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Factors influencing the recovery from potentially lethal damage (PLD) in mammalian cells in vitro and in vivo
Ca.cer TreatmentRe:ir:,s ( 19.qt); 7, ! - 13
Factors influencing the recovery from potentially lethal damage (PLD) in mammalian cells in v i t r o and in Vtvo' Marcelle Bertrand
Brain Tumor Research Center, Department oJ'aVeurologicalSurgery, School of 3Iedicine, The Lrniversity of California, .Van Francisco, Califortda 94143, U.S.A. and Department q]" A[edical Oneologv, Cit~, of IIope .\rational 3[edical Centre, Duarte, California 91010, U.S.A. a n d D e n n i s F. Deen=f
Brahz Tumor Research CenteG Department of .,Veurological Sur~,err, attd the Departmeizt of Radiation Oncolog~, School of .~Iedicine, The University of California, San Francisco, California, 94143, U.S.A.
Int r o d u c t i o n All too fi-cquently n e i t h e r d r u g n o r radiation t r c a t m e m is able to sterilizc m a l i g n a n t turnors completely. A l t h o u g h the reasons for this failure a r e multiple, a m a j o r fitctor is the ability of t u m o r ce!ls to r e c o v e r from the d a m a g e inflicted on t h e m . Potentially lethal d a m a g e is most easily discussed in terms o f a m o d e l proposed by Phillips et al. in 1966 (7,1.). (:ells exposed to a shtgle dose of X - r a y s i n c u r two types o f d a m a g e : lesions that irrcversibly inhibit the ability o ( cells to proliferate ( " l e t h a l d a m a g e " ) a n d lesions that are unstable, whose u l t i m a t e fate. ~s ,?ctcrrnined b y postt r e a t m e n t condition ( " p o t e n t i a l l y lethal d a m a g e " , P L D ) . F;~'.,..~zflc p o s t - t r e a t m e n t conditions lead to a:l enhancemer~t o f cell survival ("recove~3,"): while u n f a v o u r a b l c conditions result in a d e c r e a s e in cell survival ( " f i x a t i o n " of d a m a g e ) . R e c o v e r y fi'om P L D must be distinguished from r e c o v e r y li'om sublethal d a m a g e ( S L D ) , w h i c h is cat,setl by t r e a t m e n t widlfractionated doses o f r a d i a t i o n (11, 32, 41, 53, 57, 77, 84, 87, 95, 102, 103) or d r u g (3, 5, 6, 9). T h e relationship b e t w e e n P L D a n d S L D r e m a i n s controversial (I I, 16, 29, 32, 41, 95, 102, 103) a n d S L D will not be discussed at length in this review. As suggested b y H a h n (37-39), " r e c o v e r y " will be used in a purely 1"Address reprint requests to: Dennis F. Deen, Ph.D., Brain Tumour Re.search Ceture, 783 ]IS'W, Department of Neurological SurgeD', University of California, San Francisco, CA 94 t43, U.S.A. Tiffs work was supporte d in part by Grant CA 13525 from the National Institutes of Heahh (U.S.A.), and a grant from the Foundation-Rose and.Jean Hoguet (Belgium).
0305-7372/80/010001 + 15 $02.00/0
~, 1980 Academle Press Inc. (London) Ltd.
2
,Xl. BERTKAND AND D. F. DEEN
operational way while "repair" will be used to indicate known modification of specific maeromolec,~: s. In vitro reco,~,:et%,fi'om PLD has been observed after a wide variety of treatments, in dividing and win-dividing cells, and in oxic and hypoxic cells. T h e conflicting rcsuhs reported in th,.: J.iterature on the influence of various factors on the recovery process suggest that the ,~vaagnitude of the p h e n o m e n o n is as dependent on the cell line as on the treatment arid pTJst-treatment conditions. Furthermore, the exact target as well as the repair mechanism involved in the recove13" front PLD remains unknown, although m a n y studies have suggested a repair mechanism perhaps enzymatic, acting at the D N A level. Because recova 3, fi'om PLD also occurs in vivo and enhances the tumor rcsistance to treatment, the study of recovery processes and particularly the study of their inhibition are of considerable interest. We will review thctors influencing the recovery phenomenon in vitro and also t'i~.e in vlvo data reported. "vVe ,,rill emphasize recovery fi'om PLD Following exposure to ionizing radiation and antimitotle drug,s.
In vitro s t u d i e s Effects o f recoveO, medium~
Recovery from PLD under suboptimal growth conditions such as depicted m e d i u m (DM) (40, 41, 46, 47, 49, 57-59, 79), serum free m e d i u m (SFM) (40, 46, 78) or balanced salt solution (BSS) (11, 12, 33, 37.-40, 44, 59), and inhibition of recovery by fresh m e d i u m (FM) (40,44, 46~ 47, 49) were widely reported after X-ray treatment (Table 1). Recover 5, is not an "all or n o n e " p h e n o m e n o n because file magnitude of the process was dependent on the radiation dose (40, 59) and on the medium. Plateau phase and exponentially growing C h a n g liver cells (LICH) recovered preferentially in D M (59), which is perhaps related to a factor secreted by L I G H cells into the m e d i u m (58, 59), while plateau phase Chinese hamster ovarian cells (HA-l) showed a lligher recovery in BSS (40) dlan i n SFNI. No recovery was observed i n exPonentially growing HA-! cells held in BSS (40), a n d recovery in irradiated I~louse embryonic fibroblasts (3T3) was higher in D M than in BSS o r . S F M (46). Some recovery data reported for Xirradiated Chinese hamster lung fibroblasts (V79) conflicted, indicating that the results varied from laboratory to laboratory (Table I). Surprisingly, D u r a n d et al. Were unable to show a n y recovery in either d i v i d i n g or non,divlding V79 cells p0st-treated with v a r i o u s m e d i a (30), while Piro et al. observed an increase in survival in plateau phas e cultures held in either DM, BSS or FM, although recovery was delayed in cells held in F M (75). F u r t h e r m o r e , Mauro et al. observed recovery in V79 cells exposed t o BSS at 23°C before and after radiation; nO recovery was Found in these cells held in F M before radiafi0n (66). Recovery from ~-ray induced PLD, was similarly observed i n plateau phase C H O (34) and E M T 0 cells(36, 65) held in m e d i a that inhibit cellular proliferation. T h e data reported after high linear energy transfer ( L E T ) r a d i a t i o n (fast neutron and charged particle) conflict (Table 2). I n t h e s e studies, the a u t h o r s compared m o d e l systems in w h i c h recovery From X-ray or y-ray induced~PLD were observed. 1"(1) Depleted (p/~tteau)me(tlumor conditionedmedium is'mediumharvestedfromplateau phase culture. However, ~ome investigatox"Suse the term *'conditioned medium" for serum~-ee medium (40,78); (2) balanced salt solutions used are either Hanks', Eai'le's, Or Dulbecco's; (3)fresh, complete, or full medium is a freshl~]prepared-medhimcontaining fetal calf serum.
R E C O V E R Y FROI~I P L D IN .MAMMALIAN CELLS
3
T a b l e 1. R e c o v e r y f r o m X - r a y i n d u c e d P L D in ritro I/,ecovery~ Cell line
Dividing cells
Non-divlding cells
+ (BSS), -- (FM) + (SFM)
CHO
EMT, HA-1
-(Bss)
LICH
+ (DM) + (DM),
- -
(BSS, F M ) )
l~iorris rat hepatoma
+ (DM), -- (FM)
3T3
+ (DM > B S S > S F M ) --(EM)
V79
+ (ass)
+ (SFM)
Hall et aL (44) Raju et at. (78)
+ (DM)
Rasey et al. (79)
+ (BSS > S F M ) , -- (FM) + 03ss)
H a h n el al. (,10) Evans et aL (33), H a h n (37--39)
+(DM) + (DAI>BSS)~ -- (FAI)
H a h n eta/. (41), Little (57, 58) Little (59) Horowitz et al. (49)
+ (DM > BSS > SFM) - (v'M)
-(BSS, DA, I, F~%I)
- - ( a s s , DM, FM)
+ (DM), -- (Fh,l) + (DM), --(FM)
+ (DM), -- (~'M)
+ (~ss, 2soc)~ - ( F M , 23°C),
References
HetzeI el al; (46) Belli el al. (I1, 12) Durand et al. (30) Hetzel et al. (47) Horowhz el al. (49) Mauro et al. (66) Piro et al. (75)
+ (BSS, DM, FM)
"1"+ Recovery, -- no recovery or inhibition of recovery from PLD; 0 incubation medium after treatment: (BSS) balanced salt solution; (DM) depleted medium; (FM) fresh medium; (SFM) serum free medium. ~: See text.
T a b l e 2. R e c o v e r y ~'rom h i g h l e t r a d i a t i o n i n d u c e d P L D in vitro Recovery1" Treatment
Cell llne
a Particle
CHO
Helium ion
EMTn
Fast neutron
ENIT 6
Dividing cells -- (SFM)
Non-dividlng ceils
References
-- (SFM)
R ~ u a a/. (78)
+ (D.M)
Guichard et al. (36) A'lalalse et al. (65)
-- (DM)
Gragg et at. (34.) Hall et aL (44) Rainy et al. (79~
- (FM, J3SS)
+ (DM)
I" + Recovery, - - n o recovery from P L D ; 0 incubation medium after treatment: (BSS) balanced salt solution; (DM) depleted medium; (FM) fresh medium; ( S F M ) s e r u m free medium.
4
M. I|ER'I'I~AND ANI) D. 1". DI'EN
T a b l e 3 s t m m m r i z c s t h e in vitro r e p o n s e of m a m m a l i a n cells to P I , D i n d u c e d tW various d r u g s with reference to Llle m e d i a a n d cell line used. It shows t h a t recovery from P L D was very d e p e n d e n t o n the d r u g . N o recovery was observed after exposure to a d r i a n t v c i u (.-\D~I) (3, 313) or nitrosom'eas (37, 38), whicl, a c t u a l l y resulted in d e c r e a s e d cell survival (3. 9). Because i n h i b i t i o n of recovt:rg after D N A inhibitors has bccn obser.ved (see n e x t s e c t i o n ) , B a r r a n c o (3, 9) s u g g e s t e d t h a t the d e c r e a s e in survival f o l l o w i n g t r e a t m e n t with n i t r o s o u r e a s m i g h t b e r e l a t e d to i s o c y a u a t e , brcakdoxw~ p r o d t t c t s o£ these a n t i t m n o r a g c n t s . I s o c y a n a t c s i n h i b i t e d b o t h D N : \ synthesis (2) a n d ~he r e p a i r o f I ) N ; \ single s t r a n d b r e a k s (51). R.ccovcry f r o m B l c o m y c i n ( B L M ) - i n d u c c d I ' L D o c c t t r r c d c h a r a c t e r i s t i c a l l y for e x p o t m n t i a l l y g r o w i n g a n d p l a t e a u p h a s e cells in e i t h e r D:~I (9, 92), BSS (37-3.0, 81) or FM? (9, 37, 38, 92). R e c o v e r y in F~g mig]tt rcllect cell T a b l e ,~. R e c o v e r y
P L D in rltro
from drug-induced
Recovery'l" "l're.ttment Adriamycitl (ADM i
Cell line CliO
Dividing ccll~ -- (1)311
Non-divlding cc115 --(1)3I)
--(1:31, BSS)
ll.eference~
kt:trranco (3) Hahn (38)
Actinomycin I) (Art I))
CItO
--(F.\I), + (DM)
-- (D.M)
lktrranto (3) Barranco et al. (5"1
Bleomvcin (BLM)
Clio EMT~ HA-t
+ (F.M, D.M) + (F,M, DM)
-t- (DM) -4 (1:31, D*I) + (FM, BSS) 5- (BSS)
Barranco e t a l . (9) Twentyman et al. (92) Iiahn (37, 38) I-tahn (39), Ray tt al. (81)
CHO
(D.".I) -- (D,Xb
-t- (DM) -- (FM, D.M)
Fktrranco
5-Fh,orouracil (5-FU)
]tA- 1
-b (I;SS)
+ (BSS)
Ray
1Mechlorethamine (tiNt)
H'A- 1
+ (BSS)
4-(1:M, BSS) + (IISS)
Itahn (37, 38) Hahn (39), Ray el al. (81 )
--
Diauhydrogatacfitol (DAG) I.ow do~e
High dose
McCCNU-CCNU
ClIO
--
BCN U
I I:\- I 9L
--(FM, BSS)
Mcthyhnethane sulfonatc (MMS)
HA-I
Spirohydantoin mustard (SH.'Xl)
9I.
Sulfur mustard (SM)
V79
(D.'Xl)
+ ( I ) M , BSS) - - (F,M)
+ ( B s s (26oc)§
--~FM) (.6 c)§ •
rl
(D.M)
el at.
et al.
(6)
(81)
--(I:M, BSS)
llarranco (3) Barranco el al. (9) H:tlm (37, 38) K . T . Whccler~
q- (BSS)
ltahn (37-391
-I- ( D M , BSS) -- (1:.', l)
Bertraml et al. (13) Dcen rt al. (19) ~M;,uro ¢t
al.
(6t3)
O
"1" -i. Recovery, - - n n r e c o v e r y from PLD; 0 incubation medium after trcammnt: (BSS) balanced salt solution; (DM) depleted medium; (F.M) fresh medium. * Unpublished data. § See text'.
RECOVERY FROXI PLD IN .XlA.~IMALIAN {;F.LI.S progression d e l a y i n d u c e d by BLlk! (8) a n d inhibition o f m a c r o m o l e c u l a r synthe.~is (9); r e c o v e r y observed in D,Nf might result front a eonlblnation of the.-e eve,its a n d .subo p t i m a l g r o w d l conditions (9). T h e m a g n i t u d e of recovery from ~pirohytlantoin m u s t a r d (SFINI) i n d u c e d d a m a g e was strongly c o n c c m r a t i o n d e p e n d e n t (13, 19), as was die recovt:ry from d i a n h y d r o g a l a c t i t o t ( D A G ) that o c c u r r e d only at tow d r u g o ) n c e n t r a t i o n s in plateau phase Chinese h a m s t e r ovarian cells ( C H O ) h e l d ill l),Xl (6). Exposure to high conctmtrations of D A G decreased the survival of c i t e cells h e l d in either F/k{ or DN[ (6). l~.ecovery from Actinom)'cin J) (,\ct D) i n d u c e d PI.D was fimnd only ill e x p b n e n t i a l l y g r o w i n g C H O cells held in DR'I (3, 5). R e c o v e r y was observed in V79 cells exposed to BSS at 26°C before a n d after stllftlr nltlstard (S~I) ; Ilt~ recovel'y xv,"lS found in the cells p r e t r c a t e d with BSS at 26°C a n d ]told thereafter ill l:Xl ;I.t 26°(: (6~.:,). H e a t injured cells call recover fi'om PI,D. Palzcr et al. (72) sllowcd that e x p o n e n t i a l l y g r o w i n g l-l'ela cells recovered fi'om s u p r a n o r m a l t e m p e r a t u r e d a m a g e (-t20C:) if they w e r e i n c u b a t e d in mc(liurrt c o n t a i n i n g a n inhibitor of protein or 1-)NA symhesis (see l l ( : x l section) at nell-toxic co1~centratlons. T h e ma.t,mitudc of tile r e c o v e r y of dc,lsity inhibited HA-1 cells exposed to 43cC d e p e n d e d slg'nificantly txpon the nutrient conditions a n d pI I d u r i n g testing a n d was less atTccted t)y postheating'milieu (56). R e c o v e r y after u.v. ilTadiatlon of H A - I cell.; d e p e n d e d on nutritional ~letors both l)cfol'c and after t r e a t m e n t a n d o c c u r r e d equally in eitht.r BSS ~r IvM (37--39). Ill 1975, H a h n suggested that tile cell rccov{.gl'}" a~tCl" a variety of insults could he a n a l y z e d in terms o f X - r a y or u.v.-tikc d a , n a g c (37-39) or a u n i q u e system of recovery following h e a t shock (37, 38). I lahn':: system ,~vas hascd on the h a l f time (it) nf recovery, whick was ,-,2 h For X - r a y d a m a g e (37-39), ---13 h For u.v. d a m a g e (G. M. H a h n , personal c o m m u n i c a t i o n ) , a n d less thml 30 rain after exposure to sup,'anormal temperatures (37). Tile kinetics of recovery after X - r a y or u.v. trxposur¢ suggested a repair m e c h a n i s m acting at the D N A level, a l t h o u g h recovery kinetics a n d the kinctic.s of D N A repair after X - r a y a n d u.v. exposure (82) w e r e not identical (37-39). T h e d e m o n stration that cells deficient in D N A repair ability such as x e r o d c r m a l)igntcntosum skin fibroblasts exposed to u.v. a n d ataxia telangicctasia skin fibroblasts exposed to Xirradiation wert" also u n a b l e to recover efficiently fi'om P L D c o n f i r m e d this hypothesis (98). T h e Fast ,'ecovcry after h e a t shock suggested that proteins might be a m o r e likely target for the repair (100). R e c o v e r y after e.xposure to m e c h l o r e t h a m i n e (fiN.,) or BL,N'[ was similar to X - r a y recovery, while recovery after exposure to n t e t h y l m e t h a n e sulfonatc (I~IA,IS) or b r o m o d c o x y e y t i d i n c (l'~Cdlt.) a n d visible light had con'qmnents similar to both X - r a y a n d u.v. recovery (37-39). 9L cells treated with S H N I h a d a r e c o v e r y q. of ---.4 h, w h i c h was close to that of X-rays (13). H o w e v e r : usiIIg dif~:t'ent cell lines a n d , in some cases, ditTcrent r e c o v e r y conditions such as low t e m p e r a t u r e (102, 103), some investigators found that recovery after X - i r r a d i a t i o n occuneed m u c h m o r e rapidly, w i t h a t~ of--- 15 rain (1 I, 12, 102, I03), a n d p r e l i m i n a r y kitletic studies with a mouse fibroblast cell line (10 T~) exposed to u.v. radiation showed that the r e c o v e r y process took place largely within tlle first 6 h after exposure (17). An etdlancem e n t in tlae frequency of m a l i g n a n t transformatio,a paralleled tile e n h a n c e m e n t of survival in die 10 T t cell line after X - r a y (60, 89) a n d u.v. t r e a t m e n t (17, 61) d u r i n g tile early recovery period. H o w e v e r , transformation d e c l i n e d with longer recovery intervals.
Cell cycle phase dependence S o m e a u t h o r s }lave shown that r e c o v e r y is a cell c2/clc phase specific process. C o m p a r e d with e x p o n e n t i a l l y g r o w i n g cultures, the m a j o r i t y of cells in plateau phase cultures are
6
~I. IIERTRANI) A N D D. F. DEEN
in G 1 p h a s e o f t h e cell c y c l e (41). It h a s b e e n s u g g e s t e d t h a t this s y s t e m is a b e t t e r in vitro m o d e l for h u m a n t u m o r s t h a n e x p o n e n t i a l l y g r o w i n g c u l t u r e s (41). T h e fact t h a t t h e m a g n i t u d e o f r e c o v e r y w a s h i g h e r in p l a t e a u p h ~ e C H O (78) o r L I C H (59) cells aft,:r X - r a y t r e a t m e n t a n d t h a t n o r e c o v e r y w a s o b s e r v e d in X - i r r a d i a t e d e x p o n e t n i a l l y g r o w i n g H A - 1 culls (40) i n d i c a t e d t h a t cells in G j m i g h t b e p r i m a r i l y i n v o l v e d in t h e r e c o v e r y p r o c e s s (40, 59, 78). L i t t l e c o n f i r m e d this h y p o t h e s i s b y d e m o n s t r a t i n g Oaat r e c o v e r y o c c u r r e d in p l a t e a u p h a s e L I C ; H c u l t u r e s in w h i c h S cells w e r e s e l e c t i v e l y killed (62). T h i s f i n d i n g w a s f u r t h e r c o n f i r m e d b y e x p e r i m e n t s w i t h s y n chronized L I C I - I cells o b t a i n e d f r o m e x p o n e n t i a l l y g r o w i n g c u l t u r e s (62). H o w e w : r , in "v"-o / . , cells r e c o v e r y o c c u r r e d p r i m a r i l y in S p h a s e cells (12, 47), w h i l e r e c o v c t T o c c u r e d in C H O cells t r e a t e d w i d l e i t h e r B L M (4) o r X - r a y s ( I 0 3 ) in botJi G t a n d S p h a s e . F u r t h e r m o r e , Vehittnorc el al. o b s e r v e d r e c o v e r y fi'om P L D in all phases o f t h e c y c l e o f m o u s e L cells, bttt rt'cover 3, w a s m a x i m u m in cells i r r a d i a t e d in e a r l y S p h a s e (102). T a b l e 4. Effects o f m e t a b o l i c inhibitors on recovery" f r o m PLD ;a ~itta ~. ff~ ¢ •
Inhibltors DNA synthesis inhibitor
Treatment X-ray
Cell line
*:!
: e.:-, ,'er.v+.
References
l-[eLa
--
Bleomycin (BLM)
HeLa V79 /riA-I "F" CHO
-No effect No effect No efrcct
Cis-tHatinum complex
X-ray
V79
--
Doupte et al. (28)
P,NA synthesis inhibitor
X-ray
V79
--
HA-!
,N'o effect
Elkind el el. (32) Piro et al. (75) Evans tt el. (33)
),-ray
"F"
--
Arlctt
Bleomycin (BLM)
CHO
--
Barranco el al. (7)
X-ray
HcLa
)'-ray
*'F'"
,No effect
Arlett (1)
X-ray
HcLa 1~-1 "F"
4No effect No effect Slightly+
l'hillips et al. (74) Evans et aL (33) Arlett (!) Barranco et al. (7)
y-ray
(Actlnomycin D)
Ribosomal RNA synth~is inhibitor (Puromycin) Protein synthesis inhibitor (Cyclohexam ide)
y-ray Bleomycln (BLM)
CHO
r'hillips tt at. (74) Weiss el al. (99) Djordjevic tt al. (23) Sinclair (85) Evans el al. (33) .,Lrlett(1) Barranco el el. (7)
(I)
Djordjevic et el. (23, 24)
Phcncthyl alcoho|
X-ray
L
+
Whltmore et el. (]02)
Dinitrophenol
X-ray
L LlCHflqt
+
Dalr3"mpte et al. (18) Little eta/. ( 6 4 )
Caffeine
X-ray '),-ray
Various Various "F" L V791T-I
Enhancement of recovery; ~hddbltion of recovery from PLD.
--
No effect ~X'oeffect
Bussc a al. (15) ~raldrcn ¢t al, (96) Arlett ( 1) Rauth (80) Schroy et al. (83)
RECOVER~" I:R05[ PI.D IN ~IAMhlALIAN CELLS
7
O n the other h a n d , cell kinetic studies p e r f o r m e d d u r i n g the p o s t - t r e a t m e n t period after e i t h e r X - r a y (59) or S H M exposure (13) in a s y n c h r o n o u s cultures s h o w e d that t h e recow.'ry process o c c u r r e d p r i m a r i l y in cells held in GI phase of the cell cycle. T h e i n c u b a t i o n of p l a t e a u phase c u h u r e s or e x p o n e n t i a l l y g r o w i n g cultures in either D M or BSS i m p e d e d the progression o f cells from G l into S a n d increased the survival, whereas conditions such as FN[ allowed d m ceils to m o v e into S a n d decreased survival (13, 59). F u r t h e r m o r e , G u i c h a r d et aL f o u n d that ia EM'Fn cells allowed to recover iu their o w n m e d i u m after y-irradiation, the .'tmplittlde o f recovery was also a fitnctiotl o f ~:c11 t u r n o v e r rate a n d increased with c u l t u r e age, while the fi'action o f cells ia S phase b e c a m e smaller (35). "Yhey also s h o w e d that the delay tteccssary to obtain m a x i m u n l :~'ecove~T increased proga'essively with c u l t u r e age (35). ~ffects o f metabolic inhibitor drugs
Modificalio~ of survival in X - r a y o r B L ~ ! t r e a t e d m a m m a l i a n celts in c u l t u r e has l,ren achieved by p o s t - t r e a t m e n t exposure to several m e t a b o l i c inhibitors at non-toxic concentrations ( T a b l e 4). T h e d a t a r e p o r t e d in t h e literature are very conflicting a n d , therefore, no definitive conclusion could bc d r a w n from these studies. o n r e c o v e r y f r o m P L D in vitro
T a b l e 5. EiYect or" r e d u c e d t e m p e r a t u r e .
.
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.
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.
.
Cell line
~
.
.
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.
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.
. . . . . . . . . . . . . .
F HA-I
>'-ray X-ray X-ray X-ray X-ray Spirohydantoin mt~tard (SI-[M) X-ray X-ray
~__
~
:
. . . . . . . . . . . . . . .
~
IIIIIIIIII
.............
.
.
.
.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effect on recovery[
X-ray
I0 T.~ V79 ~_
.
CblO
L L5178"2" 9L
~
.
Treatment
HeLa
~
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.
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.
.
.
:~
Z
.....................
:
.
.
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.
.
,,
,
,,
.,,,,,,,
Rel'erenc~
+ (20~C~
.
,,,,,,,,,,
-- (4":C) No effect (25"C) --(4~'C)*,. -- (29"C) 4- (5:C) H-(34"C) -- ("--230C}.*
Dettor etal. (20) Dewey et at. (21 ) Winans etal. (103) ~,Vinans etal. (103) tM'lett (1) Evans et at. (33) Phillips et el. (74) ~Arhltmoreet el. (I02) Beer et al. (10) Bertrand et at. (13)
--(5~C) No effect (24"C)
Durand et el. (30)
.
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Little (60) .
.
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m
mm I
III
III
--inhibition of recovery; 0 recovery temperature. Recover3" decreased as the temperature was lowered and was inhibited at the temperature indicated ia 0. t q-Enhancement;
I n 1966, Phillips et aL r e p o r t e d t h a t exposure to ird,ibitors o f D N A synthesis (5fluorodeoxy'uridine a n d h y d r o x y u r e a ) resulted in a lower survival in X - i r r a d l a t e d cervical c a r c i n o m a cells ( H e L a ) , suggesting t h a t the r e p a i r process nfight involve Some type o f D N A synthesis (74). O t h e r e x p e r i m e n t s using the s a m e cell system with a variety o f D N A inhibitors s h o w e d that the m a g n i t u d e o f the effect was i n h i b i t o r specific a n d n o t related to the degree of i n h i b i t i o n of D N A synthesis (99). Tile i n t e r p r e t a t i o n of these d a t a is c o m p l i c a t e d b y the fact that using t h e s a m e cell line Djordjevic et el. o b t a i n e d conflirting results (23) a n d because n o effect o f D N A inhibitors w a s f o u n d by o t h e r investigators using v a r i o u s cell lines (1, 7, 33). P r e l i m i n a r y d a t a suggest t h a t a cisp l a t i n u m complex, w h i c h is also a n inhibitor o f D N A synthesis (50), i n h i b i t e d recovery fi'om X - r a y i n d u c e d P L D in V 7 9 cells (28).
8
.~I. B E R T R A N D
A N D D. F. D E E N
Exposure of cells to Act D (1, 7, 32. 75), ed3idium b r o m i d e (1), or acriflavine ( ] ) ; drugs that b i n d to D N A a n d inhibit D N A - d i r e c t e d R N A synthesis, resulted in decreased cell ~urvival, a l t h o u g h this observation was not universal (33). Because the i m e r a e t i o n of ;'~ct D widl c h r o m a t i n inhibited the repair of X - r a y d a n m g e to a DNA comples (31), the b i n d i n g of Act D to D N A a n d not specific interference with R N A synthesis might be p r e s u m e d to cause the inhibition of recovery from P L D (7), but the r e q u i r e m e n t of R N A synthesis tbr such rccovel T cannot be ruled out (7). l : u r t h e r n m r e , the activity of a n y e n z y m e that repairs r a d i a t i o n lc~ions in nucleic acid m a y be i m p e d e d in a steric r a m m e r by the d r u g - D N A c o m p l e x (!). In contrast, exposure of cells to inhibitors of protein wnthesis r e s u h e d in an e n h a n c e m e n t of survival after X - i r r a d i a t i o n (74) a n d p r o d u c e d m i n o r e n h a n c e m e n t of recovery from P L D after B L M t r c a m w n t (7). Phillips el al. suggested that by inhibiting protein syn,hesis, cyclohcximide mighl delay the expression of d a m a g e w i d m u t sigMficantly a h e r i n g the repair process, w h i c h allowed m o r e d a m a g e to be r e p a i r e d (74). These d a t a suggest that protein synthesis might not be rcqttired for r e c o v e r ' / f r o m P L D (7). H o w e v e r , Djordjcvic tt al. (23, 24) rcportt'd a decrease in survival of X - i r r a d i a t e d H e L a cells e.xposed to p u r o m y c i n , an i n h i b i t o r ,)f ribosontal R N A syaathesis. T a b l e 6. Efl'ect o f h y o e r t h e r m i a
o n r e c o v e r y f r o m P L D ~ in rltro
Gell line
Treatment
" P r e h e a t i n g " ,+
HA-I
Bleomycin /BL.M)
-~- (43°(:)
"Postheatlng"~
-t (41°C)
R c ference~,
Braun et M. (141
-- (43oc)
-- (43°C)
V79
X-ray
-'- (43¢C)
X-ray X-ray X-ray
-v. ( 4 l c C )
- (43:C) -- (41 cC) -- (42/43°C)
J~Iahn et al. (39) H a h n el td. (39) l.i et at. (55) .~Iurthy el at. (71) l Iarris el al. (45) D o u p l e (27)
~" + R e c o v e r y ; - - i n h i b i t i o n o f recovery. Exposure to h e a t before ( " p r e h e a t i n g " ) or a f t e r l r e m m e n t ( " p o s t h e a t i n g " ) .
Increase in survival o f . K - i r r a d i a t e d cells wa:~ also r e p o r t e d after exposure to p h c n e t h y l alcohol, an aspecific m e t a b o l i c inhibitor (102), a n d to d i n i t r o p h e n o l , w h i c h inhibits cellular A T P by u n c o u p l i n g m i t o c h o n d r i a l oxidative p h o s p h o r y l a t i o n (18, 64-). T h e s a m e effect was observed w h e n a subcellular fraction, or D N A , or its precursors were a d d e d to file m e d i u m of mouse L cells after X- (48, 68, 69, 73) or u.v. (25) irradiation. Caffeine was r e p o r t e d to h a v e either no effect (1, 80) or to inhibit recovery (15, 83, 96) from r a d i a t i o n i n d u c e d P L D in a variety of r o d e n t a n d h u m a n cells.
Effects of recovery temperature Conflicting d a t a h a v e been r e p o r t e d on tile influence of r e d u c e d p o s t - t r e a t m e n t t e m p e r a t u r e on file recovery from P L D ( T a b l e 5). I n c u b a t i o n at low t e m p e r a t u r e e i t h e r £ w o r e d recovery (10, 20, 21, 102, 103), inhibited r e c o v e r y (13, 33, 60, 74), or h a d no effect on the process (1, 30). R e c o v e r y from X - r a y d a m a g e o c c u r r e d at 5°C in m o u s e L cells (102) a n d at 34eC in l e u k e m i a cells (10); in C H O cells recovery o c c u r r e d at 20°C, but was inhibited at 4°C (I03). I n contrast, r e c o v e r y was i n h i b i t e d in H e L a
RECOVERY FRO*I I'LD IN MAMMAI.IAN CELLS
9
cells h e l d at 2 9 ° C (74) while , h e i n c u b a t i o n o f i r r a d i a t e d H A - I cells at either 25°C or 4¢C r c d u c e d or i n h i b i t e d t h e recovel T (33); t h e l a t t e r i n h i b i t i o n was reversilfle u p o n r e s t o r i n g the cells to 3 7 ° C (33). Vee f o u n d t h a t the m a g a f i t u d e of r e c o v e r y of 9 L cells after SH.'k~ t r e a t m e n t d e c r e a s e d as the t e m p r a t u r e was l o w e r e d a n d was i n h i b i t e d at 2 3 ° C (13). T h i s suggested t h a t a n e n z y m a t i c process was re.,pon.qblc for the r e c o v e r y process (1 3, 33). R e c e n t l y , h y p c r t h e r m i a has received c o n s i d e r a b l e a t t e n t i o n as an a d j u v a n t m o d a l ity to c a n c e r t h e r a p y . Its ability to interfere w i t h t h e r e c o v e r y process ( T a b l e 6) is therefore o f s o m e interest. S h o r t e x p o s u r e to t e m p e r a t u r e ~ 41 °G after t r e a t m e n t ( " p o s t h e a t i n g " ) usually i n h i b i t e d t h e recoverg"fi'om X - r a y (27, 39, 45, 55) a n d BL.XI (14, 39) i n d u c e d P L D . T h e rcversc s e q u e n c e ( " p r e h e a t i n g " ) p o t e n t i a t e d t h e effect o f t r e a t m e n t , b u t d i d n o t inhibit t h e recover T f r o m P L D (14, 39, 55, 71), a h h o u g h t h e kinetics o f recovery were slower (39, 55). T h e s e d a t a suggest that i n h i b i t i o n o f an e n z y m a t i c r e p a i r process was unlikely, a n d t h a t p o s t h c a t i n g c o n v e r t e d p o t e n t i a l l y lethal lesions into lethal ones (14, 55). O n the o t h c r h a n d , p o s t l l e a t i n g progn'esslvely r e d u c c d t h e m a g n i t u d e a~tl t h e rate o f r e c o v e r y (14, 55) ; r e c o v e r y f r o m X - r a y a n d IIL~.I i n d u c e d d a m a g e were aboli.~hcd after 60 m l n ( 5 5 ) a n d 1 0 0 r a i n (14), respectively, of h e a t i n g . Effect o f various other f a c t o r s
Several o t h e r factors w e r e r c p o r t c d to influence t h e recovery proccss after i r r a d i a t i o n ( T a b l e 7), b u t h a v e n o t yet been extensively s t u d i e d . I n h i b i t i o n o f recover 5, f r o m r a d i a t i o n d a m a g e o c c u r r e d in cells m a i n t a i n e d in single cell suspension after t r e a t m e n t , suggesting t h a t r e c o v e r y was d e p e n d e n t u p o n cell to cell c o n t a c t (33, 4-7, 59). T h e s e d a t a are, h o w e v e r , difficuh to i n t e r p r e t b e c a u s e t~-psinization a n d r e s u s p e n s i o n in D M p r o b a b l y a l t e r e d t h e cell m e m b r a n e p e r m e a b i l i t y a n d g r e a t l y e n h a n c e d t h e surlhce available to n u t r i e n t s (59). E v a n s et al. s h o w e d t h a t the rate o f D N A synthesis in the dispersed p o p u l a t i o n o f cells was m o r e t h a n twice Table 7. Effect of various conditions on recovery f r o m PLD fn ritro Conditions
Cell.line
Treatment
HA-1 LICH V79
X-ray X-ray X-ray
Mild h~,T,oxia
HA-I V79
X-ray X-ray
Extreme hypoxia
HA- i
X-ray
Koch tt al. (53)
Extremely hypertonlc solutioa
CHO
X-ray
Dettor tt al. (20) Raaphorst et at. (77) V¢inans ¢t at. (103)
Extremely hypotonic solution
V79 GHO V 79
X-ray X-ray X-ray
Utsumi et o2. (95) Raaphorst tt at. (711) Ufsumi et al. (95)
Single cell suspension
J" -l- Recovery;
-
-
irdaibhion of recovery.
Eft•ct on recovery~f
R efcren ce--.
F.va3~s et at. (33) Little (59)
Hetze! et at. (47) + +
Hahn tt al. (40) Douple (27)
I0
NI. BERTI~kND AND D. F. DEEN
t h a t in cells left in m o n o l a y e r culture (33). F u r t h e r m o r e , recovery occurrcd as well in low density m o n o l a y e r cultures (59, 78, 92). Also, unlike Hetzel et al. (47), S u t h c r l a n d et al. (87) a n d D o u p l e (26) did not observe recovery fi'onl X - r a y i n d u c e d d a m a g e in V79 cells g r o w n in s p h e r o i d cultm'e, a system in w h i c h cell to cell contact plays an i m p o r t a n t role, A l t h o u g h i r r a d i a t e d H A - I (40) and V79 (27) cells r e c o v e r e d u n d e r m i l d h y p o x i c a n d aerobic conditions, e x t r e m e l y hypoxic conditions inhibited recovery fi-om both P L D a n d S L D in HA-1 ceils (52, 53). However, a d d i t i o n o f e x t r a glucose to the m e d i m n allowed some r e c o v e r y fi'om P L D , indicating that at least p a r t o f the recovery process r e q u i r e d e n e r g y (53). I n h i b i t i o n of recovery after radiation t r e a t m e n t was also r e p o r t e d in cells exposed to extremely anisotonic solutions (Table 7), the efl~:cts of ~daich seemed to d e p e n d o n the sequence of exposure. R a a p h o r s t et al. reported that salt t r e a t m e n t of C H O cells before irradiation did not inhibit r e c o v e r y fi'om either P L D or S L D , whereas salt t r e a t m e n t d u r i n g or after i r r a d i a t i o n did inhibit both types of d a m a g e (77). H o w e v e r , D e t t o r et al. r e p o r t e d an i n h i b i t i o n of recovery fi'om P L D in C H O ceils held in h y p e r t o n i e sotution before, during, a n d after X-irradiation (20). U t s u m i et al. fi:mnd a n i~zibitiort of P I , D in V79 ceils exposed to anisotonic solutions after X - r a y t r e a t m e n t (95). Because of osmotic changes p r o d u c e d by anisotonic t r e a t m e n t , fixation of P L D m i g h t be the result o f a destabitization o f the structural relationship b e t w e e n D N A a n d the n u c l e a r envelop a n d / o r D N A a n d the n u c l e a r protein m a t r i x (95). Osmotic changes m i g h t also prevent a r e p a i r e n z y m e from acting at the D N A level (77). T h e trypsinization p r o c e d u r e used after t r e a t m e n t for both in vitro a n d in vivo systems prior to assay for sm'vival by colony formation as described by P u c k et aL (76), suggests tliat the recovery p h e n o m e n o n m i g h t be an artifact of this procedure. H o w e v e r , studies in w h i c h trypsinization was not necessary i n d i c a t e d trypsinization not to be a n artifact (11, 33, 38, 44, 67, 78, 88, 92). In ff)'O s t u d i e s
Conditions t h a t allow recovery from P L D in vitro are fairly drastic a n d are unlikely to be ever duplicated in vivo. H o w e v e r , several studies h a v e d e m o n s t r a t e d t h a t both ascites a n d solid r o d e n t tumors t r e a t e d in vivo a n d allowed to r e m a i n in situ for several hours after t r e a t m e n t r e c o v e r e d f r o m P L D (see T a b l e 8) a n d S L D (I 1, 22, 63, 84, 86) after a variety of treatments. R e c o v e r y from P L D a f t e r X-irradiation o c c u r r e d in solid t u m o r s a n d in old slow growing ascites tumors (I I, 43, 63) and was f o u n d to be dose d e p e n d e n t (42). T h e s e findings, with the observation t h a t the m a g n i t u d e a n d kinetics o f recovery w e r e similar to those observed in X - i r r a d i a t e d plateau phase ceils in culture, suggest that recove R • o c c u r s p r i m a r i l y in non-proliferating cells a n d p a r t i c u l a r l y in highly radioresistant hypoxic cells (11, 43, 63). T h e recovery process w o u l d a c c e n t u a t e the radioresistance of the tumor. I n a clinical setting, the a m o u n t of r e c o v e r y p r o b a b l y would c h a n g e d u r i n g a course o f p r o t r a c t e d r a d i a t i o n therapy as o x y g e n a t e d a n d cycling cells are killed a n d resting cells are r e c r u i t e d into the proliferative pool. I f one assumes that recovery f r o m P L D is different, b u t has a d d i t i v e effects (4I, 63) to recovery from SLD, fractionated doses m i g h t u l t i m a t e l y be m o r e efficacious. T h e s e observations were later c o n f i r m e d by t h e recovery f r o m P L D f o u n d in acute a n d chronically hypoxic solid rat s q u a m o u s c a r c i n o m a cells (NP,--S I) after X-irradiation (94) a n d m " large, ¢ " Lewis " lung necrotm
RECOVERY FROXI PI,D IN NIANINIALIAN CELLS
II
c a r c i n o m a ceils a f t e r y - i r r a d i a t i o n (84). F u r d a e r m o r e , t h e l a t t e r t u m o r r e c o v e r e d fi'om p o t e n t i a l l y l e t h a l lesions as ,,veil a s f r o m s u b l e t h a l lesions a f t e r ) , - i r r a d i a t i o n , b u t n o t a f t e r n e , t r o n i r r a d i a t i o n (84). T h i s suggests t h a t a d e c r c a s c d c a p a c i t y for r e c o v e r y m i g h t b e a n i m p o r t a n t r a d i o b i o l o g i c a l d i f f e r e n c e b e t w e e n t h e eWect o f l o w L E T ( X - r a y , y - r a y ) a n d h i g h L E T (84) r a d i a t i o n . H o w e v e r , r e c o v e r y fi'ont P L D w a s also r e p o r t e d in vivo a f t e r h i g h L E T r a d i a t i o n (36, 101) ( ' F a b l e 8).
T a b l e 8. R e c o v e r y
f r o m P L D in rlro
Treatment
(_:ell liner
Recovery$
BCNU
9L ElklT6
-+
Bleomycin (BL~I)
E1MT6
+
Ehrlicil ascit~ tumor I~tk/'F6 LNI C t EI~,IT,
+ -b +
C y c | o p h o s p h a m i d e (CP~Xl)
5-Fluorouracil (fi-FU) Carbon ion 7-ray Helium ion Neutron X-ray
BCN U + heat Neomycin+ heat X-ray+heat
+
9L
+
ENITo
+ + -b
Lewis lung carcinoma EMT n Lewis hmg carcinoma ENFF~ 9L NITG-B N C T C (Ascites/solid)
+ -% -b +
NR-S 1 P-388 (Ascitcs)
+ +
EMT 6 EMT 6 ENIT0
-b Inhibition Inhibition
l~,efert:nces lkl. L. l~,osenblum§ "Fwentyman et al. (93) H a h n et al. (42), l-ta|m (39) Twentyman et al. (9 I, 92) Takadc et al. (88) /-/,aim et al. (42) Moore et al. (70) Hahtt et al. (~t2) Wheeler et al. (I01) Guiehard el al, (36) Shipley et al. (84) Guiehard et aL (36) Shipley et at. (8,1,) Hahn (39), Hahrt el at. (43) "Wheeler et aL (101) Douple (26) Hahn et al. (43) Little et al. (63) Urano et al. (94) Belli et al. (I l) Twentyman et al. (93) Braun et at. (t4) Leith et at. (54)
1" Solid tumor except when mentioned, .++ Recovery; -- no recovery from PLD. § Unpublished data. T h e l i t e r a t u r e c o n t a i n s few d a t a o n t h e in viva r e s p o n s e to d r u g i n d u c e d P L D . T o d a t e , o n l y f o u r d r u g s h a v e b e e n studied.: B L M , c y c l o p h o s p h a m i d e ( C P M ) , 5 - f i u o r o u r a c i l ( 5 - F U ) , a n d B C N U ( T a b l e 8). A d e c r e a s e in s u r v i v a l s i m i l a r to t h a t o b s e r v e d in Vitro a f t c r e x p o s u r e to n i t r o s o u r e a s ( 3 , 9, K . T . W h e e l e r , u n p u b l i s h e d d a t a ) w a s ob~;erved ill 9 L r a t b r a i n t u m o r cells a f t e r t r e a t m e n t w i t h B C N U ( M . L. R o s e n b l u m , u n p u b t i s l l e d d a t a ) . Surprisingly~ T w e n t y m a n et al. f o u n d a n e n h a n c e d s u r v i v a l in E h ' I T B t u m o r s t r e a t e d w i t h tile s a m e d r u g , s u g g e s t i n g t h a t " e i t h e r t h e s u r v i v i n g f r a c t i o n w a s a r t e f a c t u a l l y l o w 2 h a f t e r B C N U t r e a t m e n t o r t h a t r e c o v e l T f r o m P L D w a s o p e r a t i n g to a I a r g e e x t e n t b e t w e e n 2 a n d 24 h ' " (93). R e c o v e r ) , f o l l o w i n g t r e a t m e n t w i t h e i i h e r B L M , C P M , o r 5 - F U s u g g e s t e d t h a t in a c l i n i c a l setting, f r a c t i o n a t e d doses m i g h t be m o r e e f f i c a c i o u s i n vivo t h a n a single l a r g e d o s e ( 4 2 ) . H o w e v e r , f r a c t i 0 n a t e d : d o s e s d i d n o t p r e v e n t in vivo r e c o v e r y f r o m PLY) i n d u c e d b y B L M (90). r w e n t y m a n el al. S h o w e d
12
,Xl. BF,RTILAND AND D. F. DEEN
that different sized E M T 6 tumors recovered from B L M d a m a g e (91); furthermore, they found a much. higher increase in survival (92) t h a n previously described (42). T a k a d e et al. also observed recovery from P L D in Ehrlieh ascites tumor, a system that did not r e q u i r e trypsinizalion before the survival assay, ,,daich ruled out trypsin as an artifact (88). Recently lk,Ioore et aL reported recovery from PL D in a solid rat mammal',/ carcinoma (LMC1) after treatment with C P M (70). Cellular repopulation from the resistant component that recovered fi'om P L D after a dose of 150 mg/kg was delayed lbr 6 or more days; restoration to near pretreatment status was achieved by 10 days, suggesting that a further dose of CPA'I would be more efficacious a t longer than at shorter intelwals (70). Preliminary data has suggested that recovery fi'om BLh,~ (14) an d X-ray (54) induced damage in E M T 8 solid tumors might be inhiMted by adjuvant heat treatment, duplicating in vivo the observations m a d e in vitro (Table 6). T w e n t y m a n et al., however, found that adjuvant heat did not inhibit recovery from B C N U induced PLD i n the same tumor system, although the combined modality was more efficacious than BCNU alone (93).
Conclusion
In summary, recovery from P L D is a n important p h e n o m e n o n that has occurred in m a n y experimental systems, and probably enhances the resistance of m a n y malignant tumor cells to treatment. For e x a m p l e , the radioresistance of h u m a n osteosarcoma might be c a u s e d not by an intrinsic resistance of the cells to X-ray ti'eatment, b u t rather by an efficient recovery from P L D (97). Although a substantial literature on P L D has evolved during the past decade, it is not u n c o m m o n for different authors to report conflicting results for similar experimental conditions in the same system. Because the PL D p h e n o m e n o n m a y be strongly dependent on r a t h e r subtle differences in cell tine and/or culture materials and meflaods, t h e relevance of any particular P L D phenomenon to a tumor's recovery capability in situ becomes suspect. Studies should b e designed not o n l y to observe a P L D effect, but to analyze tlae,causallty of the observation: Appropriate models to study chnlcally relevant PLD should allow differentiation of possible artifacts from true biologica 1 responses. For example, if cell lines a r e used that gn-0w both in vitroand in vivo, then various in vitro, in vivo-in Vitro, a n d hz vivoassays Can be employed] I f recovei~, (o1" the lack of recovery) from P/SD is not indicated by a l l the assays, an d logical explanations are not available, then artifacts seem likely. F r o m the experinaental data reported here; w e can conclude: (1) T h e relevance of an in vitro model to radiation t h e r a p y in vivo becomes obvious when it was showh that recovery comparable in m a g n i t u d e and[kinetics to that found in vitro occurred a!so in vivo for experimental tumors grown in both ascitiC and solid forms. Therefore, recovery from radiation-induced PLD is certainly a factor to b e considered in treating h u m a n tumors; (2) The relevance of in Vitro models to in vivochemotherapy is less Obvious because circulating d r u g metabolites m a y interfere~ with the recovery process in vivo. Furthermore, tlae drug levels used i n experimental models in vivo and in bitro are generally well a bove those likely used clinically a n d : m a y influence the recovery process.
R E C O V E R Y FRO.'~I PLD I N M A M M A L I A N CEI.I.S
13
Acknowledgments W e w i s h to t h a n k K . T . '~Vheeler a n d lkIi L . R o s e n b l u m f o r a l l o w i n g us to u s e t h e i r unpublished data, J. L. Wolf for assistance with the bibliography and the tables, Neil B u c k l e y f o r e d i t o r i a l a s s i s t a n c e , a ~ d ,~Iarilyt~ M ' i n n a a r f o r t y p i n g t h e m a t ~ t t s c r i l ) t .
References i. Arlett, C. F. (1970) bzt..7. Radiat. Biol. 17: 515. 2. Baril, B. B., Baril. E. F., Laszlo, J. & ~,Vh~.~ler, G. P. (1975) Cancel lies. :15: I. 3. llarranco, S. C:. (I976) Cancer "I'~eat. Rep. 60: 1799. 4. Barranco, S. C. & Bohon, ~V. E. (1977) Cancer Res. 37: 2589. 5. Barranco, S. C. & Flournoy, D. R. (1976) Cancce ICes. 36: 163-1-. 6. Barranco, S. C. & Flournoy, D. R. (1977) 07- .,VatI. Cancer h!st. 58: 657. 7. Barranco, S. C., Haenelt, B. R. & Bolton, ~,V. E. (1977) 07...Vatl. C'anrrr hist. 59: 1685. IL Barranco, S. C. & H u m p h r e y , R. M. (1971) Cancer Res. 31: 1218. 9. Barranco, S. C., Novak, J. K. & HumI)hrey, R. M. (1975) Canter Res. 35:119-t. 10. Beer, J. Z ' , Leer, J. T. & Alexander, P. (1963) a\'ature (London) 199: 193. 11. Belli,J. A.. l)icus, G . J . & Nagle, ~V. (1970) Front. Radiat. Ther. One: 51~10. t2. Belli, J . A. & Shelton, M. (1969) Science 16,5: 490. 13. Ilerlrand, M., Deen, D., Hoshlno, T. & l¢.1~ebei. K. (1980) Cancer Treat. Re/,. I,i I)r~:s~. 14. B r a u n , J . & l'Ialm, G./~[. (1975) Cancer Res. 35: 2921. 15. Busse, P. M.. Bose, S. K., Jones, R. ~,V. & Tohnach, L..]. (1977) Radiat. Res. 7 l : 66(i. 16. Chadwick. K. If. & Leenhouts, H. P. (1975) Radiat. and Eat'bantu. Bioplo.s. 11: 319. 17. Chan, G. L. & l.itll%J. B. (1979) Abstract o f the 6th Intern. Congre~ of Radiation Research: 179. 18. Dalrymple, G. \;., Sanders, J. L. & Baker, M. L. (1967) Nature (London) 216: 708. 19. Deen, D. F., tfoshir,o, T., V¢illiams, NI. E.. Nomura, K. & Battle, P. Nt. (1979) Cancer i?e$. "19 : 4336. 20. Dettor, C. NI.. Dewey, W. C., ~,Vinas, L. F. & N o e l , J. S. (1972) Radiat. Res. 52: 352. 21. Dewey, V¢. C., NIiller, H. H . & Leeper, D, B. (1971) Proceed. a\'atl. Acad. Scle,ces 68: 667. 22. De~,Vys, "0,'. D. & Kight, N. (1969)07..,Vatl. Cancer b~st. 4°-: 155. 23. Djordjevie, B. & Kim, J . H. (I969) RadiatRes. 37: .t35. 24. Djordjevie, IL, Kim, J . H. & Kim, S. H. (19t38)-h~]J. Radiat. Biol. 14: 1. 25. Djordjevic, O., Kostie, L. & Kanazir, D. (1962) Nature (Londo,0 193: 614. 26. l)ouple, E. B. (1975) Radiat. Res. (Abstract) 62: 552. 27, Douple, E. B. (1976)Radlat. Res. (Abstract) 67: 576. 28. D o , de, E. B. & Linquist, K. (1979) Abstract o!'6111 h ltern. Congr~.-ss of l~.:ldiation l~est'arch: 220. 29. Dritsclaiio, A., Pica, A . J . & Belli,J. A. (1976) lht. 07. Radiat. Bio/. 30: 565. 30. l)urand, R. E. & Sutherland, R. ~I. (1973) Radiat?~es. 56: 513. 31. Elki~ad, Xl. ,'~I. & Chang-Liu, G. (1972) Int. 07. Radia{.Biol. 2~: 75. 32. Etkind, ..X.l. ~I.. Sutton-Gilbert, H., Moses, \V. B. & Kamper, C. (1967) ~'ature (Ler~don) 214: 1088. 33. l'~vans~ I~.. G., Bagshaw, .Xl. A., Gordon, L. F., Kurkjian, S. D. & H a h n , G. M. (1974) RadCat. Res. ,59: 597. 34. Gragg, R. I.., Humphrey, R. ~[. & Xfeyn. R. 1"2. (1977) Radiat. Res. 71: 461. 35. Gulch~rd, M., Tublana, ~I. & NIalaise, E. P. (1979) Int. 07. Radial. Biol. 35: i 11. 36. Guichard, XI., Lather, B. & Malaise, E. P. (1977) Radiat. Res. 71: 413. 37. Hahn, G. M. (1975) in ,~folecular~Wechanismfor RepairofD..~:l. (Eels. P. C/. l.I.anawalt & R. B. Setlow). Plenum Press, New York and London: 601. 38. ]-lahn, G. iXI..(1975) Radiat. Res. 64: .533. 39. Hahn, G. M. (1976) Cancer Treat. Rep. G0: 1791. 40. H a h n , G. ~I., Bagshaw, M. A., Evarts, 1~.] G. & Gordon, L. F. (1973) Radiat. Res. "55: 280. 41. Hahn~ G. ~[. & Little, J . B. (1972) Curt. Top. Radiat. Res. 8: 39. 42. Hahn, G. NI., Ray, G. R., Gordon, L. F. & Kallman, R. F. (1973)07. Natl. Cancer b~st. 50: 529. 43. Hahn, G. *I., Rockwell, S., Kallman, R. F., Gordon, L. F. & Fri~adel, E. (1974)CancerRes. 34: 351. 4-4. Hall, E . J . & Kralievle, U. (1976) Radiology 1"-'1: 731.
14 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97.
1M. BERTRAND AND D. F. DEEN l-Iarris, J. R., Murdiy, A. ]q. & Belli, J. A. (1976) Radiology 119" 227. Hetzel, F. ~V. & Kolodny, G. M. (1976) Radiat. Res. 68: 490. I Ietzel, F. x,V., Kruuv, J. & Frcy, H. E. (I976) Radiat. Res. 68: 308. Horikawa, RI., Sugahara, T. & Doida, Y. (1964) F.xpt. Cell Res. 34- 198. Horowitz, I. A., NorwL,al, H. & Hall, E . J . (1975) Radiology 114: 723. Kowle, J. A. & Gale, G. R. (1970) Biochem. Pharmac. 19: 27.57. Kann Jr., H. E., Kohn, K. V¢. & L y l ~ , J . M. (1974) Cancer Res. 84: 398. Koch, C . J . 8,: Kruuv, J. (1971) Radiat. Res. 48: 74. Koch, C . J . , , M e n ~ , J . J . & Harris, J. %V. (1977) Radiat. Re~. 70: 542. Lelth, J. T., Miller, R. C.: Gerner, E. W. & Boone, M. L. l~I. (1977) Cancer 39- 766. Li, G. C., Evans, R. G. & Hahn, G..'kl. (1976) Radiat. Res. 67: 491. Li, G. C., Sapareto, S. A., Shiu, E. G. & Hahn, G. M. (1979) Abstract o f t h e 6th Intern. Congress of Radiation Research : 125. Little, J. B. (1969) feature (London) 224: 804. Little, J. B. (1971) hd. ff. Radiat. Biol. 20: 87. Little, J. B. (1973) Radlat. Res. 56: 320. IAttle, J. B. (I977) Radiat. Res. (Abstratt) 70: 705. Little, J. B., Chan, L. G. & Nagasawa, H. (1979) Abstracts of the 6th Intern. Congress of Radiation Research : 26. Littte, J. B. & Hahn, G. M. (1973) Int. ~. Radiat. Biol. '23: 401. Little, J. B., Hahn. G. M., Frindel, E. & Tubiana, IV[. (1973) Radiology 106: 689. Little, J. B. & ~,Villiarrts,J. R. (1976) Radial. Res. 6t3: 90. Malaise, E. P. & Guichard, 3,I. (1976) In Particle Radiation TheraI~¢. Proceedings of International IVor'l~hop, Kfy BiscaA'ne, Florida. (Ed. V. SmiLh). Am. Coll. Radiol., Philadelphia: 385. Mauro, F. & Elkind, 3,I. M. (1967) Science 155: 1561. Mauro, F. & Little, J. B. (1970) In Advances in Radiatiot, Research. (Eds. J. F. Duplan & A. Shapiro) 2 : 949. Miletie, B., Petrovle, D., Han, A. & Zajec, L. (1964) Radiat. Res. 23: 94. Miletie, B., Pctrovie, D. & Zajec, L. (1963) aVature (London) ]97: 90. Moore, J. V. & Dixon, B. (1978) Europ. o7. Cancer 14: 91. Murthy, A. K., Harris, J. R. & Belli, J. A. (1977) Radiat. Res. '70: 241. Palzer, R . j . & Heldelberger, C. (1973)Cancer R#s. 33: 415. Petrovic, M., Miletie, B., Ferle-Vidovie, A. & I-Inn, A. (1966) Radiat. Res..°7: 41. Phillips, R. A. & Tolmach, L . J . (1966) Radlat. Res. 29= 413. Piro, A..][., Taylor, C. O. & Belli, J. A. (1975) Radiat. Res. (Abstract) 62: 55l. Puck, T. T., Marcus, P. I. & Cieciura, S . J . (1956)o7. Expel. 3Ied. 103: 273. Raaphorst, G. P. & Dewey, ~V. C. (1979) Radiat. Res. 77: 325. Raju, .M.R., Frank, J. P., Bain, E., Trujillo, T. T. & Tobey, R. A. (1977) Radiat. Res. '/1: 614. Rasey, J. S., Nelson, N . J . & Carpenter, R. E. (1978) Int. oT. Radlat. Ont. 4: 1023. Rauth, A. M. (1967) Radiat. Res. 31: 121. Ray, G. I*.., Hahn, G. M., Bagshaw, .~I. A. & Kurkjian, S. (t973) Canter Chem. Re#. 57: 473. Regan,J. D. & Setlow, R. B. (1974) Canter Res. 34: 3318. Schroy, (3. B. & Todd, P. (1979) Radlat. Res. "/8: 312. Shipley, W. O., Stanley, J. A., Courtenay, V. D. & Field, S. B. (1975) Canter Res. 85: 932. Sinclair, W. K. (1968) Cancer Re:. 28: 198. Suit, H. D. & Urano, 2M. (1969) Radlat. Res. B'/: 423. Sutherland, R. b,~. & Durand, R. E. (1973) Int. 07. Radlat. Biol. 23: 235. Takabe, Y., Watanaber M., .Miyamoto, T. & Terasima, T. (1974) GA.N'Ar65: 559. Terzaghl, M. & Little, J. B. (1975) Arature 253: 548. Twentyman, P. R. (1976) Cancer Treat. Rep. 60: 259. Twentyman, P. R. & Bleehen, N. M. (I974) Br. 07. Cam:er 80: 469. Twentyman, P. R. & Bleehen, N. M. (1975) Br. 07. Cancer 32: 491. Twemtyman, P. R., Morgan, J. E. & Donaldson, J. (1977) Cancer Treat. Re#. 62- 439. Urano, M., Nesumi, N., Ando, K., Koike, S. & Ohnuma, N. (1976) Radiology 118: 447. Utsuml, H. & Elkind, M. 3d. (1979) Radiat. Re~. 77: 346. Waldren, C. A. & Rasko, I. (1978) Radiat. Res. '/3: 95. ~Veichselbaum, R., LittJe, J. B. & Nove, J. (1977) Int. oT. Radiat. Biol. 31: 295.
R E C O V E R Y F R O M PLD IN ~ i A M M A L I A N CELLS
98. 99. I00. I0l. I02. I03.
Wcichsclbaum, It. R., Nove, J. &Littlc, J. B. (1978) ,VatuTe (London) 271 : 261. Wclss~ B. G. & Tolmach, L..]. (1967) Bioph)'s.~7.7: 779. Wcslra, A. & D c w c y , W. C. (1971) Int..]. RMiat. Biol. 19: 467. ~Vhcclcr, K. T., Dccn, D. F., Lcith,J. T. & Norton~ K. L. (|979) Int..7. Rad. Biol. In prc~. ~,V}dtmorc, G. F. & Gulyn-~, S. (1967) NoR. Cancer ]~I. 3fonogr. 24: 141. Winans, L. F., Dcwcy, W. C. & Dcttor, C. M. (1972) Radiat. P~s. 52: 333.
15
Factors influencing the recovery from potentially lethal damage (PLD) in mammalian cells in v i t r o and in Vtvo' Marcelle Bertrand
Brain Tumor Research Center, Department oJ'aVeurologicalSurgery, School of 3Iedicine, The Lrniversity of California, .Van Francisco, Califortda 94143, U.S.A. and Department q]" A[edical Oneologv, Cit~, of IIope .\rational 3[edical Centre, Duarte, California 91010, U.S.A. a n d D e n n i s F. Deen=f
Brahz Tumor Research CenteG Department of .,Veurological Sur~,err, attd the Departmeizt of Radiation Oncolog~, School of .~Iedicine, The University of California, San Francisco, California, 94143, U.S.A.
Int r o d u c t i o n All too fi-cquently n e i t h e r d r u g n o r radiation t r c a t m e m is able to sterilizc m a l i g n a n t turnors completely. A l t h o u g h the reasons for this failure a r e multiple, a m a j o r fitctor is the ability of t u m o r ce!ls to r e c o v e r from the d a m a g e inflicted on t h e m . Potentially lethal d a m a g e is most easily discussed in terms o f a m o d e l proposed by Phillips et al. in 1966 (7,1.). (:ells exposed to a shtgle dose of X - r a y s i n c u r two types o f d a m a g e : lesions that irrcversibly inhibit the ability o ( cells to proliferate ( " l e t h a l d a m a g e " ) a n d lesions that are unstable, whose u l t i m a t e fate. ~s ,?ctcrrnined b y postt r e a t m e n t condition ( " p o t e n t i a l l y lethal d a m a g e " , P L D ) . F;~'.,..~zflc p o s t - t r e a t m e n t conditions lead to a:l enhancemer~t o f cell survival ("recove~3,"): while u n f a v o u r a b l c conditions result in a d e c r e a s e in cell survival ( " f i x a t i o n " of d a m a g e ) . R e c o v e r y fi'om P L D must be distinguished from r e c o v e r y li'om sublethal d a m a g e ( S L D ) , w h i c h is cat,setl by t r e a t m e n t widlfractionated doses o f r a d i a t i o n (11, 32, 41, 53, 57, 77, 84, 87, 95, 102, 103) or d r u g (3, 5, 6, 9). T h e relationship b e t w e e n P L D a n d S L D r e m a i n s controversial (I I, 16, 29, 32, 41, 95, 102, 103) a n d S L D will not be discussed at length in this review. As suggested b y H a h n (37-39), " r e c o v e r y " will be used in a purely 1"Address reprint requests to: Dennis F. Deen, Ph.D., Brain Tumour Re.search Ceture, 783 ]IS'W, Department of Neurological SurgeD', University of California, San Francisco, CA 94 t43, U.S.A. Tiffs work was supporte d in part by Grant CA 13525 from the National Institutes of Heahh (U.S.A.), and a grant from the Foundation-Rose and.Jean Hoguet (Belgium).
0305-7372/80/010001 + 15 $02.00/0
~, 1980 Academle Press Inc. (London) Ltd.
2
,Xl. BERTKAND AND D. F. DEEN
operational way while "repair" will be used to indicate known modification of specific maeromolec,~: s. In vitro reco,~,:et%,fi'om PLD has been observed after a wide variety of treatments, in dividing and win-dividing cells, and in oxic and hypoxic cells. T h e conflicting rcsuhs reported in th,.: J.iterature on the influence of various factors on the recovery process suggest that the ,~vaagnitude of the p h e n o m e n o n is as dependent on the cell line as on the treatment arid pTJst-treatment conditions. Furthermore, the exact target as well as the repair mechanism involved in the recove13" front PLD remains unknown, although m a n y studies have suggested a repair mechanism perhaps enzymatic, acting at the D N A level. Because recova 3, fi'om PLD also occurs in vivo and enhances the tumor rcsistance to treatment, the study of recovery processes and particularly the study of their inhibition are of considerable interest. We will review thctors influencing the recovery phenomenon in vitro and also t'i~.e in vlvo data reported. "vVe ,,rill emphasize recovery fi'om PLD Following exposure to ionizing radiation and antimitotle drug,s.
In vitro s t u d i e s Effects o f recoveO, medium~
Recovery from PLD under suboptimal growth conditions such as depicted m e d i u m (DM) (40, 41, 46, 47, 49, 57-59, 79), serum free m e d i u m (SFM) (40, 46, 78) or balanced salt solution (BSS) (11, 12, 33, 37.-40, 44, 59), and inhibition of recovery by fresh m e d i u m (FM) (40,44, 46~ 47, 49) were widely reported after X-ray treatment (Table 1). Recover 5, is not an "all or n o n e " p h e n o m e n o n because file magnitude of the process was dependent on the radiation dose (40, 59) and on the medium. Plateau phase and exponentially growing C h a n g liver cells (LICH) recovered preferentially in D M (59), which is perhaps related to a factor secreted by L I G H cells into the m e d i u m (58, 59), while plateau phase Chinese hamster ovarian cells (HA-l) showed a lligher recovery in BSS (40) dlan i n SFNI. No recovery was observed i n exPonentially growing HA-! cells held in BSS (40), a n d recovery in irradiated I~louse embryonic fibroblasts (3T3) was higher in D M than in BSS o r . S F M (46). Some recovery data reported for Xirradiated Chinese hamster lung fibroblasts (V79) conflicted, indicating that the results varied from laboratory to laboratory (Table I). Surprisingly, D u r a n d et al. Were unable to show a n y recovery in either d i v i d i n g or non,divlding V79 cells p0st-treated with v a r i o u s m e d i a (30), while Piro et al. observed an increase in survival in plateau phas e cultures held in either DM, BSS or FM, although recovery was delayed in cells held in F M (75). F u r t h e r m o r e , Mauro et al. observed recovery in V79 cells exposed t o BSS at 23°C before and after radiation; nO recovery was Found in these cells held in F M before radiafi0n (66). Recovery from ~-ray induced PLD, was similarly observed i n plateau phase C H O (34) and E M T 0 cells(36, 65) held in m e d i a that inhibit cellular proliferation. T h e data reported after high linear energy transfer ( L E T ) r a d i a t i o n (fast neutron and charged particle) conflict (Table 2). I n t h e s e studies, the a u t h o r s compared m o d e l systems in w h i c h recovery From X-ray or y-ray induced~PLD were observed. 1"(1) Depleted (p/~tteau)me(tlumor conditionedmedium is'mediumharvestedfromplateau phase culture. However, ~ome investigatox"Suse the term *'conditioned medium" for serum~-ee medium (40,78); (2) balanced salt solutions used are either Hanks', Eai'le's, Or Dulbecco's; (3)fresh, complete, or full medium is a freshl~]prepared-medhimcontaining fetal calf serum.
R E C O V E R Y FROI~I P L D IN .MAMMALIAN CELLS
3
T a b l e 1. R e c o v e r y f r o m X - r a y i n d u c e d P L D in ritro I/,ecovery~ Cell line
Dividing cells
Non-divlding cells
+ (BSS), -- (FM) + (SFM)
CHO
EMT, HA-1
-(Bss)
LICH
+ (DM) + (DM),
- -
(BSS, F M ) )
l~iorris rat hepatoma
+ (DM), -- (FM)
3T3
+ (DM > B S S > S F M ) --(EM)
V79
+ (ass)
+ (SFM)
Hall et aL (44) Raju et at. (78)
+ (DM)
Rasey et al. (79)
+ (BSS > S F M ) , -- (FM) + 03ss)
H a h n el al. (,10) Evans et aL (33), H a h n (37--39)
+(DM) + (DAI>BSS)~ -- (FAI)
H a h n eta/. (41), Little (57, 58) Little (59) Horowitz et al. (49)
+ (DM > BSS > SFM) - (v'M)
-(BSS, DA, I, F~%I)
- - ( a s s , DM, FM)
+ (DM), -- (Fh,l) + (DM), --(FM)
+ (DM), -- (~'M)
+ (~ss, 2soc)~ - ( F M , 23°C),
References
HetzeI el al; (46) Belli el al. (I1, 12) Durand et al. (30) Hetzel et al. (47) Horowhz el al. (49) Mauro et al. (66) Piro et al. (75)
+ (BSS, DM, FM)
"1"+ Recovery, -- no recovery or inhibition of recovery from PLD; 0 incubation medium after treatment: (BSS) balanced salt solution; (DM) depleted medium; (FM) fresh medium; (SFM) serum free medium. ~: See text.
T a b l e 2. R e c o v e r y ~'rom h i g h l e t r a d i a t i o n i n d u c e d P L D in vitro Recovery1" Treatment
Cell llne
a Particle
CHO
Helium ion
EMTn
Fast neutron
ENIT 6
Dividing cells -- (SFM)
Non-dividlng ceils
References
-- (SFM)
R ~ u a a/. (78)
+ (D.M)
Guichard et al. (36) A'lalalse et al. (65)
-- (DM)
Gragg et at. (34.) Hall et aL (44) Rainy et al. (79~
- (FM, J3SS)
+ (DM)
I" + Recovery, - - n o recovery from P L D ; 0 incubation medium after treatment: (BSS) balanced salt solution; (DM) depleted medium; (FM) fresh medium; ( S F M ) s e r u m free medium.
4
M. I|ER'I'I~AND ANI) D. 1". DI'EN
T a b l e 3 s t m m m r i z c s t h e in vitro r e p o n s e of m a m m a l i a n cells to P I , D i n d u c e d tW various d r u g s with reference to Llle m e d i a a n d cell line used. It shows t h a t recovery from P L D was very d e p e n d e n t o n the d r u g . N o recovery was observed after exposure to a d r i a n t v c i u (.-\D~I) (3, 313) or nitrosom'eas (37, 38), whicl, a c t u a l l y resulted in d e c r e a s e d cell survival (3. 9). Because i n h i b i t i o n of recovt:rg after D N A inhibitors has bccn obser.ved (see n e x t s e c t i o n ) , B a r r a n c o (3, 9) s u g g e s t e d t h a t the d e c r e a s e in survival f o l l o w i n g t r e a t m e n t with n i t r o s o u r e a s m i g h t b e r e l a t e d to i s o c y a u a t e , brcakdoxw~ p r o d t t c t s o£ these a n t i t m n o r a g c n t s . I s o c y a n a t c s i n h i b i t e d b o t h D N : \ synthesis (2) a n d ~he r e p a i r o f I ) N ; \ single s t r a n d b r e a k s (51). R.ccovcry f r o m B l c o m y c i n ( B L M ) - i n d u c c d I ' L D o c c t t r r c d c h a r a c t e r i s t i c a l l y for e x p o t m n t i a l l y g r o w i n g a n d p l a t e a u p h a s e cells in e i t h e r D:~I (9, 92), BSS (37-3.0, 81) or FM? (9, 37, 38, 92). R e c o v e r y in F~g mig]tt rcllect cell T a b l e ,~. R e c o v e r y
P L D in rltro
from drug-induced
Recovery'l" "l're.ttment Adriamycitl (ADM i
Cell line CliO
Dividing ccll~ -- (1)311
Non-divlding cc115 --(1)3I)
--(1:31, BSS)
ll.eference~
kt:trranco (3) Hahn (38)
Actinomycin I) (Art I))
CItO
--(F.\I), + (DM)
-- (D.M)
lktrranto (3) Barranco et al. (5"1
Bleomvcin (BLM)
Clio EMT~ HA-t
+ (F.M, D.M) + (F,M, DM)
-t- (DM) -4 (1:31, D*I) + (FM, BSS) 5- (BSS)
Barranco e t a l . (9) Twentyman et al. (92) Iiahn (37, 38) I-tahn (39), Ray tt al. (81)
CHO
(D.".I) -- (D,Xb
-t- (DM) -- (FM, D.M)
Fktrranco
5-Fh,orouracil (5-FU)
]tA- 1
-b (I;SS)
+ (BSS)
Ray
1Mechlorethamine (tiNt)
H'A- 1
+ (BSS)
4-(1:M, BSS) + (IISS)
Itahn (37, 38) Hahn (39), Ray el al. (81 )
--
Diauhydrogatacfitol (DAG) I.ow do~e
High dose
McCCNU-CCNU
ClIO
--
BCN U
I I:\- I 9L
--(FM, BSS)
Mcthyhnethane sulfonatc (MMS)
HA-I
Spirohydantoin mustard (SH.'Xl)
9I.
Sulfur mustard (SM)
V79
(D.'Xl)
+ ( I ) M , BSS) - - (F,M)
+ ( B s s (26oc)§
--~FM) (.6 c)§ •
rl
(D.M)
el at.
et al.
(6)
(81)
--(I:M, BSS)
llarranco (3) Barranco el al. (9) H:tlm (37, 38) K . T . Whccler~
q- (BSS)
ltahn (37-391
-I- ( D M , BSS) -- (1:.', l)
Bertraml et al. (13) Dcen rt al. (19) ~M;,uro ¢t
al.
(6t3)
O
"1" -i. Recovery, - - n n r e c o v e r y from PLD; 0 incubation medium after trcammnt: (BSS) balanced salt solution; (DM) depleted medium; (F.M) fresh medium. * Unpublished data. § See text'.
RECOVERY FROXI PLD IN .XlA.~IMALIAN {;F.LI.S progression d e l a y i n d u c e d by BLlk! (8) a n d inhibition o f m a c r o m o l e c u l a r synthe.~is (9); r e c o v e r y observed in D,Nf might result front a eonlblnation of the.-e eve,its a n d .subo p t i m a l g r o w d l conditions (9). T h e m a g n i t u d e of recovery from ~pirohytlantoin m u s t a r d (SFINI) i n d u c e d d a m a g e was strongly c o n c c m r a t i o n d e p e n d e n t (13, 19), as was die recovt:ry from d i a n h y d r o g a l a c t i t o t ( D A G ) that o c c u r r e d only at tow d r u g o ) n c e n t r a t i o n s in plateau phase Chinese h a m s t e r ovarian cells ( C H O ) h e l d ill l),Xl (6). Exposure to high conctmtrations of D A G decreased the survival of c i t e cells h e l d in either F/k{ or DN[ (6). l~.ecovery from Actinom)'cin J) (,\ct D) i n d u c e d PI.D was fimnd only ill e x p b n e n t i a l l y g r o w i n g C H O cells held in DR'I (3, 5). R e c o v e r y was observed in V79 cells exposed to BSS at 26°C before a n d after stllftlr nltlstard (S~I) ; Ilt~ recovel'y xv,"lS found in the cells p r e t r c a t e d with BSS at 26°C a n d ]told thereafter ill l:Xl ;I.t 26°(: (6~.:,). H e a t injured cells call recover fi'om PI,D. Palzcr et al. (72) sllowcd that e x p o n e n t i a l l y g r o w i n g l-l'ela cells recovered fi'om s u p r a n o r m a l t e m p e r a t u r e d a m a g e (-t20C:) if they w e r e i n c u b a t e d in mc(liurrt c o n t a i n i n g a n inhibitor of protein or 1-)NA symhesis (see l l ( : x l section) at nell-toxic co1~centratlons. T h e ma.t,mitudc of tile r e c o v e r y of dc,lsity inhibited HA-1 cells exposed to 43cC d e p e n d e d slg'nificantly txpon the nutrient conditions a n d pI I d u r i n g testing a n d was less atTccted t)y postheating'milieu (56). R e c o v e r y after u.v. ilTadiatlon of H A - I cell.; d e p e n d e d on nutritional ~letors both l)cfol'c and after t r e a t m e n t a n d o c c u r r e d equally in eitht.r BSS ~r IvM (37--39). Ill 1975, H a h n suggested that tile cell rccov{.gl'}" a~tCl" a variety of insults could he a n a l y z e d in terms o f X - r a y or u.v.-tikc d a , n a g c (37-39) or a u n i q u e system of recovery following h e a t shock (37, 38). I lahn':: system ,~vas hascd on the h a l f time (it) nf recovery, whick was ,-,2 h For X - r a y d a m a g e (37-39), ---13 h For u.v. d a m a g e (G. M. H a h n , personal c o m m u n i c a t i o n ) , a n d less thml 30 rain after exposure to sup,'anormal temperatures (37). Tile kinetics of recovery after X - r a y or u.v. trxposur¢ suggested a repair m e c h a n i s m acting at the D N A level, a l t h o u g h recovery kinetics a n d the kinctic.s of D N A repair after X - r a y a n d u.v. exposure (82) w e r e not identical (37-39). T h e d e m o n stration that cells deficient in D N A repair ability such as x e r o d c r m a l)igntcntosum skin fibroblasts exposed to u.v. a n d ataxia telangicctasia skin fibroblasts exposed to Xirradiation wert" also u n a b l e to recover efficiently fi'om P L D c o n f i r m e d this hypothesis (98). T h e Fast ,'ecovcry after h e a t shock suggested that proteins might be a m o r e likely target for the repair (100). R e c o v e r y after e.xposure to m e c h l o r e t h a m i n e (fiN.,) or BL,N'[ was similar to X - r a y recovery, while recovery after exposure to n t e t h y l m e t h a n e sulfonatc (I~IA,IS) or b r o m o d c o x y e y t i d i n c (l'~Cdlt.) a n d visible light had con'qmnents similar to both X - r a y a n d u.v. recovery (37-39). 9L cells treated with S H N I h a d a r e c o v e r y q. of ---.4 h, w h i c h was close to that of X-rays (13). H o w e v e r : usiIIg dif~:t'ent cell lines a n d , in some cases, ditTcrent r e c o v e r y conditions such as low t e m p e r a t u r e (102, 103), some investigators found that recovery after X - i r r a d i a t i o n occuneed m u c h m o r e rapidly, w i t h a t~ of--- 15 rain (1 I, 12, 102, I03), a n d p r e l i m i n a r y kitletic studies with a mouse fibroblast cell line (10 T~) exposed to u.v. radiation showed that the r e c o v e r y process took place largely within tlle first 6 h after exposure (17). An etdlancem e n t in tlae frequency of m a l i g n a n t transformatio,a paralleled tile e n h a n c e m e n t of survival in die 10 T t cell line after X - r a y (60, 89) a n d u.v. t r e a t m e n t (17, 61) d u r i n g tile early recovery period. H o w e v e r , transformation d e c l i n e d with longer recovery intervals.
Cell cycle phase dependence S o m e a u t h o r s }lave shown that r e c o v e r y is a cell c2/clc phase specific process. C o m p a r e d with e x p o n e n t i a l l y g r o w i n g cultures, the m a j o r i t y of cells in plateau phase cultures are
6
~I. IIERTRANI) A N D D. F. DEEN
in G 1 p h a s e o f t h e cell c y c l e (41). It h a s b e e n s u g g e s t e d t h a t this s y s t e m is a b e t t e r in vitro m o d e l for h u m a n t u m o r s t h a n e x p o n e n t i a l l y g r o w i n g c u l t u r e s (41). T h e fact t h a t t h e m a g n i t u d e o f r e c o v e r y w a s h i g h e r in p l a t e a u p h ~ e C H O (78) o r L I C H (59) cells aft,:r X - r a y t r e a t m e n t a n d t h a t n o r e c o v e r y w a s o b s e r v e d in X - i r r a d i a t e d e x p o n e t n i a l l y g r o w i n g H A - 1 culls (40) i n d i c a t e d t h a t cells in G j m i g h t b e p r i m a r i l y i n v o l v e d in t h e r e c o v e r y p r o c e s s (40, 59, 78). L i t t l e c o n f i r m e d this h y p o t h e s i s b y d e m o n s t r a t i n g Oaat r e c o v e r y o c c u r r e d in p l a t e a u p h a s e L I C ; H c u l t u r e s in w h i c h S cells w e r e s e l e c t i v e l y killed (62). T h i s f i n d i n g w a s f u r t h e r c o n f i r m e d b y e x p e r i m e n t s w i t h s y n chronized L I C I - I cells o b t a i n e d f r o m e x p o n e n t i a l l y g r o w i n g c u l t u r e s (62). H o w e w : r , in "v"-o / . , cells r e c o v e r y o c c u r r e d p r i m a r i l y in S p h a s e cells (12, 47), w h i l e r e c o v c t T o c c u r e d in C H O cells t r e a t e d w i d l e i t h e r B L M (4) o r X - r a y s ( I 0 3 ) in botJi G t a n d S p h a s e . F u r t h e r m o r e , Vehittnorc el al. o b s e r v e d r e c o v e r y fi'om P L D in all phases o f t h e c y c l e o f m o u s e L cells, bttt rt'cover 3, w a s m a x i m u m in cells i r r a d i a t e d in e a r l y S p h a s e (102). T a b l e 4. Effects o f m e t a b o l i c inhibitors on recovery" f r o m PLD ;a ~itta ~. ff~ ¢ •
Inhibltors DNA synthesis inhibitor
Treatment X-ray
Cell line
*:!
: e.:-, ,'er.v+.
References
l-[eLa
--
Bleomycin (BLM)
HeLa V79 /riA-I "F" CHO
-No effect No effect No efrcct
Cis-tHatinum complex
X-ray
V79
--
Doupte et al. (28)
P,NA synthesis inhibitor
X-ray
V79
--
HA-!
,N'o effect
Elkind el el. (32) Piro et al. (75) Evans tt el. (33)
),-ray
"F"
--
Arlctt
Bleomycin (BLM)
CHO
--
Barranco el al. (7)
X-ray
HcLa
)'-ray
*'F'"
,No effect
Arlett (1)
X-ray
HcLa 1~-1 "F"
4No effect No effect Slightly+
l'hillips et al. (74) Evans et aL (33) Arlett (!) Barranco et al. (7)
y-ray
(Actlnomycin D)
Ribosomal RNA synth~is inhibitor (Puromycin) Protein synthesis inhibitor (Cyclohexam ide)
y-ray Bleomycln (BLM)
CHO
r'hillips tt at. (74) Weiss el al. (99) Djordjevic tt al. (23) Sinclair (85) Evans el al. (33) .,Lrlett(1) Barranco el el. (7)
(I)
Djordjevic et el. (23, 24)
Phcncthyl alcoho|
X-ray
L
+
Whltmore et el. (]02)
Dinitrophenol
X-ray
L LlCHflqt
+
Dalr3"mpte et al. (18) Little eta/. ( 6 4 )
Caffeine
X-ray '),-ray
Various Various "F" L V791T-I
Enhancement of recovery; ~hddbltion of recovery from PLD.
--
No effect ~X'oeffect
Bussc a al. (15) ~raldrcn ¢t al, (96) Arlett ( 1) Rauth (80) Schroy et al. (83)
RECOVER~" I:R05[ PI.D IN ~IAMhlALIAN CELLS
7
O n the other h a n d , cell kinetic studies p e r f o r m e d d u r i n g the p o s t - t r e a t m e n t period after e i t h e r X - r a y (59) or S H M exposure (13) in a s y n c h r o n o u s cultures s h o w e d that t h e recow.'ry process o c c u r r e d p r i m a r i l y in cells held in GI phase of the cell cycle. T h e i n c u b a t i o n of p l a t e a u phase c u h u r e s or e x p o n e n t i a l l y g r o w i n g cultures in either D M or BSS i m p e d e d the progression o f cells from G l into S a n d increased the survival, whereas conditions such as FN[ allowed d m ceils to m o v e into S a n d decreased survival (13, 59). F u r t h e r m o r e , G u i c h a r d et aL f o u n d that ia EM'Fn cells allowed to recover iu their o w n m e d i u m after y-irradiation, the .'tmplittlde o f recovery was also a fitnctiotl o f ~:c11 t u r n o v e r rate a n d increased with c u l t u r e age, while the fi'action o f cells ia S phase b e c a m e smaller (35). "Yhey also s h o w e d that the delay tteccssary to obtain m a x i m u n l :~'ecove~T increased proga'essively with c u l t u r e age (35). ~ffects o f metabolic inhibitor drugs
Modificalio~ of survival in X - r a y o r B L ~ ! t r e a t e d m a m m a l i a n celts in c u l t u r e has l,ren achieved by p o s t - t r e a t m e n t exposure to several m e t a b o l i c inhibitors at non-toxic concentrations ( T a b l e 4). T h e d a t a r e p o r t e d in t h e literature are very conflicting a n d , therefore, no definitive conclusion could bc d r a w n from these studies. o n r e c o v e r y f r o m P L D in vitro
T a b l e 5. EiYect or" r e d u c e d t e m p e r a t u r e .
.
.
.
.
.
.
Cell line
~
.
.
.
.
.
.
.
.
.
.
. . . . . . . . . . . . . .
F HA-I
>'-ray X-ray X-ray X-ray X-ray Spirohydantoin mt~tard (SI-[M) X-ray X-ray
~__
~
:
. . . . . . . . . . . . . . .
~
IIIIIIIIII
.............
.
.
.
.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effect on recovery[
X-ray
I0 T.~ V79 ~_
.
CblO
L L5178"2" 9L
~
.
Treatment
HeLa
~
.
.
.
.
.
.
.
.
:~
Z
.....................
:
.
.
.
.
.
,,
,
,,
.,,,,,,,
Rel'erenc~
+ (20~C~
.
,,,,,,,,,,
-- (4":C) No effect (25"C) --(4~'C)*,. -- (29"C) 4- (5:C) H-(34"C) -- ("--230C}.*
Dettor etal. (20) Dewey et at. (21 ) Winans etal. (103) ~,Vinans etal. (103) tM'lett (1) Evans et at. (33) Phillips et el. (74) ~Arhltmoreet el. (I02) Beer et al. (10) Bertrand et at. (13)
--(5~C) No effect (24"C)
Durand et el. (30)
.
.
.
.
.
.
.
.
.
.
.
.
.
Little (60) .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
m
mm I
III
III
--inhibition of recovery; 0 recovery temperature. Recover3" decreased as the temperature was lowered and was inhibited at the temperature indicated ia 0. t q-Enhancement;
I n 1966, Phillips et aL r e p o r t e d t h a t exposure to ird,ibitors o f D N A synthesis (5fluorodeoxy'uridine a n d h y d r o x y u r e a ) resulted in a lower survival in X - i r r a d l a t e d cervical c a r c i n o m a cells ( H e L a ) , suggesting t h a t the r e p a i r process nfight involve Some type o f D N A synthesis (74). O t h e r e x p e r i m e n t s using the s a m e cell system with a variety o f D N A inhibitors s h o w e d that the m a g n i t u d e o f the effect was i n h i b i t o r specific a n d n o t related to the degree of i n h i b i t i o n of D N A synthesis (99). Tile i n t e r p r e t a t i o n of these d a t a is c o m p l i c a t e d b y the fact that using t h e s a m e cell line Djordjevic et el. o b t a i n e d conflirting results (23) a n d because n o effect o f D N A inhibitors w a s f o u n d by o t h e r investigators using v a r i o u s cell lines (1, 7, 33). P r e l i m i n a r y d a t a suggest t h a t a cisp l a t i n u m complex, w h i c h is also a n inhibitor o f D N A synthesis (50), i n h i b i t e d recovery fi'om X - r a y i n d u c e d P L D in V 7 9 cells (28).
8
.~I. B E R T R A N D
A N D D. F. D E E N
Exposure of cells to Act D (1, 7, 32. 75), ed3idium b r o m i d e (1), or acriflavine ( ] ) ; drugs that b i n d to D N A a n d inhibit D N A - d i r e c t e d R N A synthesis, resulted in decreased cell ~urvival, a l t h o u g h this observation was not universal (33). Because the i m e r a e t i o n of ;'~ct D widl c h r o m a t i n inhibited the repair of X - r a y d a n m g e to a DNA comples (31), the b i n d i n g of Act D to D N A a n d not specific interference with R N A synthesis might be p r e s u m e d to cause the inhibition of recovery from P L D (7), but the r e q u i r e m e n t of R N A synthesis tbr such rccovel T cannot be ruled out (7). l : u r t h e r n m r e , the activity of a n y e n z y m e that repairs r a d i a t i o n lc~ions in nucleic acid m a y be i m p e d e d in a steric r a m m e r by the d r u g - D N A c o m p l e x (!). In contrast, exposure of cells to inhibitors of protein wnthesis r e s u h e d in an e n h a n c e m e n t of survival after X - i r r a d i a t i o n (74) a n d p r o d u c e d m i n o r e n h a n c e m e n t of recovery from P L D after B L M t r c a m w n t (7). Phillips el al. suggested that by inhibiting protein syn,hesis, cyclohcximide mighl delay the expression of d a m a g e w i d m u t sigMficantly a h e r i n g the repair process, w h i c h allowed m o r e d a m a g e to be r e p a i r e d (74). These d a t a suggest that protein synthesis might not be rcqttired for r e c o v e r ' / f r o m P L D (7). H o w e v e r , Djordjcvic tt al. (23, 24) rcportt'd a decrease in survival of X - i r r a d i a t e d H e L a cells e.xposed to p u r o m y c i n , an i n h i b i t o r ,)f ribosontal R N A syaathesis. T a b l e 6. Efl'ect o f h y o e r t h e r m i a
o n r e c o v e r y f r o m P L D ~ in rltro
Gell line
Treatment
" P r e h e a t i n g " ,+
HA-I
Bleomycin /BL.M)
-~- (43°(:)
"Postheatlng"~
-t (41°C)
R c ference~,
Braun et M. (141
-- (43oc)
-- (43°C)
V79
X-ray
-'- (43¢C)
X-ray X-ray X-ray
-v. ( 4 l c C )
- (43:C) -- (41 cC) -- (42/43°C)
J~Iahn et al. (39) H a h n el td. (39) l.i et at. (55) .~Iurthy el at. (71) l Iarris el al. (45) D o u p l e (27)
~" + R e c o v e r y ; - - i n h i b i t i o n o f recovery. Exposure to h e a t before ( " p r e h e a t i n g " ) or a f t e r l r e m m e n t ( " p o s t h e a t i n g " ) .
Increase in survival o f . K - i r r a d i a t e d cells wa:~ also r e p o r t e d after exposure to p h c n e t h y l alcohol, an aspecific m e t a b o l i c inhibitor (102), a n d to d i n i t r o p h e n o l , w h i c h inhibits cellular A T P by u n c o u p l i n g m i t o c h o n d r i a l oxidative p h o s p h o r y l a t i o n (18, 64-). T h e s a m e effect was observed w h e n a subcellular fraction, or D N A , or its precursors were a d d e d to file m e d i u m of mouse L cells after X- (48, 68, 69, 73) or u.v. (25) irradiation. Caffeine was r e p o r t e d to h a v e either no effect (1, 80) or to inhibit recovery (15, 83, 96) from r a d i a t i o n i n d u c e d P L D in a variety of r o d e n t a n d h u m a n cells.
Effects of recovery temperature Conflicting d a t a h a v e been r e p o r t e d on tile influence of r e d u c e d p o s t - t r e a t m e n t t e m p e r a t u r e on file recovery from P L D ( T a b l e 5). I n c u b a t i o n at low t e m p e r a t u r e e i t h e r £ w o r e d recovery (10, 20, 21, 102, 103), inhibited r e c o v e r y (13, 33, 60, 74), or h a d no effect on the process (1, 30). R e c o v e r y from X - r a y d a m a g e o c c u r r e d at 5°C in m o u s e L cells (102) a n d at 34eC in l e u k e m i a cells (10); in C H O cells recovery o c c u r r e d at 20°C, but was inhibited at 4°C (I03). I n contrast, r e c o v e r y was i n h i b i t e d in H e L a
RECOVERY FRO*I I'LD IN MAMMAI.IAN CELLS
9
cells h e l d at 2 9 ° C (74) while , h e i n c u b a t i o n o f i r r a d i a t e d H A - I cells at either 25°C or 4¢C r c d u c e d or i n h i b i t e d t h e recovel T (33); t h e l a t t e r i n h i b i t i o n was reversilfle u p o n r e s t o r i n g the cells to 3 7 ° C (33). Vee f o u n d t h a t the m a g a f i t u d e of r e c o v e r y of 9 L cells after SH.'k~ t r e a t m e n t d e c r e a s e d as the t e m p r a t u r e was l o w e r e d a n d was i n h i b i t e d at 2 3 ° C (13). T h i s suggested t h a t a n e n z y m a t i c process was re.,pon.qblc for the r e c o v e r y process (1 3, 33). R e c e n t l y , h y p c r t h e r m i a has received c o n s i d e r a b l e a t t e n t i o n as an a d j u v a n t m o d a l ity to c a n c e r t h e r a p y . Its ability to interfere w i t h t h e r e c o v e r y process ( T a b l e 6) is therefore o f s o m e interest. S h o r t e x p o s u r e to t e m p e r a t u r e ~ 41 °G after t r e a t m e n t ( " p o s t h e a t i n g " ) usually i n h i b i t e d t h e recoverg"fi'om X - r a y (27, 39, 45, 55) a n d BL.XI (14, 39) i n d u c e d P L D . T h e rcversc s e q u e n c e ( " p r e h e a t i n g " ) p o t e n t i a t e d t h e effect o f t r e a t m e n t , b u t d i d n o t inhibit t h e recover T f r o m P L D (14, 39, 55, 71), a h h o u g h t h e kinetics o f recovery were slower (39, 55). T h e s e d a t a suggest that i n h i b i t i o n o f an e n z y m a t i c r e p a i r process was unlikely, a n d t h a t p o s t h c a t i n g c o n v e r t e d p o t e n t i a l l y lethal lesions into lethal ones (14, 55). O n the o t h c r h a n d , p o s t l l e a t i n g progn'esslvely r e d u c c d t h e m a g n i t u d e a~tl t h e rate o f r e c o v e r y (14, 55) ; r e c o v e r y f r o m X - r a y a n d IIL~.I i n d u c e d d a m a g e were aboli.~hcd after 60 m l n ( 5 5 ) a n d 1 0 0 r a i n (14), respectively, of h e a t i n g . Effect o f various other f a c t o r s
Several o t h e r factors w e r e r c p o r t c d to influence t h e recovery proccss after i r r a d i a t i o n ( T a b l e 7), b u t h a v e n o t yet been extensively s t u d i e d . I n h i b i t i o n o f recover 5, f r o m r a d i a t i o n d a m a g e o c c u r r e d in cells m a i n t a i n e d in single cell suspension after t r e a t m e n t , suggesting t h a t r e c o v e r y was d e p e n d e n t u p o n cell to cell c o n t a c t (33, 4-7, 59). T h e s e d a t a are, h o w e v e r , difficuh to i n t e r p r e t b e c a u s e t~-psinization a n d r e s u s p e n s i o n in D M p r o b a b l y a l t e r e d t h e cell m e m b r a n e p e r m e a b i l i t y a n d g r e a t l y e n h a n c e d t h e surlhce available to n u t r i e n t s (59). E v a n s et al. s h o w e d t h a t the rate o f D N A synthesis in the dispersed p o p u l a t i o n o f cells was m o r e t h a n twice Table 7. Effect of various conditions on recovery f r o m PLD fn ritro Conditions
Cell.line
Treatment
HA-1 LICH V79
X-ray X-ray X-ray
Mild h~,T,oxia
HA-I V79
X-ray X-ray
Extreme hypoxia
HA- i
X-ray
Koch tt al. (53)
Extremely hypertonlc solutioa
CHO
X-ray
Dettor tt al. (20) Raaphorst et at. (77) V¢inans ¢t at. (103)
Extremely hypotonic solution
V79 GHO V 79
X-ray X-ray X-ray
Utsumi et o2. (95) Raaphorst tt at. (711) Ufsumi et al. (95)
Single cell suspension
J" -l- Recovery;
-
-
irdaibhion of recovery.
Eft•ct on recovery~f
R efcren ce--.
F.va3~s et at. (33) Little (59)
Hetze! et at. (47) + +
Hahn tt al. (40) Douple (27)
I0
NI. BERTI~kND AND D. F. DEEN
t h a t in cells left in m o n o l a y e r culture (33). F u r t h e r m o r e , recovery occurrcd as well in low density m o n o l a y e r cultures (59, 78, 92). Also, unlike Hetzel et al. (47), S u t h c r l a n d et al. (87) a n d D o u p l e (26) did not observe recovery fi'onl X - r a y i n d u c e d d a m a g e in V79 cells g r o w n in s p h e r o i d cultm'e, a system in w h i c h cell to cell contact plays an i m p o r t a n t role, A l t h o u g h i r r a d i a t e d H A - I (40) and V79 (27) cells r e c o v e r e d u n d e r m i l d h y p o x i c a n d aerobic conditions, e x t r e m e l y hypoxic conditions inhibited recovery fi-om both P L D a n d S L D in HA-1 ceils (52, 53). However, a d d i t i o n o f e x t r a glucose to the m e d i m n allowed some r e c o v e r y fi'om P L D , indicating that at least p a r t o f the recovery process r e q u i r e d e n e r g y (53). I n h i b i t i o n of recovery after radiation t r e a t m e n t was also r e p o r t e d in cells exposed to extremely anisotonic solutions (Table 7), the efl~:cts of ~daich seemed to d e p e n d o n the sequence of exposure. R a a p h o r s t et al. reported that salt t r e a t m e n t of C H O cells before irradiation did not inhibit r e c o v e r y fi'om either P L D or S L D , whereas salt t r e a t m e n t d u r i n g or after i r r a d i a t i o n did inhibit both types of d a m a g e (77). H o w e v e r , D e t t o r et al. r e p o r t e d an i n h i b i t i o n of recovery fi'om P L D in C H O ceils held in h y p e r t o n i e sotution before, during, a n d after X-irradiation (20). U t s u m i et al. fi:mnd a n i~zibitiort of P I , D in V79 ceils exposed to anisotonic solutions after X - r a y t r e a t m e n t (95). Because of osmotic changes p r o d u c e d by anisotonic t r e a t m e n t , fixation of P L D m i g h t be the result o f a destabitization o f the structural relationship b e t w e e n D N A a n d the n u c l e a r envelop a n d / o r D N A a n d the n u c l e a r protein m a t r i x (95). Osmotic changes m i g h t also prevent a r e p a i r e n z y m e from acting at the D N A level (77). T h e trypsinization p r o c e d u r e used after t r e a t m e n t for both in vitro a n d in vivo systems prior to assay for sm'vival by colony formation as described by P u c k et aL (76), suggests tliat the recovery p h e n o m e n o n m i g h t be an artifact of this procedure. H o w e v e r , studies in w h i c h trypsinization was not necessary i n d i c a t e d trypsinization not to be a n artifact (11, 33, 38, 44, 67, 78, 88, 92). In ff)'O s t u d i e s
Conditions t h a t allow recovery from P L D in vitro are fairly drastic a n d are unlikely to be ever duplicated in vivo. H o w e v e r , several studies h a v e d e m o n s t r a t e d t h a t both ascites a n d solid r o d e n t tumors t r e a t e d in vivo a n d allowed to r e m a i n in situ for several hours after t r e a t m e n t r e c o v e r e d f r o m P L D (see T a b l e 8) a n d S L D (I 1, 22, 63, 84, 86) after a variety of treatments. R e c o v e r y from P L D a f t e r X-irradiation o c c u r r e d in solid t u m o r s a n d in old slow growing ascites tumors (I I, 43, 63) and was f o u n d to be dose d e p e n d e n t (42). T h e s e findings, with the observation t h a t the m a g n i t u d e a n d kinetics o f recovery w e r e similar to those observed in X - i r r a d i a t e d plateau phase ceils in culture, suggest that recove R • o c c u r s p r i m a r i l y in non-proliferating cells a n d p a r t i c u l a r l y in highly radioresistant hypoxic cells (11, 43, 63). T h e recovery process w o u l d a c c e n t u a t e the radioresistance of the tumor. I n a clinical setting, the a m o u n t of r e c o v e r y p r o b a b l y would c h a n g e d u r i n g a course o f p r o t r a c t e d r a d i a t i o n therapy as o x y g e n a t e d a n d cycling cells are killed a n d resting cells are r e c r u i t e d into the proliferative pool. I f one assumes that recovery f r o m P L D is different, b u t has a d d i t i v e effects (4I, 63) to recovery from SLD, fractionated doses m i g h t u l t i m a t e l y be m o r e efficacious. T h e s e observations were later c o n f i r m e d by t h e recovery f r o m P L D f o u n d in acute a n d chronically hypoxic solid rat s q u a m o u s c a r c i n o m a cells (NP,--S I) after X-irradiation (94) a n d m " large, ¢ " Lewis " lung necrotm
RECOVERY FROXI PI,D IN NIANINIALIAN CELLS
II
c a r c i n o m a ceils a f t e r y - i r r a d i a t i o n (84). F u r d a e r m o r e , t h e l a t t e r t u m o r r e c o v e r e d fi'om p o t e n t i a l l y l e t h a l lesions as ,,veil a s f r o m s u b l e t h a l lesions a f t e r ) , - i r r a d i a t i o n , b u t n o t a f t e r n e , t r o n i r r a d i a t i o n (84). T h i s suggests t h a t a d e c r c a s c d c a p a c i t y for r e c o v e r y m i g h t b e a n i m p o r t a n t r a d i o b i o l o g i c a l d i f f e r e n c e b e t w e e n t h e eWect o f l o w L E T ( X - r a y , y - r a y ) a n d h i g h L E T (84) r a d i a t i o n . H o w e v e r , r e c o v e r y fi'ont P L D w a s also r e p o r t e d in vivo a f t e r h i g h L E T r a d i a t i o n (36, 101) ( ' F a b l e 8).
T a b l e 8. R e c o v e r y
f r o m P L D in rlro
Treatment
(_:ell liner
Recovery$
BCNU
9L ElklT6
-+
Bleomycin (BL~I)
E1MT6
+
Ehrlicil ascit~ tumor I~tk/'F6 LNI C t EI~,IT,
+ -b +
C y c | o p h o s p h a m i d e (CP~Xl)
5-Fluorouracil (fi-FU) Carbon ion 7-ray Helium ion Neutron X-ray
BCN U + heat Neomycin+ heat X-ray+heat
+
9L
+
ENITo
+ + -b
Lewis lung carcinoma EMT n Lewis hmg carcinoma ENFF~ 9L NITG-B N C T C (Ascites/solid)
+ -% -b +
NR-S 1 P-388 (Ascitcs)
+ +
EMT 6 EMT 6 ENIT0
-b Inhibition Inhibition
l~,efert:nces lkl. L. l~,osenblum§ "Fwentyman et al. (93) H a h n et al. (42), l-ta|m (39) Twentyman et al. (9 I, 92) Takadc et al. (88) /-/,aim et al. (42) Moore et al. (70) Hahtt et al. (~t2) Wheeler et al. (I01) Guiehard el al, (36) Shipley et al. (84) Guiehard et aL (36) Shipley et at. (8,1,) Hahn (39), Hahrt el at. (43) "Wheeler et aL (101) Douple (26) Hahn et al. (43) Little et al. (63) Urano et al. (94) Belli et al. (I l) Twentyman et al. (93) Braun et at. (t4) Leith et at. (54)
1" Solid tumor except when mentioned, .++ Recovery; -- no recovery from PLD. § Unpublished data. T h e l i t e r a t u r e c o n t a i n s few d a t a o n t h e in viva r e s p o n s e to d r u g i n d u c e d P L D . T o d a t e , o n l y f o u r d r u g s h a v e b e e n studied.: B L M , c y c l o p h o s p h a m i d e ( C P M ) , 5 - f i u o r o u r a c i l ( 5 - F U ) , a n d B C N U ( T a b l e 8). A d e c r e a s e in s u r v i v a l s i m i l a r to t h a t o b s e r v e d in Vitro a f t c r e x p o s u r e to n i t r o s o u r e a s ( 3 , 9, K . T . W h e e l e r , u n p u b l i s h e d d a t a ) w a s ob~;erved ill 9 L r a t b r a i n t u m o r cells a f t e r t r e a t m e n t w i t h B C N U ( M . L. R o s e n b l u m , u n p u b t i s l l e d d a t a ) . Surprisingly~ T w e n t y m a n et al. f o u n d a n e n h a n c e d s u r v i v a l in E h ' I T B t u m o r s t r e a t e d w i t h tile s a m e d r u g , s u g g e s t i n g t h a t " e i t h e r t h e s u r v i v i n g f r a c t i o n w a s a r t e f a c t u a l l y l o w 2 h a f t e r B C N U t r e a t m e n t o r t h a t r e c o v e l T f r o m P L D w a s o p e r a t i n g to a I a r g e e x t e n t b e t w e e n 2 a n d 24 h ' " (93). R e c o v e r ) , f o l l o w i n g t r e a t m e n t w i t h e i i h e r B L M , C P M , o r 5 - F U s u g g e s t e d t h a t in a c l i n i c a l setting, f r a c t i o n a t e d doses m i g h t be m o r e e f f i c a c i o u s i n vivo t h a n a single l a r g e d o s e ( 4 2 ) . H o w e v e r , f r a c t i 0 n a t e d : d o s e s d i d n o t p r e v e n t in vivo r e c o v e r y f r o m PLY) i n d u c e d b y B L M (90). r w e n t y m a n el al. S h o w e d
12
,Xl. BF,RTILAND AND D. F. DEEN
that different sized E M T 6 tumors recovered from B L M d a m a g e (91); furthermore, they found a much. higher increase in survival (92) t h a n previously described (42). T a k a d e et al. also observed recovery from P L D in Ehrlieh ascites tumor, a system that did not r e q u i r e trypsinizalion before the survival assay, ,,daich ruled out trypsin as an artifact (88). Recently lk,Ioore et aL reported recovery from PL D in a solid rat mammal',/ carcinoma (LMC1) after treatment with C P M (70). Cellular repopulation from the resistant component that recovered fi'om P L D after a dose of 150 mg/kg was delayed lbr 6 or more days; restoration to near pretreatment status was achieved by 10 days, suggesting that a further dose of CPA'I would be more efficacious a t longer than at shorter intelwals (70). Preliminary data has suggested that recovery fi'om BLh,~ (14) an d X-ray (54) induced damage in E M T 8 solid tumors might be inhiMted by adjuvant heat treatment, duplicating in vivo the observations m a d e in vitro (Table 6). T w e n t y m a n et al., however, found that adjuvant heat did not inhibit recovery from B C N U induced PLD i n the same tumor system, although the combined modality was more efficacious than BCNU alone (93).
Conclusion
In summary, recovery from P L D is a n important p h e n o m e n o n that has occurred in m a n y experimental systems, and probably enhances the resistance of m a n y malignant tumor cells to treatment. For e x a m p l e , the radioresistance of h u m a n osteosarcoma might be c a u s e d not by an intrinsic resistance of the cells to X-ray ti'eatment, b u t rather by an efficient recovery from P L D (97). Although a substantial literature on P L D has evolved during the past decade, it is not u n c o m m o n for different authors to report conflicting results for similar experimental conditions in the same system. Because the PL D p h e n o m e n o n m a y be strongly dependent on r a t h e r subtle differences in cell tine and/or culture materials and meflaods, t h e relevance of any particular P L D phenomenon to a tumor's recovery capability in situ becomes suspect. Studies should b e designed not o n l y to observe a P L D effect, but to analyze tlae,causallty of the observation: Appropriate models to study chnlcally relevant PLD should allow differentiation of possible artifacts from true biologica 1 responses. For example, if cell lines a r e used that gn-0w both in vitroand in vivo, then various in vitro, in vivo-in Vitro, a n d hz vivoassays Can be employed] I f recovei~, (o1" the lack of recovery) from P/SD is not indicated by a l l the assays, an d logical explanations are not available, then artifacts seem likely. F r o m the experinaental data reported here; w e can conclude: (1) T h e relevance of an in vitro model to radiation t h e r a p y in vivo becomes obvious when it was showh that recovery comparable in m a g n i t u d e and[kinetics to that found in vitro occurred a!so in vivo for experimental tumors grown in both ascitiC and solid forms. Therefore, recovery from radiation-induced PLD is certainly a factor to b e considered in treating h u m a n tumors; (2) The relevance of in Vitro models to in vivochemotherapy is less Obvious because circulating d r u g metabolites m a y interfere~ with the recovery process in vivo. Furthermore, tlae drug levels used i n experimental models in vivo and in bitro are generally well a bove those likely used clinically a n d : m a y influence the recovery process.
R E C O V E R Y FRO.'~I PLD I N M A M M A L I A N CEI.I.S
13
Acknowledgments W e w i s h to t h a n k K . T . '~Vheeler a n d lkIi L . R o s e n b l u m f o r a l l o w i n g us to u s e t h e i r unpublished data, J. L. Wolf for assistance with the bibliography and the tables, Neil B u c k l e y f o r e d i t o r i a l a s s i s t a n c e , a ~ d ,~Iarilyt~ M ' i n n a a r f o r t y p i n g t h e m a t ~ t t s c r i l ) t .
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