ISSN 2079�0597, Russian Journal of Genetics: Applied Research, 2014, Vol. 4, No. 1, pp. 15–18. © Pleiades Publishing, Ltd., 2014.Original Russian Text © E.N. Plyusnina, M.V. Shaposhnikov, E.N. Andreeva, A.A. Moskalev, L.V. Omelyanchuk, 2013, published in Vavilovskii Zhurnal Genetiki i Selektsii, 2013,Vol. 17, No. 3, pp. 399–403.

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INTRODUCTION

GADD45 (growth arrest and DNA�damage�inducible 45) family proteins are involved in the regu�lation of gene expression, cell cycle arrest, DNArepair, and apoptosis (Moskalev et al., 2012b). Theirexpression in mammals is induced under stress condi�tions, including oxidative and genotoxic stresses(Duan et al., 2005). GADD45 interacts with proteinsparticipating in DNA excision repair (Smith et al.,1994), as well as with proteins involved in DNA doublestrand break repair (Lee et al., 2012). In Drosophila,only one GADD45 gene homolog D�GADD45 is knownand its functions are much less investigated (Peretzet al., 2007). Nevertheless, earlier we demonstratedthat D�GADD45 overexpression in Drosophila melano�gaster results in increased longevity (Plyusnina et al.,2011).

One of the possible explanations of the corre�sponding life span increase is that GADD45 overex�pression enhances the resistance to stress. We showedpreviously that flies with overexpressed D�GADD45 aremore resistant to oxidative and genotoxic stresses, heatshock, and starvation than flies with normallyexpressed D�GADD45 (Moskalev et al., 2012a). At thesame time, the majority of known stress factors

enhance D�GADD45 expression in wild type Droso�phila melanogaster strains (Moskalev et al., 2012a).

There exist sufficient data asserting the relationshipbetween stress resistance and longevity. In the courseof the organism aging, a decrease in stress resistance isobserved (Semenchenko et al., 2004). Overexpressionof genes enhancing stress resistance leads to theincrease in the lifespan (Orr and Sohal, 1994; Gianna�kou et al., 2004). Besides that, animals with mutationsthat lead to enhanced longevity are characterized byincreased tolerance to oxidative stress and heat shock(Johnson et al., 2001; Weisman et al., 2012). At thesame time, short�lived mutants of the model organ�isms possess decreased resistance to stress factors (Ver�meulen et al., 2005). The activity of genes that facili�tate aging induces endogenous stresses (e.g., P66)(Migliaccio et al., 1999). Selection of animals forresistance to some stress factor increases resistance toa number of other factors and leads to the increase inthe life span (Harshman et al., 1999). Species, e.g.,rodents, bats, and birds, with different longevity differas well in stress resistance especially in resistance tooxidative stress (Salmon et al., 2009). The long�livingnaked mole rat (Heterocephalus glaber) possessesenhanced resistance to a wide spectrum of cytotoxins,including heat shock, heavy metals, genotoxic agents,

The Analysis of the Survivorship Curves in Drosophila melanogaster with D�GADD45 Overexpression

E. N. Plyusninaa, b, M. V. Shaposhnikova, b, E. N. Andreevac, A. A. Moskaleva, b, d, and L. V. Omelyanchukc

aInstitute of Biology, Komi Scientific Center, Ural Branch, Russian Academy of Sciences, Syktyvkar, Russiae�mail: ome@mcb.nsc.ru

bSyktyvkar State University, Syktyvkar, RussiacInstitute of Molecular and Cell Biology, Siberian Branch, Russian Academy of Sciences, Novosibirsk, Russia

dMoscow Institute of Physics and Technology (State University), Moscow, RussiaReceived July 29, 2013; in final form, August 17, 2013

Abstract—It is well known that GADD45 overexpression in the nervous system leads to the increased longevityin Drosophila. In this study, we performed an approximation of survivorship curves in Drosophila withGADD45 overexpression using the Gompertz distribution. We showed that the life span prolongation is causedby the alteration of R parameter, reflecting the mortality rate at the beginning of adult life, and not by thealteration of another Gompertz distribution parameter, α, reflecting the exponentially growing mortality rate.In addition, we analyzed the survivorship curves in the case of radiation hormesis (the phenomenon of lifespan extension under the exposure to low doses of radiation) and confirmed the regularity stated above. Onthe basis of this regularity, we concluded it promising to search for geroprotectors among the substancesdecreasing the R parameter, which is easier to assess than the full survivorship curve.

Keywords: longevity, DNA repair, GADD45, overexpression, low doses of ionizing radiation, hormesis,Drosophila melanogaster

DOI: 10.1134/S2079059714010080

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PLYUSNINA et al.

and xenobiotics (Lewis et al., 2012). The same can beseen as well in humans; for example, long�living per�sons do not necessarily have a healthy lifestyle (Raj�pathak et al., 2011). Their exceptional longevity canbe explained by increased resistance to stresses. Mod�erate stress results in the increase in the life span (Rat�tan et al., 2005). For instance, exposure to low doses ofionizing radiation leads to the life span increase inDrosophila (the hormesis effect) (Moskalev, 2007;Moskalev et al., 2011). Among the mechanisms con�tributing to the increase in longevity are the inductionof a number of protection systems such as the antioxi�dant protection system (Arking et al., 2000), DNArepair (Moskalev et al., 2013), heat shock protein syn�thesis (Zhao et al., 2005; Weisman et al., 2012),immune system activation (Amrit et al., 2010), and thedeath of damaged cells (Moskalev, 2007).

The increase in the life span is accompanied byalterations of the initial (in our terms, background, R)and exponential (in our terms, age�related, α) mortal�ity components of the Gompertz equation. Neverthe�less, the precise biological sense of R and α is not yetunderstood (Gavrilov and Gavrilova, 1991), theR parameter corresponds to the age preceding the timepoint when the exponential growth of mortality beginsand can be considered as genetically determined,while the α parameter reflects the exponential mortal�ity rate and can be considered as the consequence ofavalanche�like accumulation of molecular and cellu�lar damage in the senescent organism after reachingthe threshold age of R (Kirkwood, 2005). Hence, themathematical analysis of the corresponding parameterscould unveil the mechanisms underlying the increase inlongevity. The aim of the current work was to analyzethe R and α parameters of the Gompertz equation in theflies with increased longevity resulting from D�GADD45overexpression and radiation hormesis.

MATERIALS AND METHODS

Drosophila melanogaster strains. In our experi�ments, Drosophila melanogaster Canton�S laboratorystrain was used. UAS�D�GADD45 strain possessing anadditional D�GADD45 gene copy under the control ofUAS promoter was kindly provided by Uri Abdu, BenGurion University, Israel. 1407�GAL4 strain carryingGAL4 driver constitutively expressed in the nervoussystem was obtained from the Bloomington Droso�phila Stock Center, USA. ELAV�GS�GAL4 strain car�rying mifepristone�inducible neurospecific GAL4driver was kindly provided by Dr. Haig Keshishian,Yale University, USA.

Activation of D�GADD45 overexpression. In orderto obtain constitutive overexpression of D�GADD45 in thenervous system, UAS�D�GADD45 females were crossedwith 1407�GAL4 males. For conditional D�GADD45 over�expression in neurons, UAS�D�GADD45 females werecrossed with ELAV�GS�GAL4 males and mifepristone

RU486 (Sigma, USA) was added to feed to activateGAL4 expression in the progeny.

Irradiation conditions. Experimental Drosopfilamelanogaster strains were chronically exposed toγ irradiation from 226Ra during preimaginal stages ofdevelopment (namely, embryo, the first through thethird instar larva, pupa) at the dose rate 2.5 mGy/h.The absorbed dose equaled 40 cGy. Imagoes at an ageof no more than 24 h were exposed to acute γ irradia�tion from 137Cs. The absorbed dose equaled 30 Gy atthe exposure dose rate of 75 cGy/min.

RESULTS AND DISCUSSION

It is commonly known that the Gompertz model iswell suitable for describing the mortality process ofDrosophila individuals (Gavrilova and Gavrilova,1991). Each theoretical curve can be defined by twoparameters: R, the background mortality component,and α, the age�related mortality component. In orderto find out the decrease of which of these two mortalitycomponents leads to the increase in the life span inDrosophila with D�GADD45 overexpression in the ner�vous system, we tested two alternative hypotheses. Thefirst one states that the control and experiment do notdiffer in α, but vary in R (α�const hypothesis), and thesecond one states that the control and experiment donot differ in R, but vary in α (R�const hypothesis). Theanalysis was performed with the aid of WinModestsoftware which allows processing the data of the survi�vorship curves.

From the table, it can be seen that, in the case ofthe survivorship curves for both 1407�GAL4/UAS�D�GADD45 and UAS�D�GADD45 females and males,the hypothesis claiming constant α and variable Rproved to have the highest statistical support. In thecase of ELAV�GS�GAL4/UAS�D�GADD45 in whichoverexpression was induced in flies after eclosion, pref�erence for the constant α hypothesis was observed inmales, while there was no difference between thehypotheses in females. Actually, in the earlier experimentdemonstrating the increase in the Drosophila life span asthe result of D�GADD45 overexpression, it was observedthat the total effect was lower in ELAV�GS�GAL4/UAS�D�GADD45 flies than in 1407�GAL4/UAS�D�GADD45ones. Thus, it is reasonable to speculate that the lack ofdifference between the two hypotheses in females isdue to the low level of the corresponding effect.

Apart from the cases where increased life span isinduced by some mutations or by the overexpression ofa number of genes, the effect of radiation hormesis isalso well known. This effect consists in the increase ofthe longevity as a result of the exposure to low radia�tion doses. In order to determine if radiation hormesisacts in the same way when D�GADD45 is overex�pressed, we assessed the results of the experiment inwhich the survivorship curves of the control Canton�Sstrain were compared before and after the exposure tolow�dose γ radiation (40 cGy). The same strain

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THE ANALYSIS OF THE SURVIVORSHIP CURVES 17

exposed to a high dose (30 Gy) of radiation was treatedas the negative control. From the table, it can be seenthat, both in the case of irradiation at the dose of40 cGy and in the case of irradiation at the dose of30 Gy, the hypothesis claiming constant α and variable Rhas statistical support. At the same time, when thedose of 30 Gy was administered, a substantial increasein R was detected for both females and males of theradiation�exposed strain. When the dose of 40 cGywas administered, the reciprocal effect was observed:the low�dose irradiation led to the decrease in param�eter R in comparison with the non�radiation�exposedcontrol for both females and males. Thus, the phe�nomenon of radiation hormesis is similar to the effectof D�GADD45 overexpression; namely, the irradiationinduces the increase in longevity through the decreasein the R parameter.

Despite the fact that the precise biological signifi�cance of the R parameter and the Gompertz equationis not yet fully understood (Olshansky and Carnes,1997; Sas et al., 2012), the age R, when exponentialgrowth of mortality starts, can be considered as genet�ically determined, while age�related mortality compo�nent α can be considered as resulting from avalanche�like accumulation of molecular and cellular damage inthe senescent organism (Gavrilov and Gavrilova,1991). Considered from this point of view, the dataobtained in the current work point out that the previ�ously described increase in longevity induced by theD�GADD45 overexpression in the nervous system isconnected with the genetically determined increase inthe age when senescence begins, but not with thedecline of avalanche�like accumulation of cellulardamage in the aging organism (Plyusina et al., 2011).It was also demonstrated in the current work that thepreviously investigated effect of radiation hormesis inaging (Moskalev, 2007; Moskalev et al., 2011) is alsoassociated with the increase in the age when exponen�tial growth of genetically determined mortality begins.The results obtained here show that both investigatedphenomena of life span elongation in Drosophila

proved to be genetically predetermined. On the onehand, this raises a question as to whether this regular�ity could be universal for all mutations causing the lifespan to increase in Drosophila. On the other hand, thisregularity makes possible to specify the target forpotential anti�aging drugs. They should be selectedfrom the substances able to decrease the R parameterof the survivorship curve. It should be noted that onlythe initial part of the survivorship curve is essential forR measurement, which substantially simplifies theprocedure of anti�aging drugs detection.

ACKNOWLEDGMENTS

This study was supported by the joint project of theUral Branch of the Russian Academy of Sciences(project no. 12�C�4�1007, “Structural and FunctionalOrganization of Chromosomes during the CellCycle”) and projects of the Siberian Branch of the Rus�sian Academy of Sciences (projects nos. 81 and 82).

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Translated by E. Martynova