the human prostate carcinoma cell lines pc3 , du145 ( american type culture collection ) and pc - 3m ( a gift of dr . g . weiss , surgery branch , nci ) were maintained in ham &# 39 ; s f12k ( pc3 ), eagle &# 39 ; s modified essential medium ( du145 ), or rpmi 1640 ( pc3m ), supplemented with 7 % ( pc3 ) or 10 % ( du145 and pc3m ) fetal bovine serum , 100 units / ml penicillin , 100 mg / ml streptomycin , and 300 mg / ml glutamine . the cells were grown in a mixture of 5 % co 2 , 95 % air at 37 ° c ., and propagated by typsinization and dilution into fresh medium . cells were typically used for calcium determination two days after dilution into fresh medium . measurement of intracellular free calcium [ ca 2 + ] i ; [ ca 2 + ] i was determined by the quin2 technique of tsien et al . ( j . cell biol . 94 : 325 - 334 , 1982 ) with some modifications . briefly , cells from confluent cultures were harvested by treatment with trypsin - edta . cells were washed with calcium medium ( 1 mm cacl 2 , 1 mm kcl , 1 mm mgcl 2 , 140 mm nacl , 20 mm hepes , 5 mm dextrose , ph 7 . 4 ), and loaded with 20 mm quin2 / am ( molecular probes , eugene , oreg .) in calcium medium at 37 ° c . for 30 minutes , then diluted 5 - fold with the same medium and incubated at 37 ° c . for 60 minutes . after loading , cells were washed and resuspended in the calcium media at 1 . 5 - 2 . 0 × 10 6 cells / ml and kept on ice until use . cell viability was determined by trypan blue exclusion and was usually & gt ; 95 %. for measurement of fluorescence , 2 ml of cell suspension was transferred to a cuvette maintained at 37 ° c . fluorescence was measured in a perkin - elmer 650 - 10s fluorescence spectrophotometer , with an excitation wavelength of 336 nm , slit 5 nm , and an emission wavelength of 493 nm , slit 5 nm . [ ca 2 + ] i was estimated after calibration with triton x - 100 and egta , assuming a dissociation constant of the ca 2 + - quin2 complex of 115 nm . flow cytometric studies were performed allowing analysis of the ca 2 + response at the single cell level . these studies demonstrated purinergic receptor stimulated ca 2 + transients in prostate carcinoma cells , and showed marked heterogeneity within the pc - 3 cell line in the purinergic response . flow cytometric analysis of the ca 2 + response in single cells cells were prepared as described above . the loading procedure was performed by incubating cells with 1 um fluo - 3 ( molecular probes , eugene , oreg . ), a recently described long wavelength calcium indicator ( tsien et al ., j . biol . chem . 264 : 8171 , 1989 ). cells were incubated at room temperature for 30 - 60 min ., washed , and resuspended in calcium medium . rabbit anti - fluorescein antibody ( 1 unit / ml , molecular probes ) was added to the cell suspension to reduce fluorescence background . fluorescence was measured using a becton - dickinson facstar equipped with a 5 watt argon laser , using 488 nm excitation . parallel studies were performed by fluorescence spectrophotometry ( excitation 506 nm , slit 5 nm , emission 526 nm , slit 5 nm ). the biochemical pathway activated by purinergic receptor stimulation in prostate carcinoma cells was studied further by assay of phospholipase c activity . these studies demonstrated that purinergic receptor stimulation induces phospholipase c activity and breakdown of phosphatidylinositol bisphosphate to inositol trisphosphate , which interacts with a specific inositol trisphosphate receptor and releases ca 2 + from internal stores . cells were incubated with 3 h - inositol ( 6 μci / ml ) in inositol - free rpmi 1640 medium ( nih media unit ) without serum for 2 to 3 days at 37 ° c . under an atmosphere of 5 % co 2 , 95 % air . cells were detached by trypsinization , washed twice in phosphate buffered saline ph 7 . 4 , resuspended in pbs with 10 mm licl , and maintained for 15 minutes at 37 ° c . after that , cells were washed twice with pbs plus 10 mm licl and resuspended in calcium medium plus 10 mm licl . the cell suspension was aliquoted into eppendorf tubes , pre - warmed at 37 ° c ., and treated with reagents for different times as indicated . reactions were terminated by adding 100 % trichloroacetic acid ( tca ) to a final concentration of 10 %. after centrifugation , the supernatants were removed , extracted twice with water - saturated ether to remove tca , and stored at 4 ° c . until quantitative analysis of inositol phosphates by high pressure liquid chromatography . samples prepared as described above were applied to a whatman partisil 5 sax column . inositol phosphates were separated using an ammonium phosphate gradient ( 0 . 01m to 1 . 4m . ph 3 . 8 , 1 ml / min flow rate ) as described previously ( biochem . j . 255 : 403 - 410 , 1988 ). radioactivity was measured by use of a raytest radiodetector with a 2 ml flow cell and a ratio of 4 : 1 ( v : v ) of monoflow 4 high salt scintillant ( national diagnostics ); eluate at an efficiency of 17 %. values ( counts / min ) for each inositol phosphate were expressed as a percentage of 3 h - phosphoinositides in the sample , permitting comparison among experiments . cell growth was studied by hemacytometer counts of viable cells after incubation of cells with purinergic agonists in vitro . viability was determined by trypan blue exclusion . in addition , growth studies were performed by 3 h - thymidine incorporation . these studies showed that over a period of 5 days , purinergic agonists stimulated 3 h - thymidine incorporation on a per cell basis while inhibiting cell growth . these data suggested that purinergic agonists are stimulating dna strand breaks and dna repair . the induction of dna strand breaks in the setting of cell growth inhibition is characteristic of programmed cell death or apoptosis . pc - 3 prostate carcinoma cells were treated in vitro with the purinergic agonists atp or amp - pnp , 100 um 3 times over 7 days , and their growth in athymic mice was compared to that in untreated control cells . cells were injected subcutaneously at an inoculum of 1 . 5 × 10 6 cells / mouse in 0 . 2 ml phosphate buffered saline , and the presence of palpable tumors was determined on day 22 . a wide variety of surface receptors that induce an increase in [ ca 2 + ] i was found ( fig1 ). these are novel observations for two reasons . firstly , the presence of these receptors on prostate carcinoma cells has not been reported . secondly , these are the first studies showing hormone - activated ca 2 + transients in prostatic tissue . the first studies of the biological effects of the ca 2 + response were directed to an examination of the effect of atp on the prostate carcinoma cell lines . aside from its role in cell metabolism , atp is now known to act extracellularly at specific plasma membrane receptors called purinergic receptors or purinoceptors . as can be seen from fig1 atp produces the largest ca 2 + response of the agonists tested . this response is so large in the prostate carcinoma cells that there is almost complete depletion of internal ca 2 + stores . the concentration - dependence of the atp - stimulated ca 2 + response is shown for the androgen - independent prostate carcinoma cell lines du145 ( fig2 ), pc - 3 ( fig3 ), and pc - 3m ( fig4 ). these three cell lines are the only well characterized human androgen - independent prostate carcinoma cell lines , and all three showed a strong purinergic response . there is one hormone - sensitive prostate carcinoma cell line , lncap , which was tested and found to lack a purinergic response ( data not shown ). purinergic receptors are expressed on a wide variety of cell types , ranging from hematopoietic cells to endothelial cells . these data are the first description of purinergic receptors on benign or neoplastic prostate cells , however , and suggest that the purinergic receptors identified may be specifically expressed on advanced , hormone - refractory prostatic carcinoma cells . hormone - stimulated ca 2 + transients are thought to be induced by activation of phospholipase c , an enzyme that catalyzes the hydrolysis of phosphatidylinositol bisphosphate to diacylglycerol and inositol trisphosphate , and inositol trisphosphate - mediated release of ca 2 + from internal ca + storage vesicles . whether atp acts through stimulation of phospholipase c has been determined by high pressure liquid chromatographic analysis of inositol phosphate metabolites in metabolically labeled prostate carcinoma cells ( see above , metabolism of [ 3 h ] inositol phosphates by pc - 3 and du145 cells ). the data in table 1 show a representative experiment performed in pc - 3 cells , demonstrating atp - stimulated phospholipase c activity . these data are the first to show hormone - stimulated phosphatidylinositol turnover in prostatic cells , and identify the signal transduction pathway activated by purinergic agonists in prostatic carcinoma cells . table 1______________________________________atp - stimulated inositol phosphateproduction in pc - 3 prostate carcinoma i ( 1 , 4 , 5 ) p3 cpm % of total ip______________________________________8 sec control 569 0 . 33atp 1103 0 . 6415 sec control 318 0 . 19atp 971 0 . 5730 sec control 794 0 . 46atp 1361 0 . 78______________________________________ purinergic receptors have been divided into different subtypes pharmacologically , on the basis of the order of potency of different purine compounds . in order to characterize the purinergic receptor on the prostate carcinoma cells , a series of purines was tested for their relative potency in inducing a ca 2 + transient . these compounds included the well known , commercially available purines atp ( adenosine triphosphate ), adp ( adenosine diphosphate ), amp ( adenosine monophosphate ), and adenosine , as well as the lesser known , commercially available compounds shown in fig5 a through fig5 e . the compounds shown in fig5 b through fig5 e are adenine nucleotides with modified phosphate groups . each of these modifications should increase the resistance of the compound to hydrolysis , and thus increase the usefulness of the compound as a therapeutic agent . the results of studies measuring the ca 2 + response to these compounds are shown in fig6 ( du145 ), fig7 ( pc - 3 ), and fig8 ( pc - 3m ). the data for each of these cell lines show expression of a p 2 subtype purinergic receptor , for which the more highly phosphorylated purines are most active , following the general scheme of agonist potency atp & gt ; adp & gt ; amp & gt ; adenosine , as opposed to p 1 purinergic receptors , for which adenosine & gt ; amp & gt ; adp & gt ; atp . these figures also disclose that the non - hydrolysable analogs have agonist activity . thus , the non - hydrolysable compounds should recognize androgen - independent prostate cancer in vivo , and therefore be therapeutically useful . it was found that the increase in cytoplasmic free ca 2 + induced by purinergic agonists is derived both from release of ca 2 + from internal stores and from the opening of plasma membrane ca 2 + channels on the prostate carcinoma cells . the opening of these channels serves to prolong the ca 2 + response to purinergic agonists by allowing influx of ca 2 + from the external milieu into the cell . these channels were characterized pharmacologically because a compound that opens such channels could be used in combination with the purinergic agonist to increase the therapeutic response . the channel was characterized by using specific , commercially available ca 2 + channel blockers to inhibit the influx of extracellular ca 2 + caused by atp stimulation of the prostate carcinoma cell lines . the data in fig9 show that the channel is inhibitable by the dihydropyridine class of ca 2 + channel blocker ( nicardipine and nifedipine ), by the phenylalkylamine class of channel blocker ( verapamil ), and by a benzothiazepine ( diltiazem ), but not by the peptide ca 2 + channel blocker ω - conotoxin . these data are the first characterization of the ca 2 + channel expressed on the surface of prostate carcinoma cells . the signal transduction studies that have been performed are studies of the response of many cells in a suspension . the purinergic response of individual cells has also been assessed to determine the purinergic response of individual cells to determine the degree of intratumor heterogeneity . these experiments were performed by flow cytometric analysis of the ca 2 + response , using a laser - activated flow microfluorometer ( see above , flow cytometric analysis of the ca 2 + response in single cells ). the contour plots in fig1 b show that there is heterogeneity in the purinergic responsiveness of the pc - 3 prostate carcinoma cell line . this technique is a useful adjunct for screening for therapeutically effective compounds because it allows the analysis of signal transductions on a single cell basis . since purinergic receptor agonists induce massive mobilization of internal ca 2 + stores and prolong this response through the opening of plasma membrane ca 2 + channels , the effect of purinergic receptor stimulation on the growth of androgen - independent cells was examined . the cell lines du145 , pc - 3 , and pc - 3m were incubated in the presence of atp or the nonhydrolysable atp analog adenylylimidodiphosphate ( amp - pnp ), and cell growth was determined by hemacytometer counts of viable cells , with viability determined by trypan blue exclusion . the inhibition of growth after 5 days of treatment is shown in fig1 ( du145 ) and fig1 ( pc - 3 ). these data are the first showing that purinergic agonists inhibit the growth of prostate carcinoma cells . to further document the purinergic agonist - induced growth inhibition , thymidine incorporation studies were performed in conjunction with hemacytometer cell counts , and the thymidine incorporation per cell of atp and amp - pnp treated pc - 3 and pc - 3m cells was determined . these studies , shown in table 2 , confirmed that purinergic agonists inhibit the growth of androgen - independent prostate carcinoma cells . furthermore , these studies show that 3 - thymidine incorporation is not inhibited when calculated on a per cell basis , despite the fact that cell growth was markedly inhibited . these data suggest that purinergic agonist inhibition of the growth of the prostate carcinoma cells is associated with , and may be mediated by , an increase in dna strand breaks . it is of interest that normal prostate cells die in response to androgen withdrawal in a process called apoptosis , and this cell death resembles the cell death shown after treatment with purinergic agonists in two respects . firstly , the death of normal prostate cells after androgen withdrawal is dependent on a rise in intracellular ca 2 + . secondly , the death of normal prostate cells after androgen withdrawal is mediated by an increase in the number of dna strand breaks . thus , this technique should be therapeutically useful for circumventing the absence of androgen receptors on advanced prostate carcinoma cells . the results presented here represent the discovery of a surface receptor specifically expressed on advanced prostatic adenocarcinoma which , when activated , induces a mode of cell death that apparently utilizes that same intracellular pathway as the death that normal prostate cells undergo in response to the withdrawal of androgen . table 2______________________________________comparison of cell number and . sup . 3 h - thymidine incorporation inpc - 3 and pc - 3m prostate carcinoma cell # (× 10 - 4 ) cpm cpm / cell______________________________________pc - 3control 23 . 0 5530 0 . 02atp 7 . 5 2729 0 . 04amp - pnp 0 . 5 307 0 . 06dbcamp 1 . 5 182 0 . 01pc - 3mcontrol 17 . 0 20665 0 . 12atp 3 . 0 3307 0 . 11amp - pnp 2 . 0 2790 0 . 14ionomycin 1 . 0 3920 0 . 39______________________________________ cells treated for 5 days ( pc3m ) or 7 days ( pc3 ) with atp ( 100 μm ), amppnp ( 100 μm ), or ionomycin ( 1 μm ) versus untreated control cells in further experiments , it has been found that purinergic agonist treatment inhibited the growth of androgen - independent prostate carcinoma cells in nude mice ( fig1 ). the cells were treated in vitro for 7 days , and then an equal number of untreated control cells , atp treated , or amp - pnp treated cells were inoculated subcutaneously into nude ( athymic ) mice . further nude mouse studies are presently being performed , using an implantable alzet pump to provide the equivalent of a continuous infusion . the compounds will be administered orally , by continuous infusion , or by intermittent infusion , accompanied by pharmacologic studies to determine the optimal dose and schedule to obtain a steady state plasma concentration of approximately 100 μm . the invention being thus described , it will be obvious that the same may be varied in many ways . such variations are not to be regarded as a departure from the spirit and scope of the invention , and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims .
the instant invention is directed to the use of a pharmaceutical composition containing an effective amount of adenosine or an adenosine analog or derivative as an agent useful in the treatment of hormone - independent cancers , such as prostate cancer . the invention is also directed to diagnostic uses of these compounds to determine effective treatment regimes for specific tumors , a process for screening for new , therapeutically useful analogs of these compounds , and the use of these compounds in facilitating the extraction of intracellular components .