Patent Application: US-50247705-A

Abstract:
the present ivention relates to a high through put screening method for assaying the non -, pro -, or anti - apoptonic or proliferative or necrotic activity of test compounds in cells , using vectors coding for a specific marker protein , cell lines and spheroids transfected with said vectors .

Description:
the present invention relates to the assessment of the non -, pro -, or anti - apoptotic or proliferative or necrotic activity of test compounds on test cell systems of various origins in an industrially applicable assay for purposes such as drug screening and toxicology studies . test compounds can have different activities on the test cells , i . e . non -, pro -, or anti - apoptotic , or proliferative and / or necrotic activity . in a primary screening , the overall fluorescence activity of the test cells within a single well is measured with an appropriate fluorescence - detecting device , e . g . a fluorescence plate - reader . an appropriate cut - off value is set in order to clearly discriminate between two main groups of different activities . the first group represents the pro - apoptotic and / or necrotic activity of test compounds , whereas the other group represents the non - and / or anti - apoptotic and / or proliferative activity of test compounds . in a secondary screening , the single - cell fluorescence activity of each test cell within a population of test cells is measured with an appropriate fluorescence detecting device , e . g . flow cytometry , microfluidic devices ( chip technology ) and single cell imaging scanning systems . this measurement allows the clear discrimination between pro - apoptotic and necrotic activity , and between non -, anti - apoptotic and proliferative activity . the combination of primary and secondary screening allows the clear assessment of non -, pro -, or anti - apoptotic , or proliferative or necrotic activity of test compounds on test cells . a single cell imaging , scanning systems with an appropriate throughput capacity can also be used as fluorescence detecting device in the primary screening , in order to clearly discriminate at that level between non -, pro , or anti - apoptotic , or proliferative or necrotic activity of test compounds . test compounds comprise synthetic or natural compounds , chemical or peptide structures or a combination thereof , proteins or recombinant proteins , pure compounds or a combination of pure compounds or extracts , such as plant extracts , extracts of marine micro - and macro - organisms and extracts of microbial fermentations . test compounds are incubated with test cells either alone or in combination with known pro - or anti - apoptotic compounds or stimuli of various origins . the test cell system comprises either a single cell , a single - cell population comprising cells of identical origin , a mixed - cell population comprising cells of different origin , or cells in spheroid form either of single - cell or mixed - cell population as defined above . useful test cells are cell lines or primary cells from various origin and comprise eukaryotic and prokaryotic cells . prokaryotic cells include bacterial and cyanobacterial cells . eukaryotic cells include mammalian , fungal , insect , avian , worm , fish , crustacean , reptilian , amphibian and plant cells as well as cell lines thereof . test cells usable in the method are cells of any type , preferentially normal , i . e . genetically non - altered , infected , e . g . with virus , parasites , bacteria , fungi or prions , tumor cells or genetically manipulated or altered cells of human , animal or plant origin , which can be cultured in vitro and / or in vivo . human and animal primary cells as well as cell lines that have different origins and are derived from different tissues and / or organs such as liver , kidney , spleen , heart , lung , brain , blood , skin , muscles , bladder , myeloid and lymphoid system , reproductive system , visual system , bone marrow , gut , small intestine , mucosa , stomac , esophagous , duodenum , colon , pancreas , connective , embryonal and fetal tissue among others may be used . useful test cells are cells applicable as healthy tissue - and / or organ - models or disease models , such as cell lines and primary cells of human or animal origin isolated from healthy individuals / animals or patients / animals suffering of diseases such as cancers , autoimmune diseases , organ transplantation derived pathogenesis , cardiovascular diseases and degenerative diseases of various origin , e . g . neurodegenerative diseases , or the like or spheroids . furthermore , appropriate test cells as described above are used for the purpose of toxicological studies , e . g . hepatotoxicological studies , kidney toxicity , skin toxicity , peripheral and central neurotoxicity , embryonal and fetal toxicity , toxicity of the spleen , heart , lung , blood , skin , muscles , bladder , myeloid and lymphoid system , reproductive system , visual system , bone marrow , gut , small intestine , mucosa , stomac , esophagous , duodenum , colon , pancreas , among others , where the necrotic / toxic activity of test compounds is assayed . microplate formats useful for the method are customized formats and standard formats such as 24 -, 48 -, 96 -, 384 -, 1536 - and any intermediate size well microplates , among others , as well as chip technology , where living cells can be used and a multitude of tests can be run at the same time . therapeutic fields of interest are cancer including angiogenesis , autoimmune and transplantation derived diseases , cardiovascular and degenerative diseases of various origin , such as neurodegenerative diseases , inflammation and allergic diseases , diseases of the reproductive system , dermatological applications and related diseases , among others . in fig1 , apoptosis and necrosis was induced in a20gfp cells and determined by using a fluorescence microplate reader , which measures the overall fluorescence activity within a single well . for this purpose a20gfp cells were either incubated with fasl for induction of apoptosis or with an anti - a20 antibody plus complement in order to induce necrosis . after incubation at standard conditions , the cells have been plated out into a 96 - well plate . in the case where cell - mixtures , containing cells with different cell status , of either apoptotic and necrotic or living and necrotic cells were measured , equal amounts of apoptotic , necrotic or living - cells were pipetted appropriately into the same well ( in a ratio of 1 : 1 ). subsequently , the fluorescence activity was measured with a fluorescence microplate reader . it could be observed that the homogeneous ( unmixed ) population of necrotic a20gfp cells lost almost entirely the fluorescence activity and showed values comparable to non - transfected control cells . homogeneous ( unmixed ) living or apoptotic or necrotic cell populations can be clearly distinguished from each other due to a significant difference of the overall fluorescence activity of each cell - population , as shown in fig1 . on the other hand , cell - mixtures , i . e . subpopulations of apoptotic and necrotic or living and necrotic cells within the same well , show intermediate fluorescence activities when measured on a fluorescence microplate reader . this intermediate fluorescence activity varies depending on the contribution of each subpopulation , i . e . living , apoptotic and necrotic cells , to the overall fluorescence activity . this experiment clearly demonstrates , that homogeneous ( unmixed ) populations of apoptotic or necrotic or living cells within a single well can be clearly distinguished from each other . on the contrary , subpopulations of apoptotic and / or necrotic and / or living cells within a single well , which globally show an intermediate fluorescence activity can hardly be distinguished from each other when measured on a fluorescence microplate reader . indeed , the use of cellular assays in drug screenings / high throughput screenings under real conditions shows that in most cases intermediate overall fluorescence activities are measured , which correspond to cell - mixtures within a single well . in conclusion , the need for a reliable and industrially applicable assay able to clearly discriminate between these different activities , i . e . non -, pro -, or anti - apoptotic , or proliferative or necrotic activity of test compounds on test cells , is evident . a representative primary screening with standard experimental set - up , where 16 commercially available anti - cancer drugs have been tested on five different egfp expressing cell lines ( hela , kb , a20 , ramos and jurkat ) in 96 - well microtiter plates is shown in fig2 . differential susceptibility regarding induction of apoptosis and / or necrosis after treatement with 5 - fluorouracil ( 5 - fu ), gemzar , methotrexate , eloxantin , detimedac , endoxan , paraplatin , navelbine , velbe , taxotere , taxol , adriblastin , bleomycin , campto , etopophos and farmorubicin is shown . measurements after 24 h and 48 h were performed with a fluorescence microplate reader , which measures the overall fluorescence activity in a single well . used compound concentrations are indicated by a fraction of the original concentration . fig2 demonstrate that each tested cell line shows a specific pattern of sensitivity towards the tested drugs . a20 demonstrates the most refractory behaviour and kb the highest sensitivity towards the drug treatment . beside a clear activity - dosage relation , a specific kinetic behaviour can be observed as well . the setting of a cut - off value , corresponding to 0 . 8 in this case , allows the clear discrimination between two main groups of different activities . the first group with values under 0 . 8 represents the pro - apoptotic and / or necrotic activity of test compounds , whereas the other group with values above 0 . 8 represents the non - and / or anti - apoptotic and / or proliferative activity of test compounds . a representative example is navelbine and adriblastin , which show intermediate values of 0 . 49 and 0 . 53 , respectively , when tested on hela cells at the highest concentration , i . e . 1 / 10 k for navelbine and 1 / 5 k for adriblastin . according to these intermediate values between 0 and 0 . 8 measured by a fluorescence microplate reader , it can hardly be discriminated between the subpopulations of apoptotic and necrotic cells within the same well . the unambiguous discrimination between the pro - apoptotic and necrotic activity of anti - cancer drugs on test cells within the same well , e . g . hela cells , in relation to possible mechanisms of actions is demonstrated in fig3 . for this purpose hela cells have been treated with typical anti - cancer drugs of different mode of actions . navelbine and velbe , two typical representatives of microtubules interfering drugs and adriblastin and campto , topo - isomerase inhibitors , which interfere with dna synthesis . fig3 shows histograms of facs analysis of cells treated either with navelbine , velbe , adriblastine or campto . arrows indicate regions with either necrotic cells ( left arrow ) or apoptotic cells ( middle arrow ) or proliferating / living cells ( right arrow ). in this representative example , navelbine and adriblastin , which showed comparable overall fluorescence activities in the microplate reader as described above , demonstrate different activities when measured on a single - cell detecting device , such as flow cytometry . indeed , navelbine show a strong tendency to induce necrosis , whereas adriblastin only induce apoptosis . generally , the cell cycle inhibiting drugs , navelbine and velbe , respectively , show a strong tendency to induce necrosis in a certain fraction of cells . this fact became evident after exposure of the cells for longer than 24 h . in contrary , cells treated either with campto or adriblastin , typical dna replication interfering compounds , do not show any tendency for necrotic activity even after exposure for 48 h . this experiment shows that the use of a single - cell fluorescence detecting device , e . g . flow cytometry , allows a clear discrimination between the subpopulation of apoptotic , necrotic and living / proliferating cells within the same well , after treatment of test cells with test compounds . the differential sensitivity of the fluorescence microplate reader and flow cytometry , respectively , to changes in the fluorescence signal caused by apoptotic stimuli of various origin , is demonstrated in a representative experiment shown in fig4 . as already mentioned before , the fluorescence microplate reader measures global fluorescence activities in a single well in contrary to flow cytometry , e . g . a facscan , which measures single - cell fluorescence activities . according to this different physical property , the sensitivity to changes in the fluorescence signal vary depending on the fluorescence detecting device used . in order to investigate the correlation between the apoptotic activity measured by facscan ( reflected directly by the population which shows reduced fluorescence ) and the relative fluorescence activity measured by a fluorescence microplate reader , jurkatgfp cells have been induced to undergo apoptosis by treatment with serially diluted soluble fasl . the experiment has been carried out in 96 - well microtiter plates and facs tubes ( bd biosciences ) in parallel . the samples have been measured after 24 h and 48 h , respectively , either in a bmg fluostar fluorescence microplate reader ( rel . fluorescence activity , vertical values ) or with a facscan (% apoptosis , horizontal values ) and plotted versus each other . the correlation between apoptotic activities measured by facscan and relative fluorescence activities measured by a fluorescence microplate reader turned out to change in a time dependent manner . this is illustrated in fig4 , which demonstrates the correlation at 24 h ( fig4 a ) and at 48 h ( fig4 b ), respectively . this time depedent change parallels the differential change in sensitivity towards apoptotic activities of the fluorescence microplate reader and the facscan , respectively . a few percent of apoptotic cells ( 1 - 5 %) measured by flow cytometry at 48 h , e . g . a facscan , correlate to a reduction of approximately 20 % in the relative fluorescence activity measured by the microplate reader . in other words , a change in the fluorescence signal of 20 % measured by a microplate reader is hardly detectable by flow cytometry , e . g . a facscan , which correspond to about 5 % of apoptotic cells . a sharp change in the slope of the correlation curve is observed for the range of 5 - 65 % apoptotic cells measured by flow cytometry , which correspond to a reduction in the relative fluorescence activity measured by a microplate reader of only 10 %. this range of apoptotic activities of test compounds can be accurately measured by flow cytometry , e . g . a facscan . another drastic change in the slope of the correlation curve is observed in the range of 65 - 80 % apoptotic activity measured by flow cytometry , which correspond to a further reduction of the relative fluorescence activity measured on the microplate reader of approximately 20 %. in conclusion , small amounts of apoptotic cells within the same well containing cell - mixtures of apoptotic and living / proliferating cells can be easily detected by the use of a fluorescence microplate reader . on the contrary , small amounts of apoptotic cells within the same well containing cell - mixtures are hardly detectable by flow cytometry , e . g . a facscan . this behaviour has practical implications for the screening procedure . as already described , the primary screening step is performed with a fluorescence microplate reader . consequently , this ensures that even small amounts of apoptotic stimuli of various origin can be detected by setting appropriate cut - off values and thereby acts as a highly reliable filter tool which detects even small apoptotic activities of various origin decreasing the likelyhood of missing active test compounds . test compounds positively identified in the primary screening are then subjected to the secondary screening which is run on a flow cytometer . flow cytometry is based on single - cell fluorescence measurements and demonstrates a high and reliable sensitivity in the range of 5 - 65 % of apoptotic activity , in contrary to the microplate reader , and therefore enables the reliable quantification of apoptotic activity of test compounds . the activity of an anti - apoptotic compound , e . g . the pan caspase inhibitor zvadfmk , has been tested on jurkatgfp cells after induction of apoptosis by serially diluted soluble fasl and measured by a fluorescence microplate reader . a clear - cut dose - response behaviour , dependent on the dilution of fasl , can be observed already after 24 h of incubation at standard conditions . the pan caspase inhibitor zvad , in contrary to its control peptide zfa , shows a clear inhibitory anti - apoptotic effect on the apoptotic activity of fasl . fig5 shows representative data , which have also been observed with other egfp transfected cells . the bars indicate standard deviations , which have been determined by measuring 6 single values for each datapoint . the reproducibility and the profiling of several cell lines towards standard drugs in 96 well plates is shown in fig6 . five different cell lines have been tested several times independently in a defined experimental setup , which correspond to the standard procedure for running the tests in 96 - well plates . the following drugs were used : adriblastin , gemcitabin , detimedac and farmorubicin , respectively . used cells are the following : hela , kb , mcf7 , a20 and jurkat . mean values and standard deviations are indicated for 24 h and 48 h measurements . fig6 shows that the system is robust and provides highly reproducible results . therefore this experimental system is well suitable for high throughput screening in the 96 - well format . fig7 demonstrates the reproducibility and profiling of several cell lines towards standard drugs in 384 well plates . four different cell lines have been tested several times independently in a defined experimental setup which correspond to the standard procedure for running the tests in 384 well plates . the following drugs were used : gemcitabin ; detimedac , methotrexate and farmorubicin , tamoxifen and quercetin . used cells are the following : hela and kb ; a20 and jurkat . mean values and standard deviations are indicated for 24 h and 48 h measurements . fig7 show that the system is robust and provides highly reproducible results . therefore , this experimental system is well suitable for high throughput screening in the 384 well format . beside the various above : mentioned cell lines , spheroids as three - dimensional cell models for screening non -, pro - or anti - apoptotic or proliferative or necrotic activity of test compounds may be used as well . a ) pro - apoptotic activity of test compounds has been investigated in tumor models where cells are growing as spheroids that are nearly as heterogeneous as tumor nodules in vivo , regarding cell proliferation , resting cells , necrosis and apoptosis . there is also heterogeneity , where spatial distributions and therapeutical effects of anticancer drugs are concerned . the effects of different drugs vary dramatically between different types of spheroids and between different cell layers inside the spheroids . the influence of the oxygen pressure , ph and nutrition gradients on drug sensitivity of cells in spheroids is probably similar to the influence in tumor microregions . therefore , spheroids will be of immense interest in the field of experimental tumor chemotherapy . instead of using egfp expressing monolayer cell cultures for high throughput screening , spheroids are prepared either consisting of tumor cells only or mixed with untransfected stromal cells ( e . g . fibroblast cell lines ). as a result of the gfp reporter exclusively expressed in the tumor cells it is possible to monitor their behaviour in terms of necrosis , apoptosis and proliferation . this can be achieved by measuring the fluorescence activity in an appropriate device , e . g . in a fluorescence microplate reader or a single - cell imaging scanning system , as has been described for adherent and suspension cell cultures . b ) in order to investigate the anti - apoptotic activity of test compounds in diseases where the affected or target cells and organs are prone to undergo apoptosis ( e . g . cardiovascular diseases like stroke or degenerative diseases found in neuronal - and muscle tissue pathogenesis ), the appropriate cell models representing the disease in question can be used for preparing spheroids . by altering oxygen pressure , nutrient supply , ph or with the addition of certain active molecules ( being responsible for degenerative processes ) the egfp transfected reporter and disease representing cells can be stimulated to undergo necrosis or apoptosis which can be easily monitored by its change in fluorescence activity . compounds to be tested for anti - apoptotic or anti - necrotic activity can be added to the cells and the change in fluorescence activity can be tested . the cell lines were cultured in rpmi - 1640 tissue culture medium containing either 5 % or 10 % fetal calf serum , 0 . 05 mm 2 - mercaptoethanol , 2 mm glutamine and penicillin / streptomycin 50 g / ml ( complete medium ) ( sigma , buchs , switzerland ). general growth conditions were 37 ° c . and 7 . 5 % co 2 . the following mouse cell lines were used : a20 . 2j ( attc : tib - 208 ), pb3c ( mastocyte cell line 32 ), mc57g ( atcc : crl - 2295 ): the following human cell lines were used : hela ( atcc : ccl - 2 ), kb ( atcc : ccl - 17 ), mcf7 ( atcc : htb - 22 ), sk - br - 3 ( atcc : htb - 30 ), dm and hbl ( melanoma cell lines 37 ), sk - mel 1 ( atcc : htb - 67 ), sk - mel 28 ( atcc : htb - 72 ), hacat ( transformed keratinocytes 33 ), pc - 3 ( atcc : crl - 1435 ), sw 480 ( atcc : ccl - 228 ), nci - h460 ( atcc : htb - 177 ), nci - h1792 ( attc : crl - 5895 ), ht1080 ( attc : ccl - 21 ), jurkat ( attc : tib - 152 ), ramos ( attc : crl - 1596 ), raji ( attc : ccl - 86 ), h9 ( attc : htb - 176 ), hut78 ( attc : tib - 161 ), k562 ( attc : ccl 243 ) and hl - 60 ( attc : ccl 240 ) tumor cell spheroids have been prepared according to standard procedures 31 the vectors used in the present invention , which are either termed pegfp - n1 + molv - ltr and pbluescriptiiks (+)+ ef - 1α + egfp , respectively , have already been described elsewhere ( pct / ib99 / 00030 ). any other commercially available and for the purposes of the present invention suitable vector may also be used . the vectors have been amplified and purified according to standard procedures and by the use of commercially available purification kits . transfection of the cells has been performed either by electroporation or with the help of liposomal reagents . jurkat , a20 . 2j and the pb3c cell lines have been electroporated ( pct / ib99 / 00030 ) whereas all the other cell lines have been transfected by using dotap transfection reagent ( roche molecular biochemicals , switzerland ) according to the manufacturer &# 39 ; s protocol . the transfected cells were selected in 1 mg / ml g418 ( gibco brl , life technologies ag , basel , switzerland ) containing medium for 2 - 4 weeks . selected suspension cells have then been sorted on a facsvantage flow cytometer ( bd biosciences , allschwil , switzerland ) for high egfp expression profiles and subcloned by the limiting dilution method . adherent cells positive for egfp expression have been picked with pipette tips under sterile conditions and subsequently been subcloned twice by limiting dilution . in each case the resulting clones were expanded and subsequently characterized for homogeneous expression of egfp in a facscan ( bd biosciences ) equipped with an argon laser tuned to 488 nm to excite egfp , and a 515 / 545 bandpasss filter to monitor the green fluorescence emitted by the egfp . analysis was done by the cellquest program . in each measurement 10 . 000 events were collected . all the manipulations were done under sterile conditions . the assays have been performed in commercially available 96 or 384 well flat bottom clear microtiter plates ( greiner , germany ) respectively , which are suitable for tissue culture techniques . a defined number of adherent cells ( 96 well plates : 10 4 - 10 5 , 384 well plates : 1500 − 2 * 10 4 ) have been plated out 24 h before treatment either in 75 μl ( 96 well plates ) or 60 μl ( 384 well plates ) complete medium per well in order to ensure appropriate spreading before start of the treatment . for this purpose a peristaltic pump ( e . g . multidrop by thermo - labsystems , finnland ) or another suitable device was used . cells in suspension have been plated out according to the same procedure but 1 h prior to treatment . between seeding out and treatment or addition of compounds the cells were incubated at 37 ° c . under 7 . 5 % co 2 . subsequently , the compounds under investigation were added at defined concentrations dissolved either in 25 μl or 20 μl complete medium with an appropriate device ( e . g . liquid handling system , multichannel pipette etc ). immediately after the addition of the compounds to the cells the zero fluorescence value ( t = 0 h ) was determined by using a fluorescence microplate reader in order to be able to normalize the fluorescence activities . afterwards , the testplates were further incubated for a total of 48 h at 37 ° c . under 7 . 5 % co 2 and were shortly removed only for the purpose of measurement at 8 h , 24 h and 48 h , respectively . relative fluorescence activities of egfp in treated cells in relation to control cells were measured by using a bmg fluostar microplate fluorescence reader equipped with a filterpair for excitation / emission at 485 nm / 520 nm . the optimum signal to noise ratio was detected by using the time - resolved mode of measurement with a delay of 20 μs and an integration time over 1 ms . the gain was adjusted in such a way that the control cells produced a fluorescence activity of 90 % of the maximum . kinetics were performed by measuring the relative fluorescence activities at t = 0 h , 8 h , 24 h and 48 h . crude fluorescence activities were first individually normalized for different cell numbers seeded per well by dividing each value from t = 8 h , 24 h and 48 h by the value of t = 0 h . in a second step the resulting values , which were normalized for different cell numbers have been normalized in relation to the appropriate controls ( cells only treated with dissolving agent , e . g . 1 % dmso , at the appropriate time ) by forming the quotient . the normalization procedure , which accounts for variations of various origins ( e . g . optical characteristics of individual plates , different growth properties due to serum variations , etc ) allows to compare individually performed experiments among each other for statistical and other purposes . 1 ) four different classes of cytotoxic agents ( a : antimetabolites , b : alkylating agents , c : cell - cycle inhibitor , d : dna breaker ( topo - isomerase inhibitor , intercalator , strand breaker ), e : mixtures thereof , f : compounds interfering with the signal transduction pathway , such as caspase activity modifiers , agonists and antagonists of cell death receptors , modifiers of nucleases , phosphatases and kinases , which are commonly used in anticancer therapies have been extensively tested on several egfp expressing cell lines for apoptotic / necrotic activities . they have been provided as stock solutions in ampoules . class a : 5 - fluorouracil , icn , 50 mg / ml ; gemzar , eli lilly , 50 mg / ml ; methotrexate , spitalapotheke kantonsspital basel , 4 mg / ml . class b : eloxantin , sanofi - synthelabo , 5 mg / ml ; detimedac , medac , 10 mg / ml ; endoxan , 1 mg / ml ; paraplatin , bristol - meyers squibb , 10 mg / ml . class c : navelbine , robapharm , 10 mg / ml ; velbe , eli lily , 10 mg / ml ; taxotere , aventis , 10 mg / ml ; taxol , 6 mg / ml . class d : adriblastin , pharmacia - upjohn , 1 mg / ml ; bleomycin , asta - medica , 1 mg / ml ; campto , aventis , 20 mg / ml ; etopophos , bristol - meyers squibb ; 5 mg / ml ; farmorubicin , spitalapotheke kantonsspital basel , 2 . 5 mg / ml ; hycamtin , smith - kline beecham , 1 mg / ml . all these compounds have been used in clinical formulations as they were used for treating cancer patients . in the various tests they have been diluted with complete medium in the range of 1 / 1000 to 1 / 100000 . they were kindly provided by the department of oncology , university hospital of basel , switzerland . 2 ) dexamethasone , actinomycin d , phorbol - myristate - acetate , cyclosporin a , etoposide , quercetin , tamoxifen have been purchased by alexis corporation , switzerland . caspase inhibitor z - vad - fmk and its control z - fa - fmk have been purchased by enzyme systems , dublin , ca , usa . 3 ) cells were also treated with soluble fasl and / or trail , respectively , which is described elsewhere ( pct / ib99 / 00030 ). assessment of induced apoptosis and necrosis in a20gfp cells measured by a fluorescence microplate reader . a20gfp cells were either incubated with fasl for induction of apoptosis or with an anti - a20 antibody plus complement in order to induce necrosis . after 24 h incubation at standard conditions , 100 μl , which correspond to 200 . 000 cells have been plated out into a 96 well plate . in the case where mixed populations were measured , two times 50 μl were pipetted together into the same well . subsequently , fluorescence activity was measured with a microplate reader . primary screen of 16 commercially available anti - cancer drugs on different egfp expressing cell lines . five different cell lines ( hela , kb , a20 , ramos and jurkat ) have been tested in 96 well microtiter plates for differential susceptibility regarding induction of apoptosis towards treatment with 5 - fluorouracil ( 5 - fu ), gemzar , methotrexate , eloxantin , detimedac , endoxan , paraplatin , navelbine , velbe , taxotere , taxol , adriblastin , bleomycin , campto , etopophos and farmorubicin . measurements after 24 h and 48 h were performed with a fluorescence plate reader . used compound concentrations are indicated by a fraction ( 1 / xk meaning 1 / x000 ) of the original concentration . each tested cell line demonstrates a specific pattern of sensitivity towards the tested drugs . a20 demonstrates the most refractory behaviour and kb the highest sensitivity towards the drug treatment . beside a clear activity - dosage relation a specific kinetic behaviour could be observed as well . the results are summarized in table 1 . secondary screening : discrimination between apoptotic and necrotic activity of anti - cancer drugs in hela cells in relation to possible mechanisms of actions . hela cells have been treated with typical anti - cancer drugs of different mode of actions . navelbine and velbe , two typical representatives of microtubules interfering drugs and adriblastin and campto , topo - isomerase inhibitor , which interfere with dna synthesis . histograms of facs analysis of cells treated either with navelbine ( 1 / 1000 diluted ), velbe ( 1 / 1000 diluted ), adriblastin ( 1 / 2000 diluted ) and campto ( 1 / 1000 diluted ) are shown in fig4 . arrows indicate regions with either necrotic cells ( left arrow ), apoptotic cells ( middle arrow ) and proliferating cells ( right arrow ). the cell cycle inhibiting drugs , navelbine and velbe , respectively , show a strong tendency to induce necrosis in a certain fraction of cells . this fact became evident after exposure of the cells for longer than 24 h . in contrary , cells treated either with campto or adriblastine , typical dna replication interfering compounds , do not show any tendency for necrotic activity even after exposure for 48 h . 1 . wyllie , a . h ., et al . int . rev . cytol . 68 , 251 ( 1980 ) 2 . arends , m . j ., et al . int . rev . exp . pathol . 32 , 223 ( 1991 ) 4 . roy , c ., et al . exp . cell res . 200 , 416 ( 1992 ) 6 . watanabe , f . r ., et al . j . immunol . 148 , 1274 - 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