tumor cell lines from human ( k562 myeloid leukemia , tib - 180 erythroleukemia , lncap prostate carcinoma and mda - mb468 breast adenocarcinoma ), and murine ( yac lymphoma , b - 16 melanoma ) were used and were purchased from the american type tissue culture collection , rockville . nb2 11c rat lymphoma cells ( pines et al . ( 19 ) , were also used . the cells were routinely maintained in rpm medium containing 10 % fetal calf serum . twice a week the cells were transferred to a freshly prepared medium . as a control for tumor proliferating cells , two normal proliferating cell types were used i . e ., bone marrow cells derived from the femur of c57bl / 6j mice ; fibroblasts derived from primary cultures of newborn rat skeletal muscle cells . the cells were prepared as previously , ( djaldetti , m ., sredni , b ., zigelman , r ., verber , m ., and fishman , p . muscle cells produce a low molecular weight factor with anti - cancer activity . clin . exp . matastasis , 14 : 189 - 196 , 1996 ). also used was the l - 8 myoblast rat cell line which was purchased from the atcc . the effect of adenosine on the proliferation of rat nb2 - 11c lymphoma cells was monitored by cell counting . the nb2 - 11c cells were synchronized in the go / gl phase prior to cultivation with the cm by transferring the cells to horse serum ( biological industries , beit haemek , israel ) supplemented medium for overnight incubation . 1 . 2 × 10 5 cells / ml were cultured in 24 well plates in 1 ml rpmi medium containing 5 % horse serum and 20 , 10 , 8 , 6 , 4 , 2 and 1 μm of adenosine of adenosine ( purchased from sigma , usa ) were added . cell proliferation was initiated by the addition of human growth hormone ( biotechnology general , rehovot , israel ) to a final concentration of 2 ng / ml . the cultures were incubated at 37 ° c . at 5 % co 2 and counted in coulter counter 48 hours later . inhibition of cell proliferation was calculated as follows : c s = cell count with adenosine c nh = cell count without hormone and without adenosine c wh = cell count with hormone but without adenosine . 0 . 9 × 10 4 / well of each tumor cell line or normal cells ( bone marrow cells or fibroblasts ) were incubated with rpmi medium containing 10 % fcs and 2 μm adenosine in 96 microtiter plates for 48 hours . during the last 6 hours of incubation , each well was pulsed with 1 μci [ 3 h ]- thymidine . the cells were harvested and the [ 3 h ]- thymidine uptake was determined in an lkb liquid scintillation counter ( lkb , piscataway , n . j ., usa ). a . effect of adenosine on tumor and normal cell proliferation the growth of the nb2 - 11c rat lymphoma cells ( measured by cell count ) was markedly inhibited following incubation with different concentrations of adenosine . as can be seen in fig1 , there is a dose dependent effect of adenosine on the proliferation of the nb2 - 11c cells . adenosine in a concentration of 4 μm induced proliferation inhibition in the leukemia , lymphoma and the solid tumor cell lines , while in the same time it stimulated the proliferation of the three tested normal cells ( fig2 ): all results shown as % of control ). additional studies revealed a dose dependent stimulatory effect of adenosine on bone marrow cell proliferation ( fig3 ) with a peak activity at about 10 μm ). b . theophylline ( adenosine a1 & amp ; a2 receptor antagonist ) neutralizes the inhibitory effect of adenosine on tumor cell proliferation theophylline is an antagonist of both a1 and a2 adenosine receptors . theophylline in a concentration of 0 . 1 μm was added to a culture of k562 cells in the presence of 4 μm adenosine . culture conditions were identical to those described above . as can be seen in fig4 , while adenosine inhibited tumor cell proliferation , in the presence of theophylline , this inhibitory effect was neutralized . c . effect of theophylline ( adenosine a1 and a2 receptor antagonist ). dpcpx ( adenosine a1 receptor antagonist ) and dpspx ( adenosine a2 receptor antagonist ) on bone marrow cell proliferation proliferation of bone marrow cells in the presence of the three adenosine receptor antagonists in the absence and in the presence of adenosine was tested . the concentration of theophylline was 0 . 1 μm ; and that of dpcpx and dpspx was 0 . 01 μm . the concentration of adenosine was 10 μm . the results shown in fig5 show that all these receptor antagonists caused a small increase in proliferation of bone marrow cells , while a much higher effect on increase of bone marrow cell proliferation was observed in the presence of adenosine . these results demonstrate that the stearatory effect of adenosine on bone marrow cells is likely through a mechanism not dependent on the adenosine receptors . in addition , although not wishing to be bound by theory , it is likely that the increase in proliferation obtained in the presence of the adenosine receptor antagonists is a result of freeing a larger amount of adenosine naturally present in the extracellular environment , e . g . adenosine secreted by cells from internal pools , to act through the proliferation stimulation pathways . 40 c57bl6 / j mice were divided into 4 groups each of which were treated ( by intraperitoneal injection ) according to one of the following protocols : 1 . control group : 6 days of treatment by daily injection of 1 ml saline . 2 . adenosine and chemotherapy group : daily injection of a 1 ml 5 mm adenosine solution for 5 consecutive days . on day 6 the mice were injected intraperitoneally with cyclophosphamide . 3 . adenosine group : daily injection of a 1 ml 5 nm adenosine solution for 5 consecutive days . on day 6 the mice were injected intraperitoneally with 1 ml saline . 4 . chemotherapy group : daily injection of 1 ml saline for 5 consecutive days . on day 6 the mice were injected intraperitoneally with cyclophosphamide . five mice of each group were sacrificed 25 hrs and 96 hrs following the treatments and two parameters were evaluated : 1 . complete blood cell count . 2 . proliferation capacity of bone marrow : bone marrow was aspirated from the femur and pooled for each tested group . the cells were separated , counted and the proliferative capacity was evaluated by the [ 3 h ]- thymidine incorporation assay as described above . following treatment with cyclophosphamide , the number of white blood cells dropped after 24 hrs as is shown in table 1 , below . in the group which was treated 5 days with adenosine prior to the cyclophosphamide administration , the number of white blood cells was even higher than that of the control group . the protective effect of adenosine was also demonstrated when testing bone marrow cell proliferation , as can be seen in table 2 below . it can be seen that adenosine increased the proliferating capacity of the bone marrow even above control , even in the presence of the chemotherapeutic drug . 1 . control group : 6 days of treatment by daily injection of 1 ml saline . 2 . chemotherapy group : daily injection of 1 ml saline for 5 consecutive days . on day 6 the mice were injected intraperitoneally with cyclophosphamide . 3 . adenosine group : daily injection of a 1 ml group 5 mm adenosine solution for 5 consecutive days . on day 6 the mice were injected intraperitoneally with 1 ml saline . 4 . adenosine and chemotherapy group : daily injection of a 1 ml 5 mm adenosine solution for 5 consecutive days . i . red blood cell ( rbc ) count . ii . hemoglobin ( hgb ) level . iii . white blood cell count . iv . proportion of the three major groups of white blood cells : lymphocytes ( lym ), monocytes ( mid ) and granulocytes ( gran ). the results ( mean ± s . e .) for each of the different groups is shown in the following table 3 . the above results demonstrate that while the chemotherapeutic drug had almost no effect on the red blood cell count and on the level of hemoglobin , it caused a reduction in the amount of white blood cells . as can be seen , the white blood cell count was boosted to levels above control after administration of adenosine and when adenosine was administered together with the chemotherapeutic drug , the white blood cell count remained at levels similar to control . the above results demonstrate that while the chemotherapeutic drug had almost no effect on the red blood cell count and on the level of hemoglobin , it caused a reduction in the amount of white blood cells . as can be seen , the white blood cell count was boosted to levels above control after administration of adenosine and when adenosine was administered together with the chemotherapeutic drug , the white blood cell count remained at levels similar to control . while the chemotherapeutic drugs have a toxic effect and thus give rise to reduction in count of all white blood cells , this effect is more dominant on granulocytes . this can be seen by the fact that following administration of the chemotherapeutic drug the relative proportion of granulocytes decreases while the relative proportion of lymphocytes decreases . this effect is reversed upon administration of adenosine , namely , adenosine has a more pronounced effect in causing proliferation of granulocytes in various other types of white blood cells . in a large number of in vivo studies conducted using similar protocols , the adenosine dosage which was used was reduced to about 54 μ / kg body weight per day , with a similar effect of adenosine , in toxic effect of the chemotherapeutic drag on the count of leukocytes and peripheral blood granulocytes . additionally , in further experiments conducted in accordance with the invention it was found that in order to exert its protective effect , it is sufficient to administer the adenosine for several days prior to the treatment with the chemotherapeutic drug and this effect does not require continued administration of adenosine during the course of treatment with a chemotherapeutic drug . b - 16 - f10 melanoma cells ( 2 × 10 5 ) were intravenously injected to 40 c57bl6 / j mice . the mice were divided into 4 groups each of 10 mice which were treated by intraperitoneal administration of one of the following : 1 . control group : daily administration of 1 ml saline , per mouse from day of tumor inoculation until the mice were sacrificed . 2 . adenosine group : daily administration of 1 ml of a 5 mm adenosine solution per mouse from day of tumor inoculation until the mice were sacrificed . 3 . chemotherapy group : one injection of cyclophosphamide 24 hrs after inoculation of tumor cells and daily administration of 1 ml of saline per mouse from day of tumor inoculation until the mice were sacrificed . 4 . chemotherapy and adenosine : one injection of cyclophosphamide 24 hrs after inoculation of tumor and daily administration of 1 ml of 5 mm per mouse from day of tumor inoculation until the mice were sacrificed . following 18 days the mice were sacrificed and melanoma tumor foci were counted in the lung . the results are summarized in table 4 . in another experiment , a similar protocol was followed , with a lower dosage of adenosine ( oral administration of 0 . 5 ml of a 10 μm adenosine solution , a dose equal to about 54 μg / kg body weight of adenosine ). the results are shown in fig6 . as can be seen , here again , adenosine synergized with the chemotherapeutic drug to reduce the tumor load . 50 c57bl6 / j mice were divided into 5 groups of 10 mice each , which were treated by intraperitoneal injection with one of the following treatments : 1 . control group : 6 days of treatment by daily injection of 1 ml saline . 2 . adenosine group i : daily injection of 1 ml 5 m adenosine solution for 6 days ( a dose of about 5 μm / kg body weight ). 3 . adenosine group ii : daily injection of 1 ml 10 m adenosine solution for 6 days ( a dose of about 1 . 08 μg / kg body weight ). 4 . chemotherapy group : daily injection of 1 ml saline for 5 consecutive days . on day 6 the mice were injected intraperitoneally with cyclophosphamide . 5 . adenosine and chemotherapy group : daily injection of 1 ml 10 m adenosine solution for 5 consecutive days . on day 6 the mice were injected intraperitoneally with cyclophosphamide . the total white blood cell count and the percentage of granulocytes , in each of the groups , is shown in the following table 5 . the above results show that adenosine at both concentrations had no effect in increasing the percentage of granulocytes in the white blood cell population . in addition , while the chemotherapy caused a decreased in the number of white blood cells and in the relative proportion of granulocytes , the adenosine was capable of restoring the total white blood cell count , although not to full control levels , but more importantly , notwithstanding the chemotherapy , it yielded a very large increase in the relative proportion of granulocytes . mononuclear cells ( mnc ) were fractionated from heparinized blood of 10 healthy volunteers using ficoll - hypaque gradient . 5 × 10 6 mononuclear cells / ml were incubated with 4 μm adenosine and rpmi containing 10 % fcs with and without 0 . 0075 % of the mitogen staphylococcus aureus cowan i ( sac , calbiochem pansorbin ) for 18 hours . at the end of the incubation period the supernatant was collected , centrifuged and filtered through 0 . 22μ sterile filter and kept at − 70 ° c . until assayed . the level of il - 12 in the supernatants was analyzed using a commercial kit of r & amp ; d system . the effect of adenosine on the activity of human peripheral blood nk cells was assayed by a standard 4 h 51 cr - release assay using k562 leukemia cells as targets . peripheral blood mononuclear cells were separated from 10 healthy volunteers using ficoll - hypaque gradient . cells were cultured at a concentration of 5 × 10 5 cells / well in 96 well round bottom plates , and used as the effector ( e ) cells . the cells were preincubated with 4 μm adenosine in rpmi containing 5 % fcs for 18 hours . k562 cells were used as the targets ( t ) and were labeled with 100 μci of na 2 [ 51 cr ] o 4 at 37 ° for 1 h . after extensive washing to remove the excess cr , cells ( 1 × 10 4 ) were resuspended and mixed with the effector cells at the e : t ratio of 1 : 50 in a volume of 200 μl using triplicate assays . after 4 h of incubation at 37 ° c . in 5 % co 2 , plates were centrifuged , and the supernatants were counted in a gamma counter ( lkb ). % ⁢ ⁢ lysis = cpm ⁢ ⁢ experiment - cpm ⁢ ⁢ spontaneous cpm ⁢ ⁢ maximal - cpm ⁢ ⁢ spontaneous × 10 where cpm spontaneous and cpm maximal were determined by measuring cpm of the supernatants of the target cells alone in the presence of assay medium or in the presence of 1 % sds , respectively . spontaneous release was below 8 % of the maximal release throughout this experiment . adenosine stimulated the production of il - 12 by human peripheral blood mononuclear cells in the presence of sac mitogen ( fig7 ). in another set of experiments adenosine induced il - 12 production without the presence of mitogen in the culture system ( 19 % stimulation of il - 12 production ). nk activity was also stimulated by 32 % following the addition of adenosine to the culture system . it may be concluded that one of the potential mechanisms through which adenosine stimulates nk activity is the induction of il - 12 production which is known as an activator of nk cells . b . in vivo effect of adenosine on nk activity of mice splenocytes icr mice were treated for 5 consecutive days with 10 μm of adenosine . three days later the mice were sacrificed , spleens were taken out and mononuclear cells were separated using ficoll hypaque gradient . nk activity was measured as describe above for the human assay excluding the use of yac murine lymphoma cells as targets instead of the k562 cells . nk cells derived from adenosine treated mice were more potent in lysing the yac target cells as was seen in the two ratios of effectors : targets assayed ( fig8 ). the modulating affect of adenosine may explaine some of its in vivo anti - tumoral activity shown below .
adenosine and active agent which interact with the adenosine system are used to treat conditions of weakened , immune system , as an anti - cancer therapy and for improving the therapeutic index of a variety of therapeutic drugs .