FEMS MicrobiologyLetters 72 (]990) 159-162 Publishedby Elsevier
159
FEMSLE04191
Do heat shock proteins provide protection against freezing? Yasuhiko Komatsu, Sunil C. Kaul, Hitoshi lwahashi and Kaoru Obuchi Fermemution Research Institute. Tsukuba~ Ibarakt. Japan
Received 19 March 1990 Revisionreceived22 June 1990 Accepted 25 June 1990 Key words: Heat shock; Freezing protection; Hydrophohic hydration
1. SUMMARY Yeast cells were frozen by plunging directly into liquid nitrogen (LN2) after exposure at 43°C. Both the cells frozen without prior exposure to heat shock and those treated with cycloheximide showed almost 100% loss of viability during freezing and thawing, Heat exposure prior to freezing and thawing significantly increased the cell viability. This increase in cell viability was associated with the induction of heat shock protein synthesis, which was detected by gel eleetrophoresis. This protein may act by stabilizing the macromolecules and by increasing the hydrophobic interactions.
2. INTRODUCTION An interesting aspect of cellular response to hyperthermia is the phenomenon of thermotolerance [1-31. Cells exposed to a non-lethal heat shock acquire a transient resistance to subsequent heat challenge. The inechanism of thermotolerance is not yet known, However, this transient heat resistance coincides with the synthesis of
Komatsu. Fermentation Research Institute, 1-1-3. Tsukuba. Ibaraki 305.Japan. Correspondence to: ¥.
0378-t097/90/$03,50
!
several new proteins, termed heat shock proteins (HSPs) [4-7]. Of the several HSPs synthesized, HSP 70 is highly conserved during evolution [8]. In yeast cells, a temperature shift to a supraopritual level induces the transient synthesis of such proteins [9 13]. Several studies have shown that cryoprotectant acts as a proleetive agent during heat treatment. Glycerol increases the survival of cultured mammalian cells during hyperthermia [14-18]. Protection of cells from heat by glycerol is thought to result from its ability to stabilize cellular macromolecules [19-21]. Cryobiological and biochemical studies have also shown that glycerol can stabilize cell membranes and protein structure. One of the characteristics of the protective agents is the presence of hydrogen-bonding groups (OH, N H a, etc.) and often hydrogen binds strongly with water. There is evidence that this ability to protect cells against freeze damage is correlated with, and may, therefore, be functionally related to this hydrogen binding. A protective agent could prevent injury from ice, either by preventing intracelhilar freezing or by rendering the ice crystals harmless. The nucleation and growth of ice crystals is strongly hindered in solutions of glycerol [22]. In considering the use of glycerol as cryoprotectant and thermoprotectant, the possibility occurs that the induction t,,f HSPs and subsequent
1990Federationof European MicrobiologicalSocieties
160 acquisition of thermotolerance may be involved in the protection of the cells. This paper reports that in yeast cells the induction of HSP synthesis by preincubation at heat shock temperature confers protection against a subsequent cryoinjury by liquid nitrogen. 3. M A T E R I A L S A N D M E T H O D S The yeast Saccharomyces cerevisiae Hansen IFO-0224, which grows exponentially at 3 0 ° C , was used for this study. Selection of the heat shock intensity was such that at least 90% of the heat treated e l l s recovered. The criterion for recovery was the ability of cells to establish colonies when transfered back to 30 ° C and plated in agar medium. Cells were shocked for 10 to 180 min at preselected temperatures between 37 to 45 ° C by immersion in a water bath, and then being checked for the ability to form colonies. Mean values were calculated from two carefully operated runs under the same conditions. Thus, exponentially growing cells (100 ml) were divided into 2 parts. One part of the culture was resuspended in Y M medium (polypeptone, 1%; glucose, 1%; yeast extract, 0.6%; malt extract, 0.6%; p H 5.6) in 4 test tubes (each containing 10 ml) at about 5 x 10 ~ cells/ml. Cycloheximide, an eukaryotic protein synthesis inhibitor was added to one tube at a final concentration of 100 # g / m l . T h e tubes were heat treated for 10 to 60 min. T h e cells were then washed three times with distilled water, and finally suspended in 0.5 ml of the distilled water, then put in N u n k test tubes ( A / S N u n k Co,, Kamstrup DK-4000, Roskilde, Denmark). They were dropped directly into liquid nitrogen and stored overnight, then quickly thawed at 3 0 ° C in a water bath, The cells were then checked for viability. Adequately diluted cell suspensions were plated out on a Y M agar medium and incubated at 3 0 ° C for 1 day until colony counting became possible. Cell platings were carried out for 3 dilution series, each (10"~-10~/ml) in triplicate, Similarly, the second part of cultured cells was frozen directly in LN, without heat treatment overnight and then tested for viability, In order to demonstrate proteins newly formed in response to heat shock, gel electrophoresis [23]
was employed. Thus. 3 ml of cell suspension was removed from the tube at 4 3 ° C after 30 rain heat treatment, collected and washed thoroughly by centrifugation, The cells were frozen in a mortar with L N > then grounded into a fine powder with a pestle under air. After adding 1 ml of distilled water to the paste, the debris was removed by eentrifugation and 15/xl of supernatant were used for electrophoresis together with the controi cells which had not been heat shocked. In both supernatants, the total amount of proteins in 15 ' ! was determined as about 100 # g by the Lowry ~ e t h o d [241.
4. R E S U L T S A N D D I S C U S S I O N The effect of LN., treatment on the viability of exponentially growing cells is shown in Table 1.
Table I Effect of ben! shock on cell viability alter LN.-treatment The yeast cells were gn~'n in YM medium. Exponentially growing cells at 30°C ~ere collected and ix.suspended in a fresh YM medium (10 rail at a cell eonL~ntralion of abo~t 5 × ]0~/m] and heat treated for 10 to 60 nun at 43° C. The cells were heat treated for 30 rain. supplemented wilh cy¢loheximide (tgO btg/ml) and used for cyelohcaimide control and subsequent LN:-treatraent. Cells were then ~ashed three times vcilh distilled water and finally suspended in 0.5 ml of distilled water, then put in Hunk test tube';. The tubes were dropped directly into LN, and stored overnight, then quickly thawed at 30°C in a water bath. The cells were then checked for viability. Adequately diluted ,:ell suspensions v,'~r¢ plaled out on YM agar mediun'l and incubaled at 3O°C for one day until colony t~ounting was possible. The cell plating were done at 3 dilution series, each (10"-104/ml) in Lripheat¢. and mean va|ues x~.er¢calculated. Trealmem Viabilaies (~) Liquid nilrogen Heal shc~:k Cyclohexiraide (Mean-+S.D.) (100 ~g/ml) -
+ + + -
-
+(30 rain) {10,nin} + 30rain 60 rain +130 rain) + +(30rain) +
100
:~2
96 4-2 3_+0.5 7 ±0.5 6 +0.5 0.6_+0.2 0,3+_0.2 97 _+2
161
1
2
3
4
Fig. 1. SDS-PAGE of HSP 70 and 90. Coomassieblue Rst0inod SDS-PAGEshowingproteins produced by the Yeast Saceharom)~es cereclsiae Hanson [FO-0224 as a function of heat stress at 43~C for 30 rain Lane l is for the protein standards Lane2 is fromcontrolcellswhichdid not receivea heat shock. Lane 3 is from43°C-treated cells. Lane 4 is from 43°C-treated cellswith cycloheximidcIU]Opg/ml)~ Apparent ~,1~of HSP 90 (singlearrow) and HSP 70 {doublearrow) were determined by a comparisonwith the protein standards having a knownmolecularmass.
The viability of the cells, frozen in LN, only or supplemented with cyclohe.dLnide, were found to be less than 0.6~£. However, wuen the cells were heat shocked for 10 to 60 rain and then frozen in LN 2, 3 to 7% remained viable. The 30 rain treatmeal was the most effective, and this was accompmiied by the production of an appreciable amount of HSP 90 group of protein (Fig. 1, single arrow) and the increase in amount of HSP 70 protein (Fig. 1, double arrow). However, in the control cells or cycloheximide treated cells, no
such changes were found (lanes 2, 4). Consequently, this pre-incubation at 43°C appears to protect the cells against the lethal effects of LN_~. To investigate this phenomenon further, cells were heat shocked at different temperatures, frozen in LN 2 and their viability checked. It was found that the most suitable temperature for the induction of HSPs was also best suited for maximum protection against damage in LN 2, as mentioned above. High survival was observed after exposure of cells to heat shock temperature of 43°C for 30 rain, but the survival decreased rapidly after longer time and at higher temperature (data not shown). During heat shock, proteins become partially denatured, exposing the hydrophnbic tx'glons, which then interacted to form insoluble aggregates. By binding tightly to hydrophobic surfaces, HSP 70 limits such interactions and promotes disaggregations [25]. In separate studies, it has been proposed that heat-labile proteins may he stabilized non-specifically from thermal inactivation if heated in the presence of other heat stable proteins [26]. It has been further proposed that the function of HSPs, which are produced in such large amounts following heat shock, may nonspecifically protect other proteins. HSP 90 in calf uterus and rat liver have been shown to possess a significant hydrophobic region on the surface [27]. The mechanisms of thermotolerance are still relatively unknown. Glycerol protection against heat damage has been ascribed to its ability to increase interaction between solvent and solute macromoleculcs, such as globular proteins [28,29]. The similarities in the characteristics of glycerol protection and thermotoierance are consistent with the possibility that heat-induced thermotoleranee is mediated by an intraeelhilar protector that is capable of strengthening hydrophobic interactions [16l. However, such protector molecules can be expressed and cells require a longer time for the development of thermotoleranee than for heat protection by glycerol, which is limited only by glycerol diffusion into ceils. Both naturally occurring intracellular protectors and glycerol would then prevent cell death by stabilizing a wide spectrum of cellular macromolecules against thermal denaturation [19]. Direct support for this hypothesis is not yet available, although the larger polyols
162 a p p e a r to possess the postulated properties of a physiological and intracellular protector. T h e a u t h o r s propose that w h e n cells are frozen in L N 2 after exposure to heat shock treatment, the h i g h level of viability m a y b e associated w i t h m a c r o m o l e c u l a r stabilization a n d an increase in h y d r o p h o b i c h y d r a t i o n b y the newly synthesized H S P s . F u r t h e r studies are currently u n d e r way to o b t a i n evidence for this hypothesis.
ACKNOWLEDGEMENT S.C.K. wishes to express his gratitude to the Science a n d T e c h n o l o g y Agency, J a p a n for a Post-doctoral Fellowship.
REFERENCES [1] Gemer, E.W. and Schneider. M.J, 11975) Nature 256, 500-502. [2] LL G.C. and Mivechi, N.F. (19861 in Hyperthermia in Cancer Treatment (Anghileri, L.J. and Robert, J., eds.), pp. 59-78, CRC Press, Boca Raton, FL. [3] Carper, S.W., Daffy, J,J. and Gerner, E.W, (1987) Cancer Res. 47, 5249-5255. 141 LL G.C. and Werb, A. (19821 proc. Natl. Acad. Sci, U.S.A. 79, 3218-3222. 15] Li. G,C., Peterson, N.S. and Mitchell, H.K. (19821 Inl, J. Radiat. Oncol. BioL Phys. 8, 63-67. 16] Landry, J., Chretien, P. Bernier, D., Nicole, L,M,, Marceau, N. and Tansuay, R,M. (19821 Int. J. gad[at, Oncol. Biol. Phys. 8, 59-62. [7] Landry, J. and Chretien, P, (19831 Can, J. Biochem. Cell Biol, 61,428-437. [8] Lindquist, S, (19861 Anne. Rev, Bie~hem, 55,1151-1191,
I9] McAlistcr. L. and FinkelsTein. D.B. (1980) Bicchem. Biephys. ges. Commun. 93, 819-824. [10] Miller, M.J, Xuong, N-H. and Geiduschek. E.P. (19791 Proc. Natl. Aead. Sci. U.S.A. 76. 5222-5225. [I]] McAlister. L.. Stausherg, S.. Kulaga, A,, and Fink¢lstein. D.B. (19791 Current Genet. 1.63-74. [121 Plesset, J., Palm, C. and McLaughlin, C.S, (1982} BiGchem. Biophys. Res. Commun. 108,1340-1345. [13] Miller, M.J.. Xuong. N.-H. and Geiduschek. E.P. (19821J. Bactcriol. 131. 311-327. [141 Mclver. DJ.L., Schnrch. S. and Sridher. R. (19801 Physiol. Chem. Phys. ]2, 369-372. [15] Fischer, G.A.. Li, G.C. and Hahn. G.H. (19821 Radiat. Res. 92, 530-540. [16] Herl¢. K.J. and Wafters, R.L, (1982) Cancer Res. 42, 21"1-21"/6. [171 He~,le K.J.. Peck, J,W.. and Higashikubo. R, (19931 Cancer Res. 43.1624-1627. [18] Lin, E-S.. Hefter, K. and Ho, K.-C (1984l Cancer Res. 44. 5775-5784. [19] Henle. KJ. (19811 Radiat. Res. 88, 392-402. [201 Lin, P,-S.. Kwock, L. and Hefter, K. 119811J. Cell Physiol. 108, 439-443. [21l Massicone-Nolan, p., Glofeheski. D.J., Kruuv, J,. and Lepoek. J.R. (1981) Radiat, Res. 87, 284-299. [22l Mazur, p. (]966) in C~obiology. plx 214-310, Academic Press. London. [23l Gegenbeimer, p, (19901 in Guide to Protein Purification. Methods in Enzymology, voL 182 (De:utst:her, M.P., ed.), Academic Press. [24] Lowry, O.H., Rosbrough, N,J,, Farr. A.L. and Randall. R.J. O9511 J, Biol. Chem, 193, 265-2"/5. [25] Pelham, H.R,B. (19861 Cell 46. 959-96L [26] Minton, K,W., Karmin. p,, Hahn, G.M and Minton, A.P. (19821. [27] lwasaki, M., Saito. H,. Yamamo|o, M,, Korach, K.S.. Hirogome, T., and Segano, H. (19891 Biochim. Biophys, Acta 992, 1-8, [281 Back, J.F., Oakenfull, D. and Smith, M,B, (19791 Biechemistry 18. 5191-5196, [29] Gerlsma. S.Y, and Stuur. E.R. (19721 Int. J. Rept. Protein Res. 4, 3"/7-383.
159
FEMSLE04191
Do heat shock proteins provide protection against freezing? Yasuhiko Komatsu, Sunil C. Kaul, Hitoshi lwahashi and Kaoru Obuchi Fermemution Research Institute. Tsukuba~ Ibarakt. Japan
Received 19 March 1990 Revisionreceived22 June 1990 Accepted 25 June 1990 Key words: Heat shock; Freezing protection; Hydrophohic hydration
1. SUMMARY Yeast cells were frozen by plunging directly into liquid nitrogen (LN2) after exposure at 43°C. Both the cells frozen without prior exposure to heat shock and those treated with cycloheximide showed almost 100% loss of viability during freezing and thawing, Heat exposure prior to freezing and thawing significantly increased the cell viability. This increase in cell viability was associated with the induction of heat shock protein synthesis, which was detected by gel eleetrophoresis. This protein may act by stabilizing the macromolecules and by increasing the hydrophobic interactions.
2. INTRODUCTION An interesting aspect of cellular response to hyperthermia is the phenomenon of thermotolerance [1-31. Cells exposed to a non-lethal heat shock acquire a transient resistance to subsequent heat challenge. The inechanism of thermotolerance is not yet known, However, this transient heat resistance coincides with the synthesis of
Komatsu. Fermentation Research Institute, 1-1-3. Tsukuba. Ibaraki 305.Japan. Correspondence to: ¥.
0378-t097/90/$03,50
!
several new proteins, termed heat shock proteins (HSPs) [4-7]. Of the several HSPs synthesized, HSP 70 is highly conserved during evolution [8]. In yeast cells, a temperature shift to a supraopritual level induces the transient synthesis of such proteins [9 13]. Several studies have shown that cryoprotectant acts as a proleetive agent during heat treatment. Glycerol increases the survival of cultured mammalian cells during hyperthermia [14-18]. Protection of cells from heat by glycerol is thought to result from its ability to stabilize cellular macromolecules [19-21]. Cryobiological and biochemical studies have also shown that glycerol can stabilize cell membranes and protein structure. One of the characteristics of the protective agents is the presence of hydrogen-bonding groups (OH, N H a, etc.) and often hydrogen binds strongly with water. There is evidence that this ability to protect cells against freeze damage is correlated with, and may, therefore, be functionally related to this hydrogen binding. A protective agent could prevent injury from ice, either by preventing intracelhilar freezing or by rendering the ice crystals harmless. The nucleation and growth of ice crystals is strongly hindered in solutions of glycerol [22]. In considering the use of glycerol as cryoprotectant and thermoprotectant, the possibility occurs that the induction t,,f HSPs and subsequent
1990Federationof European MicrobiologicalSocieties
160 acquisition of thermotolerance may be involved in the protection of the cells. This paper reports that in yeast cells the induction of HSP synthesis by preincubation at heat shock temperature confers protection against a subsequent cryoinjury by liquid nitrogen. 3. M A T E R I A L S A N D M E T H O D S The yeast Saccharomyces cerevisiae Hansen IFO-0224, which grows exponentially at 3 0 ° C , was used for this study. Selection of the heat shock intensity was such that at least 90% of the heat treated e l l s recovered. The criterion for recovery was the ability of cells to establish colonies when transfered back to 30 ° C and plated in agar medium. Cells were shocked for 10 to 180 min at preselected temperatures between 37 to 45 ° C by immersion in a water bath, and then being checked for the ability to form colonies. Mean values were calculated from two carefully operated runs under the same conditions. Thus, exponentially growing cells (100 ml) were divided into 2 parts. One part of the culture was resuspended in Y M medium (polypeptone, 1%; glucose, 1%; yeast extract, 0.6%; malt extract, 0.6%; p H 5.6) in 4 test tubes (each containing 10 ml) at about 5 x 10 ~ cells/ml. Cycloheximide, an eukaryotic protein synthesis inhibitor was added to one tube at a final concentration of 100 # g / m l . T h e tubes were heat treated for 10 to 60 min. T h e cells were then washed three times with distilled water, and finally suspended in 0.5 ml of the distilled water, then put in N u n k test tubes ( A / S N u n k Co,, Kamstrup DK-4000, Roskilde, Denmark). They were dropped directly into liquid nitrogen and stored overnight, then quickly thawed at 3 0 ° C in a water bath, The cells were then checked for viability. Adequately diluted cell suspensions were plated out on a Y M agar medium and incubated at 3 0 ° C for 1 day until colony counting became possible. Cell platings were carried out for 3 dilution series, each (10"~-10~/ml) in triplicate, Similarly, the second part of cultured cells was frozen directly in LN, without heat treatment overnight and then tested for viability, In order to demonstrate proteins newly formed in response to heat shock, gel electrophoresis [23]
was employed. Thus. 3 ml of cell suspension was removed from the tube at 4 3 ° C after 30 rain heat treatment, collected and washed thoroughly by centrifugation, The cells were frozen in a mortar with L N > then grounded into a fine powder with a pestle under air. After adding 1 ml of distilled water to the paste, the debris was removed by eentrifugation and 15/xl of supernatant were used for electrophoresis together with the controi cells which had not been heat shocked. In both supernatants, the total amount of proteins in 15 ' ! was determined as about 100 # g by the Lowry ~ e t h o d [241.
4. R E S U L T S A N D D I S C U S S I O N The effect of LN., treatment on the viability of exponentially growing cells is shown in Table 1.
Table I Effect of ben! shock on cell viability alter LN.-treatment The yeast cells were gn~'n in YM medium. Exponentially growing cells at 30°C ~ere collected and ix.suspended in a fresh YM medium (10 rail at a cell eonL~ntralion of abo~t 5 × ]0~/m] and heat treated for 10 to 60 nun at 43° C. The cells were heat treated for 30 rain. supplemented wilh cy¢loheximide (tgO btg/ml) and used for cyelohcaimide control and subsequent LN:-treatraent. Cells were then ~ashed three times vcilh distilled water and finally suspended in 0.5 ml of distilled water, then put in Hunk test tube';. The tubes were dropped directly into LN, and stored overnight, then quickly thawed at 30°C in a water bath. The cells were then checked for viability. Adequately diluted ,:ell suspensions v,'~r¢ plaled out on YM agar mediun'l and incubaled at 3O°C for one day until colony t~ounting was possible. The cell plating were done at 3 dilution series, each (10"-104/ml) in Lripheat¢. and mean va|ues x~.er¢calculated. Trealmem Viabilaies (~) Liquid nilrogen Heal shc~:k Cyclohexiraide (Mean-+S.D.) (100 ~g/ml) -
+ + + -
-
+(30 rain) {10,nin} + 30rain 60 rain +130 rain) + +(30rain) +
100
:~2
96 4-2 3_+0.5 7 ±0.5 6 +0.5 0.6_+0.2 0,3+_0.2 97 _+2
161
1
2
3
4
Fig. 1. SDS-PAGE of HSP 70 and 90. Coomassieblue Rst0inod SDS-PAGEshowingproteins produced by the Yeast Saceharom)~es cereclsiae Hanson [FO-0224 as a function of heat stress at 43~C for 30 rain Lane l is for the protein standards Lane2 is fromcontrolcellswhichdid not receivea heat shock. Lane 3 is from43°C-treated cells. Lane 4 is from 43°C-treated cellswith cycloheximidcIU]Opg/ml)~ Apparent ~,1~of HSP 90 (singlearrow) and HSP 70 {doublearrow) were determined by a comparisonwith the protein standards having a knownmolecularmass.
The viability of the cells, frozen in LN, only or supplemented with cyclohe.dLnide, were found to be less than 0.6~£. However, wuen the cells were heat shocked for 10 to 60 rain and then frozen in LN 2, 3 to 7% remained viable. The 30 rain treatmeal was the most effective, and this was accompmiied by the production of an appreciable amount of HSP 90 group of protein (Fig. 1, single arrow) and the increase in amount of HSP 70 protein (Fig. 1, double arrow). However, in the control cells or cycloheximide treated cells, no
such changes were found (lanes 2, 4). Consequently, this pre-incubation at 43°C appears to protect the cells against the lethal effects of LN_~. To investigate this phenomenon further, cells were heat shocked at different temperatures, frozen in LN 2 and their viability checked. It was found that the most suitable temperature for the induction of HSPs was also best suited for maximum protection against damage in LN 2, as mentioned above. High survival was observed after exposure of cells to heat shock temperature of 43°C for 30 rain, but the survival decreased rapidly after longer time and at higher temperature (data not shown). During heat shock, proteins become partially denatured, exposing the hydrophnbic tx'glons, which then interacted to form insoluble aggregates. By binding tightly to hydrophobic surfaces, HSP 70 limits such interactions and promotes disaggregations [25]. In separate studies, it has been proposed that heat-labile proteins may he stabilized non-specifically from thermal inactivation if heated in the presence of other heat stable proteins [26]. It has been further proposed that the function of HSPs, which are produced in such large amounts following heat shock, may nonspecifically protect other proteins. HSP 90 in calf uterus and rat liver have been shown to possess a significant hydrophobic region on the surface [27]. The mechanisms of thermotolerance are still relatively unknown. Glycerol protection against heat damage has been ascribed to its ability to increase interaction between solvent and solute macromoleculcs, such as globular proteins [28,29]. The similarities in the characteristics of glycerol protection and thermotoierance are consistent with the possibility that heat-induced thermotoleranee is mediated by an intraeelhilar protector that is capable of strengthening hydrophobic interactions [16l. However, such protector molecules can be expressed and cells require a longer time for the development of thermotoleranee than for heat protection by glycerol, which is limited only by glycerol diffusion into ceils. Both naturally occurring intracellular protectors and glycerol would then prevent cell death by stabilizing a wide spectrum of cellular macromolecules against thermal denaturation [19]. Direct support for this hypothesis is not yet available, although the larger polyols
162 a p p e a r to possess the postulated properties of a physiological and intracellular protector. T h e a u t h o r s propose that w h e n cells are frozen in L N 2 after exposure to heat shock treatment, the h i g h level of viability m a y b e associated w i t h m a c r o m o l e c u l a r stabilization a n d an increase in h y d r o p h o b i c h y d r a t i o n b y the newly synthesized H S P s . F u r t h e r studies are currently u n d e r way to o b t a i n evidence for this hypothesis.
ACKNOWLEDGEMENT S.C.K. wishes to express his gratitude to the Science a n d T e c h n o l o g y Agency, J a p a n for a Post-doctoral Fellowship.
REFERENCES [1] Gemer, E.W. and Schneider. M.J, 11975) Nature 256, 500-502. [2] LL G.C. and Mivechi, N.F. (19861 in Hyperthermia in Cancer Treatment (Anghileri, L.J. and Robert, J., eds.), pp. 59-78, CRC Press, Boca Raton, FL. [3] Carper, S.W., Daffy, J,J. and Gerner, E.W, (1987) Cancer Res. 47, 5249-5255. 141 LL G.C. and Werb, A. (19821 proc. Natl. Acad. Sci, U.S.A. 79, 3218-3222. 15] Li. G,C., Peterson, N.S. and Mitchell, H.K. (19821 Inl, J. Radiat. Oncol. BioL Phys. 8, 63-67. 16] Landry, J., Chretien, P. Bernier, D., Nicole, L,M,, Marceau, N. and Tansuay, R,M. (19821 Int. J. gad[at, Oncol. Biol. Phys. 8, 59-62. [7] Landry, J. and Chretien, P, (19831 Can, J. Biochem. Cell Biol, 61,428-437. [8] Lindquist, S, (19861 Anne. Rev, Bie~hem, 55,1151-1191,
I9] McAlistcr. L. and FinkelsTein. D.B. (1980) Bicchem. Biephys. ges. Commun. 93, 819-824. [10] Miller, M.J, Xuong, N-H. and Geiduschek. E.P. (19791 Proc. Natl. Aead. Sci. U.S.A. 76. 5222-5225. [I]] McAlister. L.. Stausherg, S.. Kulaga, A,, and Fink¢lstein. D.B. (19791 Current Genet. 1.63-74. [121 Plesset, J., Palm, C. and McLaughlin, C.S, (1982} BiGchem. Biophys. Res. Commun. 108,1340-1345. [13] Miller, M.J.. Xuong. N.-H. and Geiduschek. E.P. (19821J. Bactcriol. 131. 311-327. [141 Mclver. DJ.L., Schnrch. S. and Sridher. R. (19801 Physiol. Chem. Phys. ]2, 369-372. [15] Fischer, G.A.. Li, G.C. and Hahn. G.H. (19821 Radiat. Res. 92, 530-540. [16] Herl¢. K.J. and Wafters, R.L, (1982) Cancer Res. 42, 21"1-21"/6. [171 He~,le K.J.. Peck, J,W.. and Higashikubo. R, (19931 Cancer Res. 43.1624-1627. [18] Lin, E-S.. Hefter, K. and Ho, K.-C (1984l Cancer Res. 44. 5775-5784. [19] Henle. KJ. (19811 Radiat. Res. 88, 392-402. [201 Lin, P,-S.. Kwock, L. and Hefter, K. 119811J. Cell Physiol. 108, 439-443. [21l Massicone-Nolan, p., Glofeheski. D.J., Kruuv, J,. and Lepoek. J.R. (1981) Radiat, Res. 87, 284-299. [22l Mazur, p. (]966) in C~obiology. plx 214-310, Academic Press. London. [23l Gegenbeimer, p, (19901 in Guide to Protein Purification. Methods in Enzymology, voL 182 (De:utst:her, M.P., ed.), Academic Press. [24] Lowry, O.H., Rosbrough, N,J,, Farr. A.L. and Randall. R.J. O9511 J, Biol. Chem, 193, 265-2"/5. [25] Pelham, H.R,B. (19861 Cell 46. 959-96L [26] Minton, K,W., Karmin. p,, Hahn, G.M and Minton, A.P. (19821. [27] lwasaki, M., Saito. H,. Yamamo|o, M,, Korach, K.S.. Hirogome, T., and Segano, H. (19891 Biochim. Biophys, Acta 992, 1-8, [281 Back, J.F., Oakenfull, D. and Smith, M,B, (19791 Biechemistry 18. 5191-5196, [29] Gerlsma. S.Y, and Stuur. E.R. (19721 Int. J. Rept. Protein Res. 4, 3"/7-383.
