Patent Application: US-8337998-A

Abstract:
a first embodiment of a cell culture system has a cell death accelerator comprising one or more cell death inducing substances , including serum albumin , hemoglobin , glycine and glutamic acid . in a second embodiment a cell death inhibitor comprises one or more kinds of cell death inhibiting substances which include mercapto group containing amino acids , other mercapto group containing compounds and tryptophan . in a third embodiment a cell death inhibitor comprises an inhibitor of rna or protein synthesis , optionally augmented with a thiol . the system can be applied to selectively induce death of cultured cells , such as neoplastic cell lines , or to inhibit death of other cells , such as neoplastic cell lines or non - neoplastic cells such as brain cells .

Description:
as used herein certain abbreviations and acronyms have the following meanings : ______________________________________act d actinomycin d fbs fetal bovine serum chx cycloheximide gsh glutahione reduced cys cysteine gssg glutathione oxidized cys - cys cystine 2 - me 2 - mercaptoethanol dl - hc dl - homocystine nac n - acetyl - l - cysteine dtn sodium dithionite non non - inhibitor dtnb 5 , 5 &# 39 ; dithiobis ( 2 - pbs phosphate buffered saline nitrobenzoic acid ) pmc puromycin . 2hcl dtt dithiothreitol tga thioglycolic acid eaa essential amino acids mem minimal essential medium emt emetine . 2hcl neaa nonessential amino acids et br ethidium bromide______________________________________ in one experiment , a cell system had a cytotoxic concentration of serum sufficient to induce cell death . a population of living cells was exposed to the serum , and a concentrated and refined molecular species that prevents induction of death of the cells by the serum was included in a culture medium . suitable cells were human fetal lung fibroblasts , human epithelioid carcinoma cells , and mouse melanoma cells . more particularly tig - cells , i . e ., human fetal lung fibroblasts , were seeded into a 96 - well multiplate ( 1 × 10 4 cells / well ) and incubated overnight . thereafter the culture medium was removed to be exchanged with test medium , a culture medium containing fetal bovine serum ( fbs ) and various selected reagents . six days after the exchange of the culture medium , living cells in the culture medium were counted using a dye elution method to obtain the living cell density associated with each reagent . the molecular species used was a thiol or a dithiol homo - dimer thereof , or a mixed disulfide in various concentrations which included the range of between about 1 and 10 mm . the following reagents were used : l - cystine ( l - cyscys ); d - cystine ( d - cyscys ); l - cysteine ( l - cys ); d - cysteine ( d - cys ); reduced glutathione ( gsh ); oxidized glutathione ( gssg ); dl - homocystine ( dl - hc ); n - acetyl - l - cysteine ( nac ); 2 - mercaptoethanol ( 2 - me ); thioglycolic acid ( tg ); dithiothreitol ( dtt ); 5 , 5 &# 39 ;- dithiobis ( 2 - nitro benzoic acid ) ( dtnb ); sodium dithionite ( dtn ); and l - tryptophan ( l - trp ). the concentration of each reagent was set at four levels : 0 . 01 mm , 0 . 1 mm , 1 . 0 mm and 10 mm . a result of the experience is shown in fig1 . in fig1 each reagent is indicated along the abscissa and the corresponding cell density is indicated along the ordinate . the height of each column in the graph indicates the mean of the measured cell densities of six wells , and a bar on the top of each column , if shown , indicates the standard error . according to the result , reagents which had a mercapto group in their chemical structures conduced to inhibition of the cell death . l - cyscys , d - cyscys , l - cys , 2 - me , tg and dtn showed greatly inhibiting effects . they could prevent cell death even at a concentration of 1 mm . it was recognized that mercapto group containing amino acids and other mercapto group containing compounds could inhibit cell death . experiments on cell death inducing substances which exist in serum will now be explained . a cell system was used which had a cytotoxic concentration of serum sufficient to induce cell death . a population of living cells was exposed to the serum , and the culture medium contained a concentrated and refined molecular species that accelerates induction of death of the cells by the serum . the molecular species was one of the group consisting of serum albumin , hemoglobin , glycine and glutamic acid . suitable cells were human fetal lung fibroblasts , human epithelioid carcinoma cells , and mouse melanoma cells . a low molecular weight fraction in fbs serum was freeze - dried , and a solvent then added . the insoluble fraction in was removed by centrifugation to purify the low molecular weight fraction . the soluble fraction thereby obtained was freeze - dried again , and washed in acetonitrile . the resulting freeze - dried substance was dissolved in distilled water to prepare a sample solution having an approximately 20 - fold dilution of the low molecular weight fraction with respect to fbs , supposing that recovery of the low molecular weight fraction therefrom is 100 %. thereafter , the sample solution was filtered through a filter having a 0 . 2μ mesh . this sample solution was added to hank &# 39 ; s solution so as to obtain a test medium containing 10 volume % of above mentioned sample solution , 1 volume % of fbs and 10 volume % of mem . further , each reagent or combination of the reagent and cysteine being tested was added to the test medium at reagent concentrations of 1 mm and 10 mm . tig - cells were seeded into a 24 - well multiplate ( 5 × 10 3 cells / well ) having the test medium and incubated for 48 hours . thereafter living cell density was measured using the dye elution method . according to the experimental results , glycine , glutamic acid and tryptophan showed a cell death inducing effect at 1 mm . phenylalanine , aspartic acid , asparagine and glutamine also showed a cell death inducing effect at 10 mm . on the other side , thyrosin , serine and glycylglycine did not show a cell death inducing effect , but inhibited growth and / or multiplication of cells . it was furthermore proved that hemoglobin had growth and / or multiplication inhibiting effect in the same manner . these and other experiments will now be disclosed in further detail . human fetal lung fibroblasts ( tig - 1 ), human epithelioid carcinoma ( hela ) cells , and mouse melanoma ( b16 ) cells were obtained from the japanese cancer research resources bank . all cells were maintained in eagle &# 39 ; s mem containing 10 % fbs at 37 ° c . cell cultures were examined and photographed with a nikon diaphot phase - contrast inverted microscope . amino acids : l - cysteine or l - cystine ( kanto chemical ), d - cysteine ( sigma ), d - cystine ( wako ), l - tryptophan ( kanto ), d - tryptophan , dl - homocystine ( wako ), glutathione oxidized form ( sigma grade iii ), reduced form ( merck ), dithiothreitol ( wako ), 2 - mercaptoethanol , thioglycolic acid , n - acetyl - l - cysteine , sodium dithionite ( kanto ), 5 , 5 &# 39 ;- dithiobis ( 2 - nitro benzoic acid ) ( kanto ), emetine . 2hcl ( fluka ag . ), cycloheximide , puromycin . 2hcl , actinomycin d , ethidium bromide ( sigma ). fbs was from boehringer mannheim ( lot . 614413 , 562044 , 147013 ). eagle &# 39 ; s mem &# 34 ; nissui &# 34 ; was from nissui pharmaceutical co ., eaa ( essential amino acids ) supplement for mem × 50 , neaa ( nonessential amino acids ) supplement for mem × 100 and vitamin supplement for mem × 100 were from boehringer mannheim , flow laboratories , and dainippon pharmaceutical co ., respectively . fbs was filtered through an ultrafiltration membrane ym2 ( m . w . 1 , 000 ) ( amicon co .). fbs was concentrated tenfold and diafiltered with a tenfold volume of deionized water to remove the low - molecular - weight fraction . the resultant macromolecular fraction of fbs was again concentrated tenfold and was diluted to the original fbs volume with 10 / 9 concentrated eagle &# 39 ; s mem . ph and osmolality were adjusted to 7 . 2 ± 0 . 2 and 290 ± 10 mosmol / kg . h 2 o ), as the low - molecular - weight - fraction depleted fbs . appropriate numbers of cells were seeded into 96 - well culture plates containing eagle &# 39 ; s mem , 10 % fbs ( 100 μl / well ) ( corning ). after overnight incubation at 37 ° c . in 5 % co 2 , the culture medium was removed . wells were washed with calcium and magnesium - free pbs ( cmf - pbs ), and 100 μl of test medium was added . after several days of incubation , cells were harvested by trypsinization and suspended in isotone ii ( coulter electronics ). cell number was measured with a coulter counter zm ( coulter electronics ) and displayed numbers were corrected with standard hemocytes . we previously verified that the numbers displayed on the counter were in agreement with those counted using a hemocytometer . results were expressed as the means (± se ) of six independent measurements . tig - 1 cells were seeded in 96 - well microplates in 100 μl of eagle &# 39 ; s mem , 10 % fbs . after several day &# 39 ; s incubation , medium was removed and the cells were washed with cmf - pbs . cell quantity was determined by the method of hori et al . ( 1988 ) with a minor modification . cells were stained with 50 μl of 0 . 5 % crystal violet per well in ethanol / water ( 1 : 4 ) for 10 min and then washed with water . the dye was eluted with 150 μl of 33 % acetic acid per well , and the absorbance at 600 nm was measured by a microplate reader ( corona mtp - 22 ). results were expressed as the means (± se ) of six independent determinations . we preliminarily confirmed that the absorbance at 600 nm was proportional to the density of tig - 1 cells cultured in mem , 10 % fbs . tig - 1 cells were seeded in 48 - well microplates in 100 μl of eagle &# 39 ; s mem , 10 % fbs . after incubation , cells were harvested with 0 . 1 % trypsin , in cmf - pbs , and 0 . 5 % trypan blue in pbs was added . cells that were not stained were counted with a hemocytometer . results were expressed as the means (± se ) of four independent determinations . tig - 1 cells were seeded into a 24 - well multiplate at 1 / 2 confluency ( about × 10 5 cells / well ) and incubated overnight . the next morning , the culture medium was removed and changed to test medium ( 20 μci l -[ 35 s ] methionine ( amersham ), 20 % cmf - pbs , 80 % fbs ). after appropriate time ( 0 - 16 hr ) of incubation , each culture was washed three times with 0 . 02 % edta cmf - pbs , and the cells were lysed with 1 % sds . macromolecules were precipitated with each 10 μl tca and centrifuged , and the precipitate was washed with 10 % tca . then , it was dissolved in 300 μl of 1 % sds , 100 μl of which was dissolved in 1 ml of liquid scintillation cocktail and counted ( aloka lsc - 700 ). tig - 1 cells were cultured in a 24 - well plate ( 3 × 10 5 cells / well ) in fresh medium for 24 hr . the medium was then changed to the test medium , mem plus 10 % fbs , hank &# 39 ; s balanced solution , or whole fbs supplemented with various reagents . total thiol content was determined as follows . after appropriate hr ( 0 - 16 ) of incubation , cells were washed twice with 0 . 02 % edta , cmf - pbs , and they were lysed with 200 μl / well of 1 % sds , 0 . 02 % edta , 50 mm tris - hcl ( ph . 8 . 2 ). the lysate was dyed with 10 μl of 20 mm dtnb dissolved in methanol . pre - chilled ethanol ( 600 μl ) was added to precipitate the macromolecules , and precipitates were collected by centrifugation at 13 , 000 × g for 20 min . absorbance at 415 nm of the supernatant was measured by a microplate reader ( biorad 3550 ). concentration of thiol was calculated by comparison to a gsh standard . ethanol - soluble thiol was measured as follows . after appropriate hr ( 0 - 16 ) of incubation , cells were washed twice with 0 . 02 % edta , cmf - pbs , and they were lysed with 100 μl / well of 1 % sds , 0 . 02 % edta , 50 mm tris - hcl ( ph . 8 . 2 ). macromolecules were precipitated with 300 μl of pre - chilled ethanol , and precipitates were removed by centrifugation at 13 , 000 × g for 20 min . the supernatant was dyed with 10 μl of 20 mm dtnb dissolved in methanol , and the absorbance at 415 nm of the supernatant was measured by a microplate reader . concentration of thiol was calculated by comparison to a gsh standard . thiol content was expressed as sh mol / living cell number . tig - 1 cells ( 3 . 5 × 10 5 cells / 35 mm dish ) were washed twice with cmf - pbs and lysed in 0 . 8 ml 0 . 6 % te buffer with 1 μg / ml rnase . then , 200 μl of 5 m nacl was added to the solution and stored at 4 ° c . overnight , and macromolecular dna was pelleted by centrifugation at 13 , 000 × g for 30 min . dna in the supernatant was purified by phenol - chloroform extraction , and traces of phenol were removed by extracting twice with chloroform . purified dna was collected by centrifugation at 13 , 000 × g for 20 min with 3 382 m potassium acetate and 70 % ethanol . precipitated dna was dissolved in te buffer , and 1 / 5 to 1 / 3 volume of the solution was electrophoresed in a 2 . 6 % agarose gel . gel was stained by 0 . 5 μg / ml ethidium bromide and photographed in 254 nm ultraviolet . we previously discovered that most types of cultured cells died in concentrations of serum exceeding 60 %. however , low molecular weight fraction - depleted serum whose osmotic pressure was adjusted with eagle &# 39 ; s mem ( uf - fbs ) never showed cytotoxicity . the low molecular weight fraction thus filtered was similar to mem in salt content , but it was deficient in nutrients such as vitamins and amino acids . we therefore enriched fbs with these nutrients equivalent to the amounts present in mem for cell culture . fig3 shows the effect of serum concentration on tig - 1 cell growth , wherein □ indicates unenriched fbs ; ▴ indicates fbs + aa vit ; and ∘ indicates low molecular weight fraction - depleted fbs . in developing the data illustrated in fig3 tig - 1 cells were seeded at a density of 5 × 10 3 cells / well in 96 - well microplates in eagle &# 39 ; s mem containing 10 % fbs . after overnight incubation , medium was replaced with eagle &# 39 ; s mem containing several concentrations of fbs . after 6 days , cells harvested by trypsinization were counted with a coulter counter . as shown in fig3 cells died at a high concentration of unenriched fbs ( whole fbs ), but not in the uf - fbs supplemented with mem . in addition , cell growth was suppressed in enriched fbs as the concentration was increased . nevertheless , the cells remained viable , suggesting mem contained molecules that prevented cell death . among the vitamins and amino acids tested , only the latter showed this rescue effect . one half to fourfold mem equivalent of amino acids rescued tig - 1 cells from serum - induced cell death ( data not shown ). we measured the concentration of each amino acid in fbs . among essential amino acids , l - cysteine or l - cystine and arginine were of less concentration in fbs as compared to those in mem , which were 8 - 15 vs . 24 and 1 - 6 vs . 126 , respectively . to identify the amino acids responsible for the rescue effect , each of the 20 l - amino acids involved in protein synthesis and l - hydroxyproline were tested ( data not shown ). rescue from serum - induced cell death by amino acids is illustrated in fig4 . in developing the data illustrated in fig4 tig - 1 cells were seeded in 96 - well microplates at a density of 5 × 10 3 cells / well in eagle &# 39 ; s mem containing 10 % fbs . after overnight incubation , medium was replaced with fbs containing amino acids . after 6 days , cells were counted with a coulter counter . only two amino acids , l - cysteine or l - cystine and l - tryptophan , showed ability to rescue cells from serum - induced toxicity ( fig3 ) with l - cysteine , cell death was completely prevented at 0 . 1 mm , and cells grew well at 1 mm . with l - cystine , whose maximum solubility is ˜ 0 . 5 mm , protective effect were observed beginning at 0 . 05 mm ( 0 . 1 mm cysteine equivalent ). in contrast , l - tryptophan was effective only at concentrations above 5 mm , and it only partially rescued the cells . concentrations of l - cysteine higher than 5 mm sometimes inhibited cell growth . similar to tig - 1 cells , hela cells and b16 cells were also protected by l - cysteine or cystine and l - tryptophan ( data not shown ). the rate of glutathione synthesis in cultured fibroblasts is known to depend on the cystine content of the medium ( meister and tate , 1976 ), and when glutathione was added to the medium of tig - 1 cells , death was prevented . this also occurred when oxidized glutathione ( gssg ) was tested . there was no evidence of extracellular enzymatic reduction of gssg or its transport into the cell . accordingly , the rescue activity of glutathione may not be due to its ability to conjugate to other molecules via thiol linkages . to further study the roles of cysteine and glutathione , experiments were performed as before using reducing agents , dithiols , l - amino acids , denaturants , and an active oxygen - eliminating enzyme . these reagents are listed below . reducing agents : l - ascorbic acid , l - ascorbic acid phosphate , mg , dl - α - tocopherol , dithiothreitol , 2 - mercaptoethanol , thioglycolic acid , n - acetyl - l - cysteine , sodium dithionite , potassium ferrocyanide , thiourea . dithiols : dl - homocysteine , 5 , 5 &# 39 ;- dithiobis ( 2 - nitro benzoic acid ); l - amino acids : l - tryptophan , l - methionine ; chelates : ethylene diamine tetra - acetic acid and ethylene glycol bis ( 2 - aminoethylether ). denaturants : urea , sodium dodecyl sulfate ; active - oxygen eliminating enzyme : bovine erythrocyte superoxide dismutase ( sod ) ( wako ). fig1 . illustrates rescue from serum - induced cell death by thiols . in developing the data shown in fig1 tig - 1 cells were seeded in 96 - well microplates at a density of 1 × 10 4 cells / well in eagle &# 39 ; s mem containing 10 % fbs . after overnight incubation , medium was removed , and 80 μl of fbs plus 20 μl of pbs containing cysteine ( cys ), cystine ( cys - cys ), glutathione oxidized ( gssg ), glutathione reduced ( gsh ), dithiothreitol ( dtt ), 2 - mercaptoethanol ( 2 - me ), sodium dithionite ( dtn ), thioglycolic acid ( tga ), dl - homocystine ( dl - hc ), n - acetyl - l - cysteine ( nac ), or 5 , 5 &# 39 ;- dithiobis ( 2 - nitrobenzoic acid ) ( dtnb ) were added to each well . all reagents were tested for rescue from cell death at concentrations ranging from 10 μm to 1 mm , in some cases to 10 mm . d - and l - cystine were tested in the range of 1 μm to 100 μm , because of low solubility . after 4 days , cell number was determined by the dye elution method . reagents were tested in concentrations from 1 μm to 1 mm or 1 μm to 10 mm , except in the case of sod , which was tested in the range 10 1 - 10 4 units / ml . only rescue activity - positive data from independent experiments are depicted in fig1 . all reagents bearing sh or cleaved dithiols possessed the rescue activity , and cysteamine and cystamine also were effective in preventing cell death . in contrast , the nonthiol - reducing agents were inactive ( data not shown ). to determine whether death was due to apoptosis or necrosis , various inhibitors of protein synthesis and rna synthesis were tested for the rescue activity , since it is generally accepted that in apoptosis specific protein ( s ) are synthesized . the procedure is explained with reference to fig5 . which illustrates rescue from serum - induced cell death by inhibitors of protein and rna synthesis . the ordinate in fig5 represents the number of viable tig - 1 cells after 24 hr incubation . open columns indicate reagent only . striped columns indicate reagent + 1 mm n - acetyl - l - cysteine ( nac ). non , pbs only ; chx , 1 μg / ml cycloheximide ; pmc , 1 μg / ml puromycin . 2hcl ; emt , 1 μg / ml emetine . 2hcl ; l - trp , 10 mm l - tryptophan ; d - trp , 10 mm d - tryptophan ; act d , 10 μg / ml actinomycin d ; et br , 25 μg / ml ethidium bromide . tig - 1 cells were seeded in 48 - well microplates at a density of 3 × 10 4 cells / well in eagle &# 39 ; s mem containing 10 % fbs . after 3 days incubation , medium was replaced with 200 μl of fbs plus 50 μl of pbs containing each reagent . after 24 hr , the number of viable cells was determined by a hemocytometer . all the reagents were tested for rescue from cell death at concentrations ranging from 0 . 1 μg / ml to 10 μg / ml except for d - and l - tryptophan . data at the most effective concentration are shown in this graph . all reagents were tested at concentrations ranging from 0 . 01 to 10 μg / ml , and in a separate group 1 mm nac was added as a positive control . protein synthesis inhibitors chx , pmc and emt , and rna synthesis inhibitors act d and et br , prevented death of cells in these cultures , suggesting that serum - induced toxicity requires protein synthesis . inhibitors of protein and rna synthesis protected cells against seruminduced toxicity similar to thiols , yet they never showed a growth - promoting effect . accordingly , these inhibitors may act via a molecular mechanism different from that of thiols . to determine whether thiols inhibited protein synthesis , l -[ 35 s ] methionine uptake into tig - 1 cultured in fbs was measured . a typical result from three independent experiments is presented in fig6 . to develop the data shown in fig6 tig - 1 cells were seeded into a 24 well - multiplate at a half - confluency ( about 6 × 10 4 cells / well ), and medium was replaced with fbs containing 20 μci l -[ 35 s ] methionine . after incubation for each indicated time , cells were lysed and macromolecules were precipitated with tca . precipitates were dissolved in buffer and counted by liquid scintillation . tig - 1 cells cultured in fbs showed substantial uptake of l -[ 35 s ] methionine , suggesting that cells vigorously synthesized protein prior to death . l -[ 35 s ] methionine uptake into tig - 1 cells in all three experiments was completely inhibited by 1 μg / ml chx and partially inhibited by 5 mm l - tryptophan ( 30 - 50 %). by comparison , 1 mm nac did not inhibit l -[ 35 s ] methionine uptake . we interpreted these results to indicate that supplemental thiols prevent cell death by maintaining intracellular thiol levels . we found , however , that acid - soluble thiol content of tig - 1 cells cultured in fbs quickly decreased whether or not the medium was supplemented with thiol ( data not shown ). thereafter , total and ethanol - soluble thiol content were measured in subsequent experiments . as shown in fig7 a and 7b , both thiol contents were conservative when 1 mm nac was added to fbs , whereas protein synthesis inhibitor chx did not affect thiol content either total and ethanol - soluble . the following procedure was followed in developing the data plotted in fig7 a and 7b : after several hours incubation in test medium , cells were lysed in 1 % sds , 0 . 02 % edta , 50 mm tris - hcl ( ph . 8 . 2 ). either the total lysate or the lysate after precipitation of macromolecules by × 3 volume of ethanol ( ethanol - soluble ) was dyed with 20 mm dtnb . absorbance at 415 nm was measured by microplate reader 3550 ( biorad ). concentration of thiol was calculated by comparison to a gsh standard . thiol content was expressed as sh mol / living cell number . fig7 a and 7b are to be interpreted in conjunction with the following key : ______________________________________  10 % fbs containing ea - ▴ fbs + 1 mm nac ; gle &# 39 ; s mem ; ∘ 1 μg / ml chx ; □ hank &# 39 ; s balanced solution ; ▪ fbs + 10 mm l - trp . δ fbs ; ______________________________________ another distinguishing feature of apoptosis , namely , dna fragmentation , was evaluated by agarose gel electrophoresis of low molecular weight dna from dying cells at various stages of serum toxicity . living cell ratios were measured by the method of trypan blue exclusion , as illustrated in fig8 . the chart shown in fig8 reflects the result of the following procedure : tig - 1 cells were seeded into a 35 mm culture dish at a density of 2 × 10 5 cells / dish . when the density reached 3 . 5 × 10 5 cells / dish , medium was replaced with fbs . after the incubation for each indicated time , cells were harvested with 0 . 1 % trypsin in cmf - pbs , and 0 . 5 % trypan blue in pbs was added . cells that excluded the dyed were counted as living with a hemocytometer . results are expressed as the means (± se ) of four independent determinations . until 6 hr of the serum treatment , the cells appeared morphologically viable and were not stained by trypan blue . at that point , viability declined steeply , and all cells were dead by 12 hr . dna fragments ( ladder ) appeared at 6 hr of the treatment before the plasma membrane breakdown , and they persisted until 12 hr . electrophoresis of dna fragments of dying cells ( not shown ) was conducted using 2 . 6 % agarose - gel . the maximal length of the dna ladder was relatively small (& lt ; 0 . 7 kbp ), and the space between rungs of the ladder was slightly smaller than the 0 . 2 kbp value reported for the dna ladder usually observed during apoptosis ( kerr et al ., 1972 ; wyllie et al ., 1980 ; ellis et al ., 1991 ; raff , 1992 ; eastman , 1993 ). nevertheless , dna fragmentation proceeded morphological change by a few hours , and rna and protein synthesis inhibitors prevented cell death , consistent with the view that serum - induced toxicity is a type of apoptosis . in the present study , we observed that serum - induced toxicity was prevented with thiol ( sh )- related molecules . disulfide , d - cysteine or d - cystine , and dtnb were effective in rescuing cells despite the fact that these compounds are not normally present in cells and do not function as reducing agents in medium . in addition , dithionite , a reducing disulfide , was protective . the findings suggest that these reagents may promote efficient cellular utilization of sh by dithiol exchange or by a reaction involving chemical reduction . the standard cell culture system is an oxidative environment , where sh is oxidized to form dithiol . sh - compounds like cysteine and glutathione in serum are , therefore , able to form dithiol homo - dimers or mixed disulfides with low molecular weight thiols and proteins . in contrast , cells find little use for mixed disulfides . however , if molecules such as l - cysteine , reduced glutathione , or l - cystine are produced through exchange or reducing , they may be readily used by cells , resulting in promotion of intracellular sh metabolism . intracellular thiol levels were decreased in high concentrations of serum , and addition of thiols reversed the decline . thiol content began to decrease several hours prior to plasma membrane breakdown , and this suggests that the lowering of thiol content was a causal factor in serum - induced cell death rather than a result . it was not , however , the only cause of death , because hank &# 39 ; s balanced solution did not induce death even though thiol content was reduced as in the case of cells cultured in fbs . protein synthesis inhibitors prevented cell death , but they had no affect on intracellular thiol content , suggesting that protein synthesis was necessary for serum - induced toxicity . taken as a whole , the evidence is consistent with two hypotheses for serum - induced cell death , schematically illustrated in fig9 a and 9b . the first is that a decrease in thiol content directly induces synthesis of proteins required for programmed call death ( fig9 a ). the second is that a decrease in thiol content affects a variety of intracellular processes that occur during cell death including protein synthesis ( fig9 b ). whether either of these hypotheses can be experimentally verified remains to be determined . during protein synthesis , de novo rna synthesis may not be necessary , since rna synthesis inhibitors only partially protected against death of hela cells and low density cultures of tig - 1 cells . high concentrations of l - and d - tryptophan showed that equal ability to rescue cells . to our knowledge , inhibition of protein synthesis by tryptophan has not been reported , yet our preliminary study showed that l - tryptophan incompletely inhibited l -[ 35 s ] methionine uptake by cultured tig - 1 cells ( fig6 ). this may indicate that l - and d - tryptophan can act as protein synthesis inhibitors in some cases . addition of exogenous thiol compounds maintained the intracellular level of total thiol , including the protein fraction against depletion by serum - induced toxicity . the level of acid - soluble thiol was , however , not always restored by the addition , whereas that of ethanol soluble thiol moderately increased . ethanol is known to solubilize short peptides more efficiently than tca , and this may be the reason why the ethanol soluble thiol level was higher . these results suggest that supplemental thiol was predominantly incorporated into protein fractions that maintained the total thiol level . it is possible that thiols contained in protein fractions may act as modifiers of cysteine residues . for example , some enzymes require modification of their cysteine residues by glutathione for activation ( ziegler , 1985 ). serum - induced toxicity may reflect such a functional disorder caused by a shortage of thiols . a second major observation was that dna was cleaved into fragments beginning a few hours before plasma membrane breakdown . the electrophoretic behavior of the dna differed from ordinary apoptosis in that the fragments were smaller , and sometimes the dna appeared to be randomly digested , showing a smeared pattern rather than discrete bands . nevertheless , a characteristic feature of apoptosis was evident , namely , dna fragmentation prior to membrane disintegration . abrams , j . m ., white , k ., fessler , l . i ., and steller , h . 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( 1984 ) cell death in invertebrate nervous system . ann . rev . neurosci ., 7 : 171 - 188 . wyllie , a . h ., kerr , j . f . r ., and currie , a . r . ( 1980 ) cell death : the significance of apoptosis . int . rev . cytol ., 68 : 251 - 306 . yonish - rouach , e ., resnitzky , d ., lotem , j ., sachs , l ., kimchi , a ., and oren , m . ( 1991 ) wild - type p53 induces apoptosis of myeloid leukemic cells that is inhibited by interleukin 6 . nature , 352 : 345 - 347 . ziegler , d . m . ( 1985 ) role of reversible oxidation - reduction of enzyme thiolsdisulfides in metabolic regulation . ann . rev . biochem ., 54 : 305 - 329 . while this invention has been explained with reference to the structure disclosed herein , it is not confined to the details set forth and this application is intended to cover any modifications and changes as may come within the scope of the following claims :