Algunas concepciones de Emprendimiento

In this section, th e biochem ical techniques th a t have been u sed to m easu re the in d u ctio n an d rep air of DNA dsb in m am m alian cells are described and th e resu lts characteristically obtained w ith th ese m ethods are d iscussed. U nlike cytogenetic or recom binant DNA assay s, th ese tech n iq u es m easu re th e cum ulative (bulk) effect of rad iatio n on a large n u m b er of cells (in th e order of 1.10^ - 1.10® cells/sam p le). C aution n eed s to be adopted w hen com paring the resu lts of different a ssa y s if non-isogenic cell types have been used. C hrom atin stru c tu re can differ m arkedly betw een cell lines an d th is could determ ine th e resp o n se of

cells to ionising rad iatio n {e.g. W heeler an d W ierowski 1983; Koval and K azm ar 1988a).

The n e u tra l velocity se d im e n ta tio n tec h n iq u e (L ehm ann an d Stevens 1977; B locher 1982) is, a t p resen t, th e only assay th a t enables direct m easu rem en t of th e m olecular w eight of the DNA (based on the d istan ce sedim ented by th e DNA in a n e u tra l su cro se g rad ien t u n d e r centrifugation) an d th u s of th e actual n u m b ers of dsb presen t in the DNA. The technique u n fo rtu n ately h a s th e d isadvantages of being extrem ely tim e consum ing an d n o t p articu larly sensitive i.e. the doses u sed are generally outside the ’biologically relevant’ range. Using th is technique, several au th o rs have found th a t dsb are formed as a linear function of dose in b acteria (K rasin an d H u tch in so n 1977), y east (Resnick an d M artin 1976; F rankenberg-Schw ager et al. 1979) and in m am m alian cells (Corry an d Cole 1973; L ehm ann and Stevens 1977; Blocher 1982; A hnstrôm and B ryant 1982). The fact th a t n e u tra l velocity sedim entation stu d ies have found dsb to be linearly dependent on dose (except a t very high doses of -4 0 0 0 Gy w here ssb are so frequent th a t they by chance in teract to form a dsb) rep resen ts stro n g evidence in favour of th e ’single-track’ theory of dsb in d u ctio n referred to earlier. The n e u tra l velocity sed im en tatio n technique is therefore generally regarded as the m ost reliable a ssa y for dsb.

The DNA unw inding technique em ploys alkali lysis conditions (pH 12) w hich cau ses the DNA stra n d s to separate and unw ind from points of b reak ag e. U n d er carefu lly controlled lysis co nditions th e e x ten t of unw inding is proportional to the n u m b er of stra n d breaks. This m ethod therefore detects a to tal n u m b er of b reak s [i.e. ssb p lu s dsb), b u t B ryant an d B locher (1980) devised an experim ental protocol by w hich DNA unw inding can be u se d to follow the kinetics of d s b repair. In th is resp ect they found good sim ilarity betw een rep air kinetics a s m easured

by th e DNA unwinding and n e u tra l velocity sedim entation techniques. The DNA unw in d in g tech n iq u e h a s th e ad v an tag e of its tech n ical sim plicity as well as good reproducibility.

B oth th e above techniques d etected first-order repair kinetics i.e.

an exponential decrease in th e n u m b er of dsb rem aining w ith tim e. The half-tim es ( t i/2) of repair were estim ated a t -3 .5 h (Lehmann and Stevens 1977) an d 1 3 7 = 2-4 h depending o n c u ltu re conditions (B ryant and B locher 1982; Blocher an d Pohlit 1982). T hese resu lts th u s im plied a single, relatively slow repair com ponent w hich in tu rn signified th a t there m ay only be one m echanism of d sb repair. U sing the DNA sy n th esis inhibitor a ra A (9-p-D -arabinofuranosyladenine) B ryant and Blocher (1982) show ed th a t th is repair m echanism h a d a n absolute requirem ent for DNA polym erization. T his w ould su p p o rt th e notio n of a recom bination m echanism of dsb repair as against ligation.

A nother rapid and sim ple m ethod for detecting DNA breaks is the DNA precipitation assay of Olive (1988). This technique is based on the differential precipitibility of varying DNA fragm ent sizes (large fragm ents precipitate while the sm aller fragm ents rem ain in the su pernatant), b u t a m ajor draw back of th is technique is its insensitivity for the detection of dsb. U nder the n eu tral conditions u sed to assay for dsb the percentage of DNA precipitated decreases linearly a s a function of X-ray dose, indicating a linear increase of dsb w ith dose albeit a t relatively large X-ray doses (40 - 200 Gy). No dsb repair stu d ies have yet been u n d ertak en u sin g this precipitation technique.

R ecently B locher et al. (1989) show ed th a t th e new ly developed CH EF (clam ped h o m o g en eo u s e le ctrica l field) gel e le ctro p h o resis technique w as a suitable m ethod for m easuring dsb in m am m alian DNA th a t is b ased on the m ovem ent of negatively charged DNA m olecules in an electric field. T his a ssa y ex h ib its good sen sitiv ity an d allow s the

detection of dsb down to ~2 Gy of X -rays. In th eir initial rep o rt Blocher

e t a l (1989) observed n o n -lin e ar induction of dsb w ith dose, b u t in a su b seq u e n t article (Blocher 1990) he explains th a t th is m ay be due to an a rte fa c t of th e tech n iq u e, viz. th e extraction p ro p erties of th e DNA m olecules out of the well, an d once corrected for th e a ssa y gave linear in d u ctio n of dsb. S tam ato an d D enko (1990) u sing the asym m etric field in v ersio n gel electro p h o resis (AFIGE) tech n iq u e have also rep o rted a lin e ar induction of d sb w ith dose of ionising radiation. The d sb repair ra te a s m easured by CHEF electrophoresis is considerably faster th a n th at m easu red by the n eutral velocity sedim entation or DNA unw inding, w ith a re p a ir tim e co n stan t t i/ 2 of 30-40 m in (Blocher et a l 1989). A kinetic s tu d y revealed biphasic ra th e r th a n first-order rep air kinetics, b u t this h a s as yet not been repeated by other investigators.

The n o n -d en atu rin g filter elution technique of B radley an d Kohn (1979) is cu rren tly the m o st widely u sed a ssa y for DNA dsb an d th is technique exhibits non-linear dsb induction and biphasic rep air kinetics w ith a t i/ 2 of 30-40 m in (Kemp et a l 1984). T his technique an d these re su lts are discussed in greater detail in the following section (1.6).

The above tech n iq u es are q u an titativ e a ssa y s of th e in d u ctio n frequency of dsb and the extent of dsb repair th a t can n o t com m ent on the type of rep air effected or w h eth er different types of DNA term in i are m ore pro n e to erro n eo u s rep air. R ecom binant DNA tec h n iq u e s are required to investigate th ese asp ects of the fidelity of dsb rep air (Thacker

1986).

It is fairly obvious th a t th e resu lts of studies of induction and repair of dsb depend largely on the assay th a t w as employed. This leaves one to q u estio n the accuracy a n d /o r validity of each a ssa y an d th e possible in terp retatio n of the resu lts, especially in the case of the non-denaturing filter elution assay.

1.6 The controversy surrounding the non-denaturing filter