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Cancer and oxidative stress
Cancer and Oxidative Stress
18.1
18.3
167
CANCERAND OXIDATIVE STRESS Peter Cerutti, Alexander Peskin, Girish Shah and Paul ~ s t a d , Department of Carcinogenesis, ISREC-1066 Epalinges, Switzerland. Growth promotion by oxidants is observed with fibroblasts as well as epidermal cells. I t is expected to play a role in inflammation, fibrosis and tumorigenesis. Indeed, oxidants trigger (patho)physiological reactions which resemble those induced by growth- and differentiation factors. They cause S6-phosphorylation, activation and translocation of protein kinase C, Ca2+- dependent DNA breakage and transcriptional activation of the growth competence genes c-fos and c-myc. For the induction of c-fos message protein phosphorylation and poly ADP-ribosylation are required. In fact FOS-protein which acts as a transcriptional regulator is poly ADP-ribosylated in response to oxidants. Deletion analysis of the 5'-regulatory sequences of the c-fos gene indicates that the dyad symmetry axis element and the cyclic AMP responsive element serve as enhancers in c-fos induction by oxidants. The c e l l u l a r antioxidant defences are bound to affect the consequences of oxidant exposure. Overproducers of Cu,Zn-SOD are sensitized to the toxic effects of extracellular O~ plus H202 while overproducers of catalase (CAT) are protected. Both SOD and CAT attenuate the induction of c-fos. Overproducers of both SOD plus CAT resemble wild-type c e l l s . It is concluded that a fine balance of the multiple components of the antioxidant defence determines the biological effects of oxidative stress.
HYDROGEN PEROXIDE CAUSES AMPLIFICATION OF VIRAL AND CELLULAR GENES. S.A. Weitzman, P.W. Turk, K. Rundell, Northwestern University Cancer Center, Chicago, IL 60611, U.S.A.
MOLECULAR MECHANISMS OF DAMAGE TO E I O L O G I C A L M E M B R A N E S BY FREE R A D I C A L S Y u r y A. V l a d i m i r o v 2nd M o s c o w M e d i c a l Institute, Moscow, 117513, USSR
INTRACELLULAR ACTIVE OXYGEN SPECIES IN THE PROMOTION AND SUPPRESSION OF TUMOUR CELL GROWTH Roy H. Burdon, Vera Gill & Catherine Rice-Evans* University of Strathclyde, Todd Centre, Glasgow G4 ONR, U.K. *Royal Free Hospital School of Medicine, London NW3 2PF, U.K.
Free radical reactions of lipid peroxidation (LPO) were i n i t i a t e d by UV-irr a d i a t i o n of a r t i f i c i a l a ~ b i o l o g i c a l m e m b r a n e s or by a d d i n g Fe ions to the m e m b r a n e suspensions. The k i n e t i c s of LPO was m o n i t o r e d by c h e m i l u m i n e s c e n c e a c c o m p a n y i n g the process and by accumu l a t i o n of d i f f e r e n t LPO products. Artificial p l a n a r p h o s p h o l i p i d m e m b r a n e s (BLM), liposomes, i s o l a t e d mitochondria, red cell m e m b r a n e s have been studied. Three types of early changes were o b s e r v e d in the membranes: (i) O x i d a t i o n of SH-groups f o l l o w e d by increase of c a t i o n i c permeability; (2) A p p e @ r a n c e of se~ctive permeability to H-(OH-) and Ca-- ions; (3) D e c r e a s e in m e m b r a n e electric stability, i.e. lowering the b r e a k d o w n potential. The latter event is c o n s i d e r e d as a m a i n m e c h a n i s m by w h i c h the b a r r i e r function of b i o l o g i c a l m e m b r a n e s is i m p a i r e d in disease, in particular, under the action of free radicals on cellular stuctures. On the other hand, LPO modifies the p h y s i c a l p r o p e r t i e s of the m e m b r a n e lipid b i l a y e r , i n c r e a s i n g micr o v i s c o s i t y and n e g a t i v e surface charge density.
18.2
Amplified genes may be responsible for certain types of drug resistance and may be important in carcinogenesis. Furthermore, amplification may be coupled to chromosomal deletions, translocations, and other abnormalities. Since gene amplification can be induced in mammalian cells by a variety of agents,including drugs,carcinogens, and radiation (U.V. or X-ray), we queried whether reactive oxygen species might be able to induce this process as well. Based on methods of Lavi using carcinogens (PNAS 78:6144, Mol. Cell. Biol. 6:1958), we exposed cells to varying concentrations of H202 and subsequently prepared both chromosomal and low molecular weight extrachromosomal DNA, since extrachromosomal elements are found in amplification. In SV40 transformed Chinese hamster embryo cells (CO60) we observed consistent amplification of SV40 viral sequences in extrachromosomal DNA after exposure, and sometimes observed amplification of these sequences in the chromosomal DNA. In C3H mouse 10TI/2 cells we observed consistent extrachromo-
somal amplification of the dihydrofolate reductase gene. Conclusion: H202 can cause amplification of endogenous and viral gene sequences in mammalian cells; this may be yet another potential mechanism by which reactive oxygen species contribute to cancer development.
Oncogenes are involved in processes leading to excessive, or inappropriate, cell proliferation. Other genes however act in normal cells to suppress proliferation. We find t h a t serum contains factors that negatively regulate the rate of intracellular O~ formation in normal and malignant cells. Such O~ generatlon is relevant to tumour cell prollferat~on and experiments w i t h DDC, paraquat and MTT indicate that low levels of endogenous 0~- (or H~O 2) stimulate growth, particularly of c e 6 1 s 'transformed' to malignancy with~ specific oncogenes. Cell proliferation can nevertheless be suppressed by higher levels of H~02, or by withdrawal of serum factorS, oWhic5 causes greatly enhanced intracellular 02 generation. Notable outcomes of growth suppression by these means are increased cellular lipid peroxidation and progressive induction of 'growth arrest genes'. One of these encodes a RNase, possibly responsible for the reduction of ribsosomes and the depressed level of protein synthesis in growth arrested cells. Use of scavengers indicates that radicals may be involved in mechanisms that 'trigger' this growth arrest gene. e -
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18.4
18.1
18.3
167
CANCERAND OXIDATIVE STRESS Peter Cerutti, Alexander Peskin, Girish Shah and Paul ~ s t a d , Department of Carcinogenesis, ISREC-1066 Epalinges, Switzerland. Growth promotion by oxidants is observed with fibroblasts as well as epidermal cells. I t is expected to play a role in inflammation, fibrosis and tumorigenesis. Indeed, oxidants trigger (patho)physiological reactions which resemble those induced by growth- and differentiation factors. They cause S6-phosphorylation, activation and translocation of protein kinase C, Ca2+- dependent DNA breakage and transcriptional activation of the growth competence genes c-fos and c-myc. For the induction of c-fos message protein phosphorylation and poly ADP-ribosylation are required. In fact FOS-protein which acts as a transcriptional regulator is poly ADP-ribosylated in response to oxidants. Deletion analysis of the 5'-regulatory sequences of the c-fos gene indicates that the dyad symmetry axis element and the cyclic AMP responsive element serve as enhancers in c-fos induction by oxidants. The c e l l u l a r antioxidant defences are bound to affect the consequences of oxidant exposure. Overproducers of Cu,Zn-SOD are sensitized to the toxic effects of extracellular O~ plus H202 while overproducers of catalase (CAT) are protected. Both SOD and CAT attenuate the induction of c-fos. Overproducers of both SOD plus CAT resemble wild-type c e l l s . It is concluded that a fine balance of the multiple components of the antioxidant defence determines the biological effects of oxidative stress.
HYDROGEN PEROXIDE CAUSES AMPLIFICATION OF VIRAL AND CELLULAR GENES. S.A. Weitzman, P.W. Turk, K. Rundell, Northwestern University Cancer Center, Chicago, IL 60611, U.S.A.
MOLECULAR MECHANISMS OF DAMAGE TO E I O L O G I C A L M E M B R A N E S BY FREE R A D I C A L S Y u r y A. V l a d i m i r o v 2nd M o s c o w M e d i c a l Institute, Moscow, 117513, USSR
INTRACELLULAR ACTIVE OXYGEN SPECIES IN THE PROMOTION AND SUPPRESSION OF TUMOUR CELL GROWTH Roy H. Burdon, Vera Gill & Catherine Rice-Evans* University of Strathclyde, Todd Centre, Glasgow G4 ONR, U.K. *Royal Free Hospital School of Medicine, London NW3 2PF, U.K.
Free radical reactions of lipid peroxidation (LPO) were i n i t i a t e d by UV-irr a d i a t i o n of a r t i f i c i a l a ~ b i o l o g i c a l m e m b r a n e s or by a d d i n g Fe ions to the m e m b r a n e suspensions. The k i n e t i c s of LPO was m o n i t o r e d by c h e m i l u m i n e s c e n c e a c c o m p a n y i n g the process and by accumu l a t i o n of d i f f e r e n t LPO products. Artificial p l a n a r p h o s p h o l i p i d m e m b r a n e s (BLM), liposomes, i s o l a t e d mitochondria, red cell m e m b r a n e s have been studied. Three types of early changes were o b s e r v e d in the membranes: (i) O x i d a t i o n of SH-groups f o l l o w e d by increase of c a t i o n i c permeability; (2) A p p e @ r a n c e of se~ctive permeability to H-(OH-) and Ca-- ions; (3) D e c r e a s e in m e m b r a n e electric stability, i.e. lowering the b r e a k d o w n potential. The latter event is c o n s i d e r e d as a m a i n m e c h a n i s m by w h i c h the b a r r i e r function of b i o l o g i c a l m e m b r a n e s is i m p a i r e d in disease, in particular, under the action of free radicals on cellular stuctures. On the other hand, LPO modifies the p h y s i c a l p r o p e r t i e s of the m e m b r a n e lipid b i l a y e r , i n c r e a s i n g micr o v i s c o s i t y and n e g a t i v e surface charge density.
18.2
Amplified genes may be responsible for certain types of drug resistance and may be important in carcinogenesis. Furthermore, amplification may be coupled to chromosomal deletions, translocations, and other abnormalities. Since gene amplification can be induced in mammalian cells by a variety of agents,including drugs,carcinogens, and radiation (U.V. or X-ray), we queried whether reactive oxygen species might be able to induce this process as well. Based on methods of Lavi using carcinogens (PNAS 78:6144, Mol. Cell. Biol. 6:1958), we exposed cells to varying concentrations of H202 and subsequently prepared both chromosomal and low molecular weight extrachromosomal DNA, since extrachromosomal elements are found in amplification. In SV40 transformed Chinese hamster embryo cells (CO60) we observed consistent amplification of SV40 viral sequences in extrachromosomal DNA after exposure, and sometimes observed amplification of these sequences in the chromosomal DNA. In C3H mouse 10TI/2 cells we observed consistent extrachromo-
somal amplification of the dihydrofolate reductase gene. Conclusion: H202 can cause amplification of endogenous and viral gene sequences in mammalian cells; this may be yet another potential mechanism by which reactive oxygen species contribute to cancer development.
Oncogenes are involved in processes leading to excessive, or inappropriate, cell proliferation. Other genes however act in normal cells to suppress proliferation. We find t h a t serum contains factors that negatively regulate the rate of intracellular O~ formation in normal and malignant cells. Such O~ generatlon is relevant to tumour cell prollferat~on and experiments w i t h DDC, paraquat and MTT indicate that low levels of endogenous 0~- (or H~O 2) stimulate growth, particularly of c e 6 1 s 'transformed' to malignancy with~ specific oncogenes. Cell proliferation can nevertheless be suppressed by higher levels of H~02, or by withdrawal of serum factorS, oWhic5 causes greatly enhanced intracellular 02 generation. Notable outcomes of growth suppression by these means are increased cellular lipid peroxidation and progressive induction of 'growth arrest genes'. One of these encodes a RNase, possibly responsible for the reduction of ribsosomes and the depressed level of protein synthesis in growth arrested cells. Use of scavengers indicates that radicals may be involved in mechanisms that 'trigger' this growth arrest gene. e -
•
L
.
.
.
18.4