ANÁLISIS E INTERPRETACIÓN DE LOS RESULTADOS

4.2.1 EAT cell culture, labelling and irradiation

Ehrlich ascites tu m o u r (EAT) cells are a well characterised m ouse tu m o u r cell line th a t can be grown in suspension. The com position of the m edium for suspension cu ltu res is described in Iliakis and Pohlit (1979). Cells were kept in exponential grow th by reducing the cell concentration from 8.1 0®/ml to 2.1 0®/ml daily.

Cells a t 2.10®/m l (in 40 ml) w ere in cu b ated in vertically standing 75 cm2 plastic flask s (Sterilin) for 24 h in th e presen ce of ®H-TdR (tritiated thym idine, 1.59 TB q/m m ol). 37-74 kBq p er flask (in 40 ml) w as u sed for the DNA unw inding experim ents an d 74-148 kBq for the n e u tra l elution experim ents. E qui-m olar am o u n ts (1-2 pmol/1) of cold thym idine w ere added to facilitate th e uniform u p tak e of radioactive thym idine.

Cells were diluted to a concentration of 2-10.10®/m l in m edium and 3 m l aliquots in 5 ml plastic bijou bottles were irradiated a t a dose-rate of 5.8 G y/m in. In the case of dose-response experim ents, cell sam ples were held on ice for 0.5 - 1 h before irradiation, irradiated a t 0 °C and returned to ice u n til th ey could be processed further. For th e DNA unw inding experim ents th e cells w ere ch ased in non-radioactive m edium prior to irra d iatio n , w hich involved centrifuging th e cells o u t of th e labelled m edium and replacing it w ith unlabelled m edium for 4-5 h. This reduced th e observed b ackground level of b reak s by m inim ising th e detection of unw inding from replication forks (Costa 1987).

For th e sh o rt-term (0-3 h) rep air experim ents th e cell sam ples (3 m l in bijou bottles) w ere h eld in a 37 oc w ater b a th for 0.5-1 h before irrad iatio n , irrad iated a t 37 °C an d retu rn ed to th e w ater b a th for the appropriate repair tim es. Cells were irradiated to 8 Gy for th e short-term DNA unw inding rep air experim ents and to 30 Gy for th e n eu tral elution re p a ir ex p erim en ts. T he irra d ia tio n s w ere stag g ered su c h th a t all sam p les w ere collected a t a com m on tim e point, a t w hich tim e the cells w ere poured in to ice-cold PBS (neutral elution) or ice-cold saline (DNA unw inding), and were k ep t on ice u n til further processing.

For the DNA unw inding long-term (2-8 h) rep air experim ents, the cells w ere irradiated to 50 Gy a t 37 oC and th en plated o u t a t 2.10® cells p er petri dish (non-tissue culture) in 5 ml of m edium . T he sam ples were irrad iated consecutively, p lated o u t an d placed in a hum idified, 5 % CO2

in cu b ato r a t 37 oC. After th e appropriate incubation tim e, sam ples were rem oved and once th e cells h a d been loosened by pipetting, each sam ple w as divided into 2 centrifuge tu b es containing 5 ml ice-cold saline and processed further.

The re p a ir in h ib ito rs, a ra A (Sigma) an d a ra C (Sigma) were dissolved in HBSS (Hanks balanced salts solution) a t a concentration of 10

m m ol/1. Ara C w as sufficiently soluble in HBSS a t th is concentration, b u t 0 .0 7 5 m ol/1 HCl w as needed to dissolve the ara A. These stock solutions w ere routinely stored a t 4 oC. The rep air inhibitors w ere added to the cell su sp en sio n s 1 h before irradiation to allow for phosphorylation. The in h ib ito r c o n c e n tra tio n w as k e p t th e sam e a t e ac h stag e of the experim ent Le. during the pre-irrad iatio n incubation, irradiation and the rep a ir tim e following irradiation.

4.2.2 DNA unw inding a ssa y and hydroxylapatite chromatography

Lysing solution th a t contained 0.03 mol/1 NaOH (pH 12) and either 0 .1 5 m ol/1 or 1 m ol/1 NaCl, w as freshly p rep ared ju s t prior to each experim ent an d k ep t on ice. The cell sam ples (at 1.10® cells/sam ple) in ice-cold saline solution w ere centrifuged to a pellet an d th e su p e rn ata n t asp irated . After vortexing, 0.5 ml of ice-cold lysing solution w as forcefully ad d ed to each sam ple u sin g a m icropipette. Sam ples w ere left on ice u n d istu rb e d an d u n d e r conditions of low light for 1 h. Subsequently, sam p les w ere n eu tralised by adding 1 . 1 ml of 0 . 0 2 m ol/ 1 Na2H P0 4 and im m ediately sonicated for 6-10 s. After addition of 0.2 ml 0.85 mol/1 SDS (25%), sam ples w ere vortexed and deep frozen (-20 °C) overnight.

After defrosting the sam ples w ere applied to sm all affinity colum ns com p risin g approxim ately 0.2 g of hydroxylapatite gel (Biorad, 50:50 m ix tu re of Bio-Gel HTP an d DNA grade HTP) held a t 60 ^c. Before and after sam ple application, th e hydroxylapatite w as w ashed w ith 2.5 m l of 0 .0 1 2 5 mol/1 SPB (sodium p h o sp h ate buffer) a t pH 6 .8. Single-stranded DNA (ss) w as th en eluted w ith 2x 2.5 m l of 0.125 mol/1 SPB and double­ stra n d e d DNA (ds) w ith 2x 2.5 m l 0.25 mol/1 SPB. 0.3 m l 5 m ol/1 HCl a n d 5 m l scintillation cocktail (O ptiphase MP, LKB) w ere added to each e lu te d sam ple an d after vortexing th e radioactivity p er sam ple w as determ ined by liquid scintillation counting.

The relative m a ss fractio n of DNA in d o u b le -stra n d e d form (m ds/îîids +mss) w as obtained from th e ratio Ads/(Ads +Ass). w here Ads is th e activity (dpm) m easu red in the double-stran d ed fraction and Ags the activity m easu red in th e sin g le-stran d ed fraction. T his relative m ass fraction is indicative of th e a m o u n t of undam aged or u n b ro k en DNA. D ose-response curves w ere obtained by plotting the relative m ass fraction ag ain st dose. In the rep air experim ents the relative m ass fraction values w ere converted to 'dose' values u sin g a dose-response curve. These dose values, expressed in Gy, reflect the am o u n t of the X-ray dam age rem aining after the various repair tim es.

4.2.3 Non-denaturing filter elution a ssa y (pH 9.6)

The n eu tral elution procedure is described in detail in C hapter 2 (section 2.2.2) an d w as perform ed a t pH 9.6 an d u n d e r conditions s tip u la te d in sectio n 2 .4 . The p ro ced u re follow ed in th e rep a ir experim ents is described in detail in C hapter 3 (section 3.2.2).

4.2.4 DNA synthesis a ssa y

U nlabelled EAT cells a t a concentration of 5-6.10®/m l were placed in glass bottles in a w ater b a th and allowed to equilibrate to 37 oc. A ra A or a ra C, in the form of a 10 mmol/1 stock solution in HBSS, w as added to various final concentrations. At chosen tim e intervals after the addition of inhibitor, a 1 ml aliquot of cells w as rem oved to w hich 18.5 kBq of ®H- TdR w as rapidly added. After an incubation tim e of exactly 5 m in a t 37 °C, 5 ml of cold saline w as forcefully added to the sam ple, w hich w as then p u t on ice. T his p ro cess w as rep eated a t 10, 20. 40 an d 60 m inute intervals following the addition of a ra A or ara C and control sam ples were ru n sim ultaneously. O nce all the sam ples had been accum ulated on ice, th ey were centrifuged an d the su p e rn a ta n t aspirated. After vortexing the

pellets, 1 ml of 0.03 mol/1 NaOH w as added, followed by 1.5 ml of 0.61 m ol/1 TCA (trichloracetic acid) 10 m in later. Sam ples w ere stored a t 4 overnight to allow full precipitation of DNA.

T he p recip itated DNA w as collected on to g lass-fib re filters (W hatm an), rinsed twice w ith ice-cold 0.31 mol/1 TCA an d finally w ith ice-cold absolute ethanol. 4 m l of scintillation cocktail (O ptiphase MP, LKB) w as added to the dried filters an d radioactivity p er filter determ ined b y liquid scintillation counting.

The m ean value of the ®H-activity per filter a t the various sam pling tim e s w as determ ined for th e co n tro l sam ples an d th is value w as norm alised to unity. The incorporation of ®H-TdR in th e presence of the in h ib ito r w as th en calculated relative to th is norm alised m ean control v alu e an d plotted a g ain st th e tim e elapsed betw een ad d itio n of the inhibitor and sampling.

4.3 Results

4.3.1 Inhibition o f DNA syn th esis

The inhibitory action of the nucleoside analogues a ra A and a ra C on DNA synthesis w as tested by m easu rin g the extent of ®H-TdR (tritiated thym idine) incorporation into th e DNA of EAT cells during a 5 m in pulse. T he am o u n t of ®H-activity in co rp o rated into the DNA of th e control sam p les of EAT cells during a 5 m in pulse w ith 18.5 kB q ®H-TdR and tak e n a t various sam pling tim es, is given in Table 4.1.

Incubation tim e (m in) 3h- controli dpm contrôla 1 0 14 882 14 250 2 0 14 168 16 617 4 0 16 739 16 295 6 0 14 582 15 817 (mean = (m ean = 15 097) 15 745)

Table 4.1 The amount of ®H-actlvlty incorporated into the DNA of EAT cells during a 5 min pulse with ®H-TdR in the absence of the DNA synthesis inhibitors.

The incorporation of ®H-activity in the p resen ce of th e DNA synthesis inhibitors w as calcu lated relative to the m ean values determ ined for the control sam ples a s show n in Table 4.1, w here th e m ean value of the controls w as norm alised to unity. The resu lts of the DNA synthesis assay in the presence of th e drugs are given in Fig. 4.1, w here the cells were incubated in m edium a t 37 °C in the presence an d absence of (a) ara A and (b) ara C.

o

g|

If

;

T — I— 1— I— •— I— I— I— ' — r

10 20 30 40 50 60 70

0.20 0.15 0.10 0.05- (b) ara C 1 0 f i m o l / 1

•s

f

Figure 4.1 DNA synthesis assay; relative incorporation of ^H-TdR into the DNA of EAT cells incubated at 37 °C in the presence and absence of (a) ara A and (b) ara C.

|xmol/l

In the absence of th e Inhibitors the incorporation of ^H-TdR into DNA w as found to be approxim ately co n stan t over th e 1 h in cu b atio n period (Table 4.1). A stro n g d ecrease in in co rp o ratio n w as observed, th a t in creased w ith in cu b atio n tim e, after the addition of 1 0 an d 1 0 0 pm ol/l a ra A (Fig. 4.1a) or a ra C (Fig. 4.1b). An ara A concentration of 100 pmol/1 w as sufficient to alm ost com pletely inhibit DNA synthesis, as m easured by th e inco rp o ratio n of ^H-TdR, after cells h a d b e en in c u b a te d in the p resen ce of a ra A for 1 h. A ra C w as found to be m ore effective in in h ib itin g DNA sy n th esis, an d 10 pmol/1 w as sufficient to com pletely in h ib it ^H -incorporation after a 2 0 m in incubation interval. Since these w ere exponentially grow ing cells th e above re s u lts im ply th a t sem i­ conservative DNA synthesis can be virtually com pletely inhibited by either a ra A (100 pmol/1) or a ra C (10 pmol/1).

4.3.2 DNA unw inding results

A dose-response curve for EAT cells following X -irradiation obtained w ith th e DNA u n w inding m ethod u sin g 0.15 m ol/1 NaCl In the lysis solution, Is show n In Fig. 4.2.

1 •8

S

Figure 4.2 Dose-response curve for induction of DNA strand breaks in X-irradiated EAT cells in the presence and absence of 400 pmol/1 ara A measured by the DNA unwinding method (pH 12). The frequency of strand breaks is inversely related to the logarithm of the function: (mds/mds +m sj' Vertical bars represent the standard error of mean values.

The relative m ass fraction (m ds/m ds+m ss). w hich reflects th e proportion of undam aged DNA (see section 4.2.2), is plotted ag ain st dose and can be seen to decrease In a linear m an n er w ith increasing dose. The response of cells w hich h ad been Incubated in the presence of 400 jimol/1 a ra A for 1 h o u r before irrad iatio n an d irrad iated in the p resence of a ra A (400 pmol/1) is show n by th e d ash ed line. Clearly the presence of 400 jimol/1 a ra A did not affect th e Induction of DNA breaks by X -lrradlation.

The DNA unw inding dose-response relationship of X -irradiated EAT cells, following lysis in high ionic stren g th (1 mol/1 NaCl) alkaline solution (0.03 mol/1 NaOH) is show n in Fig. 4.3.

1.0 f i 0.9-

S

Figure 4,3 DNA unwinding dose-response of X-irradiated EAT cells following lysis in high ionic strength (1 mol/1 NaCl) alkaline solution (pH 12). The data points are the mean of two experiments and the vertical bars the standard error of mean values.

S tra n d se p a ra tio n of th e DNA u n d e r alk alin e co n d itio n s (pH 12) is a c c e le ra te d b y h ig h NaCl c o n c e n tra tio n s (R ydberg 1975). T his sub stan tially increases the sensitivity of th e technique and th u s facilitates dose-response m easu rem en ts in th e lower dose range of 0-12 Gy. The relative m ass fractio n (m ds/^^ds +niss) show s a lin ear decrease w ith increasing dose an d it w as w ith the aid of th is dose-response curve (Fig. 4.3) th a t th e relative m ass fractio n v alu es o b tain ed in th e rep air experim ents w ere converted to rem aining dam age values, to be expressed in Gy.

U sing th e DNA unw inding m ethod, the kinetics of DNA rep air of EAT cells w ere followed u p to 1 h post-irradiation (8 Gy) in the presence and absence of a ra A and a ra C . These short-term repair results, assum ed to reflect rapid ssb repair, are show n in Fig. 4.4.

10 1600 Hmol/1 400 I |xmol/l 1 6 0 8 0 4 0 20 0

Tim e after irradiation (min)

"#— control ara A 10 100 jrmol/1 400 p.mol/1 1 6 0 4 0 80 20 0 control ara C

Time after irradiation (min)

Figure 4.4 Kinetics of disappearance of remaining damage, interpreted as DNA ssb repair, as a function of time after X-ray exposure (8 Gy) in the presence and absence of (a) ara A and (b) ara C. Vertical bars represent standard error of mean values.

The control sam ples (in b o th (a) an d (b)) show typical b ip h asic kinetics for ssb repair, a s h as been previously reported for EAT cells (Bryant et a l

1984). Lower concentrations of a ra C w ere used, since a ra C w as found to in h ib it DNA sy n th esis m ore effectively for the sam e co n cen tratio n th an a ra A (see Fig. 4.1). From panel (a) it can be seen th a t ssb rep air was inhibited in th e presence of a ra A an d th a t the extent of inhibition was greater a t 1600 pmol/1 th a n a t 400 pm ol/l. It is obvious th a t even a t the

exceedingly high concentration of 1600 pmol/1 a ra A, ssb repair w as not com pletely inhibited. In th e case of a ra C (panel (b)) co n cen tratio n s of 100 pm ol/1 and 400 pmol/1 resu lted in more or less th e sam e extent of inhibition of ssb repair, b u t once again full inhibition of ssb repair w as not achieved.

U sing th e DNA unw inding m ethod, th e rep air of EAT cells after exposure to 50 Gy of X -rays w as followed from 2 h to 8 h post-irradiation. T hese long-term repair experim ents, assum ed to reflect dsb repair, were perform ed in the presence an d absence of (a) 400 and 1600 jimol/1 ara A an d (b) 100 and 400 |im ol/l a ra C an d the results are presented in Fig.4.5.