocytes with BrdU and Etoposide in G2 resulted in more than two times increasing of all studied criteria.

Conclusion

First of all, our analysis shows thatBrdU incorporation outside of S-phase occurs at minor DNA fraction, which presents a very insignificant part of genomic DNA. Correspondingly we can conclude that the radiosensitizing effect of BrdU is determined by not amount of incorporated BrdU but localizationat a chromosome of those DNA sequences which include in BrdU during SAS of DNA in mentioned periods of cell cycle.

It proved to be also that Ethoposyde affectsspecifically upon the character of radiosensitizing effect of BrdU in G0, G1 and G2- phases. An analysis of data obtained in present study allows us to propose that BrdU incorporates in G2 phases at DNA fractions which are placed at the nuclear matrix attachments cites or very closely to them:BrdU+Etoposide treatment of G2-cells in combination with irradiation resulted in significant increasing of DSB and ECA.

Acknowledgements

We appreciate very much the comments on the manuscript of Dr. M. Gildieva and Dr. A.Ergashev.

References

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