Patent Abstract:
the present invention relates to the signalling pathways connecting dna damage , such as that induced by ionizing radiation or alkylating agents , and phosphorylation by tyrosine kinases .

Detailed Description:
the following examples are included to demonstrate preferred embodiments of the invention . it should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention , and thus can be considered to constitute preferred modes for its practice . however , those of skill in the art should , in light of the present disclosure , appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention . alkylating agents activate the lyn tyrosine kinase and promote tyrosine phosphorylation of p34 cdc2 cell culture . hl - 60 cells were grown in rpmi - 1640 medium containing 15 % heat - inactivated fetal bovine serum ( fbs ) supplemented with 100 units / ml penicillin , 100 mg / ml streptomycin , 2 mm l - glutamine , 1 mm sodium pyruvate and 1 mm non - essential amino acids . cells were treated with mmc ( sigma chemical co ., st . louis , mo . ), adozelesin ( sigma ), cis - platinum ( sigma ), nitrogen mustard ( sigma ), genistein ( gibco / brl , gaithersburg , md . ), herbimycin a ( gibco / brl ) and h - 7 ( seikagaku america inc ., rockville , md .). cell viability was determined by trypan blue exclusion . immune complex kinase assays . cells ( 2 − 3 × 10 7 ) were washed twice with ice cold phosphate buffered saline ( pbs ) and lysed in 2 ml of lysis buffer ( 20 mm tris , ph 7 . 4 , 150 mm nacl , 1 % np - 40 , 1 mm sodium vanadate , 1 mm phenylmethylsulfonyl fluoride , 1 mm dtt and 10 mg / ml of leupeptin and aprotinin ). after incubation on ice for 30 min , insoluble material was removed by centrifugation at 14000 rpm for 10 min at 4 ° c . soluble proteins were precleared by incubating with 5 mg / ml rabbit - anti - mouse igg for 1 h at 4 ° c . and then for an additional 30 min after addition of protein a - sepharose . the supernatant fraction was incubated with pre - immune rabbit serum , anti - fyn , anti - lyn , anti - src ( ubi , lake placid , n . y .) or anti - cdc2 ( sc - 54 , santa cruz biotechnology , santa cruz , calif .) antibodies for 1 h at 4 ° c . followed by 30 min after addition of protein a - sepharose . the immune complexes were washed three times with lysis buffer and once with kinase buffer ( 20 mm hepes , ph 7 . 0 , 10 mm mncl 2 and 10 mm mgcl 2 ) and resuspended in 30 ml of kinase buffer containing 1 mci / ml [ γ - 32 p ] atp ( 3000 ci / mmol ; nen , boston , mass .) with and without 5 - 8 mg of acid - treated enolase ( sigma ). the reaction was incubated for 15 min at 30 ° c . and terminated by the addition of 2 × sds sample buffer . the proteins were separated in 10 % sds - polyacrylamide gels and analyzed by autoradiography . radioactive bands were excised from certain gels and quantitated by scintillation counting . immune complexes were also resuspended in 30 ml kinase buffer containing 1 mci / ml [ γ - 32 p ] atp and either 100 mm cdc2 peptide ( amino acids 7 to 20 ; iekigegt y gvvyk ; seq id no : 1 ) or 100 mm mutated cdc2 peptide with phe - 15 substituted for tyr - 15 ( iekigegt f gvvyk ; seq id no : 2 ). the reactions were incubated for 15 min at 30 ° c . and terminated by spotting on p81 phosphocellulose discs ( gibco / brl ). the discs were washed twice with 1 % phosphoric acid and twice with water before analysis by liquid scintillation counting . immunoblot analysis . immune complexes bound to protein a - sepharose were prepared as for the autophosphorylation assays . proteins were separated in 10 % sds - polyacrylamide gels and transferred to nitrocellulose paper . the residual binding sites were blocked by incubating the filters in 5 % dry milk in pbst ( pbs / 0 . 05 % tween - 20 ) for 1 h at room temperature . the blots were subsequently incubated with anti - cdc2 or anti - phosphotyrosine ( anti - p - tyr ; mab 4g10 , ubi ). after washing twice with pbst , the filters were incubated for 1 h at room temperature with anti - mouse igg ( whole molecule ) peroxidase conjugate ( sigma ) in 5 % milk / pbst . the filters were then washed and the antigen - antibody complexes visualized by the ecl detection system ( amersham , arlington heights , ill .). coimmunoprecipitation . immunoprecipitations were performed with anti - p34 cdc2 at 5 mg / ml cell lysate . immune complexes were collected on protein a - sepharose beads ( pharmacia ), washed three times with lysis buffer and twice with kinase buffer , resuspended in kinase buffer and then incubated for 10 min at 30 ° c . in the presence of 1 mci / ml [ γ - 32 p ] atp . one aliquot of the kinase reaction was subjected to sds - page and autoradiography . the other aliquot was washed in lysis buffer to remove free atp and then boiled in 20 mm tris - hcl , ph 8 . 0 containing 0 . 5 % sds and 1 mm dtt to disrupt protein - protein interaction . after dilution to 0 . 1 % sds , a secondary immunoprecipitation was then performed by adding anti - lyn antibody and protein a - sepharose beads . the anti - lyn immunoprecipitates were then subjected to sds - page and autoradiography . fusion protein binding assays . the plasmid encoding a glutathione s - transferase ( gst )- lyn ( amino acids 1 to 243 ) fusion protein was obtained from t . pawson , toronto , canada and transfected into e . coli dh5a ( pleiman et al ., 1993 ). the fusion protein was induced with iptg , purified by affinity chromatography using glutathione - sepharose beads ( pharmacia ) and equilibrated in lysis buffer . hl - 60 cell lysates were incubated with 50 mg immobilized gst or gst - lyn for 2 h at 4 ° c . the protein complexes were washed three times with lysis buffer and boiled for 5 min in sds sample buffer . the complexes were then separated in 10 % sds - page and subjected to silver staining or immunoblot analysis with anti - cdc2 . previous studies have demonstrated that hl - 60 cells express the p59 fyn , p56 / p53 lyn and pp60 c - src tyrosine kinases ( barnekow & amp ; gessler , 1986 ; gee et al ., 1986 ; katagiri et al ., 1991 ). in this example , the inventors have shown that certain of these tyrosine kinases are activated during treatment of hl - 60 cells with mmc . immunoprecipitates from control and mmc - treated cells were assayed for autophosphorylation . there was no detectable kinase activity in precipitates obtained with pre - immune rabbit serum ( fig1 a ). other studies with an anti - fyn antibody demonstrated that autophosphorylation of p59 fyn is decreased at 1 h of mmc treatment ( fig1 b ). similar results were obtained at multiple time points through 6 h of mmc exposure . in contrast , immunoprecipitates with anti - lyn demonstrated an increase in p56 / p53 lyn activity as a result of mmc exposure ( fig1 c ). the finding that anti - src immunoprecipitates also exhibited a decrease in pp60 c - src activity in mmc - treated cells ( fig1 d ) suggests that mmc exposure is associated with selective activation of p56 / p53 lyn . activation of p56 / p53 lyn was confirmed at different concentrations of mmc and by assaying for phosphorylation of the substrate protein enolase . increases in p56 / p53 lyn activity were found at 10 − 8 and 10 − 7 m mmc , while more pronounced stimulation of this kinase was apparent at 10 − 6 and 10 − 5 m ( fig2 a ). the results further demonstrate that p56 / p53 lyn activity is rapidly induced in mmc - treated cells . increases in mmc - induced phosphorylation of p56 / p53 lyn and enolase were first detectable at 30 min ( 4 . 2 - fold increase for enolase ) and persisted through at least 12 h ( 4 . 1 - fold for enolase ) of drug exposure ( fig2 b ). the induction of p56 / p53 lyn activity was not related to cell death since viability as determined by trypan blue exclusion was & gt ; 90 % at 12 h of mmc treatment . immunoblot analysis was also performed to determine whether the increases in p56 / p53 lyn activity were due to a greater abundance in protein . the results demonstrate similar levels of p56 / p53 lyn protein ( fig2 c ). these findings supported a rapid and prolonged activation of p56 / p53 lyn in response to mmc treatment . in order to confirm that activation of p56 / p53 lyn is associated with tyrosine phosphorylation , the anti - lyn immune complexes were assayed by immunoblotting with anti - p - tyr . the results demonstrate an increase in tyrosine phosphorylation of p56 / p53 lyn from mmc - treated as compared to control cells ( fig3 a ). analysis of the anti - lyn immunoprecipitates by immunoblotting with anti - lyn confirmed the presence of similar levels of protein after mmc treatment ( fig3 b ). the involvement of tyrosine phosphorylation was further supported by the demonstration that pretreatment of cells with the tyrosine kinase inhibitors , genistein ( akiyama et al ., 1987 ) and herbimycin a ( uehara et al ., 1989 ) completely blocks the stimulation of p56 / p53 lyn activity associated with mmc treatment ( fig4 a ). in contrast , pretreatment with the isoquinoline sulfonamide inhibitor of serine / threonine protein kinases , h - 7 ( hidaka et al ., 1984 ), had no detectable effect on the mmc - induced activity ( fig4 b ). these effects of mmc on induction of p56 / p53 lyn could be related to direct interaction of this agent with lyn kinase . however , incubation of anti - lyn immune complexes in the presence of mmc was associated with a decrease in kinase activity ( fig4 c ). taken together , these findings indicated that mmc induces the tyrosine kinase activity of p56 / p53 lyn by an indirect mechanism . the available evidence indicates that mmc acts as a monofunctional and bifunctional alkylating agent ( carrano et al ., 1979 ). consequently , adozelesin , another monofunctional but structurally distinct alkylating agent ( bhuyan et al ., 1992 ; hurley et al ., 1984 ), was investigated . the results demonstrate that treatment of hl - 60 cells with adozelesin is similarly associated with stimulation of p56 / p53 lyn and enolase phosphorylation ( fig5 a ). other studies were performed with agents that also induce the formation of dna cross - links . nitrogen mustard , an agent that forms monoadducts and dna interstrand cross - links ( ewig & amp ; khon , 1977 ; hartley et al ., 1992 ), was effective in inducing p56 / p53 lyn activity ( fig5 b ). moreover , treatment of cells with cis - platinum , an agent that forms intrastrand cross - links ( sherman & amp ; lippard , 1987 ), was associated with stimulation of the p56 / p53 lyn kinase ( fig5 c ). these findings indicated that the response of cells to diverse alkylating - type agents induces activation of p56 / p53 lyn . in order to examine the significance of p56 / p53 lyn activation , the association of this kinase with specific intracellular proteins that undergo tyrosine phosphorylation in mmc - treated cells was investigated . this issue was initially addressed using a gst - lyn fusion protein to identify molecules which interact with p56 / p53 lyn . lysates from mmc - treated cells were incubated with immobilized gst or gst - lyn . analysis of the adsorbates by sds - page and staining demonstrated the presence of a 34 kd protein . the inventors assayed the adsorbates for reactivity with anti - cdc2 . the results indicate that p34 cdc2 associates with the gst - lyn fusion protein and not the gst control ( fig6 a ). the potential interaction between p56 / p53 lyn and p34 cdc2 was further examined in coimmunoprecipitation studies . lysates of control and mmc - treated cells were subjected to immunoprecipitation with anti - cdc2 and the immunoprecipitates were assayed for autophosphorylation ( fig6 b ). one aliquot of the in vitro kinase reaction was assayed by sds - page and autoradiography . while immunoprecipitates from mmc - treated cells exhibited phosphorylation of 53 - 56 kd proteins , there was little if any of this activity in control cells ( fig6 b ). in order to determine whether the anti - cdc2 immunoprecipitates contain p56 / p53 lyn , the other aliquot of the in vitro kinase reaction was treated to disrupt protein complexes and then subjected to immunoprecipitation with anti - lyn . the results demonstrate increased levels of autophosphorylated p56 / p53 lyn when assaying mmc - treated as compared to control cells ( fig6 b ). the finding that mmc exposure induces an interaction between p56 / p53 lyn and p34 cdc2 prompted further studies to determine whether p34 cdc2 exhibits increased tyrosine phosphorylation in mmc - treated cells . immunoprecipitation of p34 cdc2 and then immunoblotting of the precipitates with anti - p - tyr demonstrated an increase in reactivity as a result of mmc treatment ( fig7 a ). reprobing the filter with the anti - cdc2 antibody demonstrated similar levels of p34 cdc2 protein ( fig7 b ). since these findings indicated that mmc treatment is associated with increased tyrosine phosphorylation of p34 cdc2 , other studies were performed to determine whether p56 / p53 lyn can phosphorylate p34 cdc2 in vitro . in order to study a potential phosphorylation site for src - like kinases located at tyr - 15 of p34 cdc2 , synthetic peptides were prepared with sequences derived from amino acids 7 to 20 of p34 cdc2 and another with substitution at tyr - 15 with phe - 15 . while anti - lyn immune complexes from control cells phosphorylated the cdc2 peptide , similar complexes from mmc - treated cells exhibited nearly a 2 - fold stimulation in this activity ( fig8 ). in contrast , there was little phosphorylation of the mutated cdc2 peptide with anti - lyn complexes from control or mmc - treated cells ( fig8 ). these findings indicated that p56 / p53 lyn phosphorylates the tyr - 15 site of p34 cdc2 . the present results demonstrate that treatment of hl - 60 cells with mmc is associated with selective activation of the p56 / p53 lyn tyrosine kinase . these findings are not limited to hl - 60 cells since other cell lines , for example u - 937 myeloid leukemia cells , also respond to this agent with increases in p56 / p53 lyn activity . the lyn gene encodes two forms of the tyrosine kinase , p56 lyn and p53 lyn , due to alternate mrna splicing ( yamanashi et al ., 1987 ; yamanashi et al ., 1989 ). as a member of the src - like family of tyrosine kinases , p56 / p53 lyn is related to pp60 c - src and p59 fyn ( cantley et al ., 1991 ). however , only p56 / p53 lyn was activated in mmc - treated cells . these kinases are often associated with cell surface receptors at the interface between the cell membrane and cytoplasm . studies of p56 / p53 lyn in b cells have demonstrated an association with the b - cell antigen receptor ( pleiman et al ., 1993 ; yamanashi et al ., 1992 ). engagement of the b - cell antigen receptor induces activation of p56 / p53 lyn , as well as other src - like kinases , and tyrosine phosphorylation of substrates that include plcg2 , map kinase and gap ( pleiman et al ., 1993 ). other studies have shown that p56 / p53 lyn associates with the 85 kda a - subunit of pi 3 - k and induces pi 3 - k activity ( yamanashi et al ., 1992 ). thus , p56 / p53 lyn is capable of associating with and phosphorylating diverse downstream effector molecules . although the cellular effects of alkylating agents such as mmc are generally attributed to dna damage , their action may be related to alkylation of rna or protein . the demonstration that mmc treatment of intact cells is associated with activation of p56 / p53 lyn raised the possibility that this effect might be due to direct alteration of lyn protein . p56 / p53 lyn activity was however decreased in vitro by incubation of anti - lyn immune complexes with mmc . in order to address the possibility that mmc - induced activation of p56 / p53 lyn is related to formation of dna lesions , another agent , adozelesin , was used that covalently binds to the n - 3 of adenine within the minor groove of dna ( bhuyan et al ., 1992 ; hurley et al ., 1984 ). adozelesin also induces p56 / p53 lyn activity . hl - 60 cells also respond similarly to other alkylating agents , such as nitrogen mustard which reacts predominantly with guanines by alkylation of their n - 7 positions or forms dna interstrand cross - links ( ewig & amp ; khon , 1977 ; hartley et al ., 1992 ). moreover , p56 / p53 lyn activity was stimulated by cis - platinum which induces intrastrand cross - links ( sherman & amp ; lippard , 1987 ). thus , structurally distinct agents that damage dna by diverse mechanisms are capable of inducing p56 / p53 lyn activity . recent studies have demonstrated that treatment of hela cells with ultraviolet ( uv ) irradiation is associated with increases in the catalytic activity of c - src and c - fyn , but not that of c - yes ( devary et al ., 1992 ). taken together with the absence of detectable pp60 c - src or p59 fyn activation in mmc - treated hl - 60 cells , these results suggest that induction of these tyrosine kinases may be cell - type or agent specific . the p34 cdc2 serine / threonine protein kinase controls entry of cells into mitosis ( nurse , 1990 ; pines & amp ; hunter , 1990 ). this kinase is regulated by networks of kinases and phosphatases that appear to respond to the state of dna replication . activation of p34 cdc2 involves association with cyclin b and posttranslational modifications of the p34 cdc2 / cyclin b complex ( norbury & amp ; nurse , 1992 ). phosphorylation of p34 cdc2 on thr - 161 is required for activation ( atherton - fessler et al ., 1993 ; desai et al ., 1992 ; solomon et al ., 1992 ), while tyr - 15 phosphorylation results in inhibition of both p34 cdc2 activity and entry of cells into mitosis ( gould & amp ; nurse , 1989 ; gould et al ., 1990 ). studies have demonstrated that treatment of mammalian cells with alkylating and other dna - damaging agents is associated with g 2 arrest ( konopa , 1988 ; lau & amp ; pardee , 1982 ; tobey , 1975 ). however , the precise mechanisms responsible for this effect have remained unclear . exposure of cells to ionizing radiation is associated with rapid inhibition of p34 cdc2 activity and g 2 arrest ( lock & amp ; ross , 1990 ). other studies have demonstrated that arrest of nitrogen mustard - treated cells at g 2 is temporally related to formation of dna cross - links and p34 cdc2 inhibition ( o &# 39 ; connor et al ., 1992 ). in the present studies , it is demonstrated that mmc treatment results in rapid tyrosine phosphorylation of p34 cdc2 . similar findings have been obtained in cells treated with ionizing radiation ( see following examples ). this modification of p34 cdc2 is associated with loss of kinase activity as determined by assaying anti - cdc2 immunoprecipitates for phosphorylation of h1 histone . thus , the phosphorylation of p34 cdc2 on tyrosine appears to represent in part the response of mammalian cells to dna damage and may contribute to g 2 arrest by inhibition of p34 cdc2 activity . the available evidence indicates that the p107 wee1 dual - specificity kinase is responsible for phosphorylation of p34 cdc2 on tyr - 15 ( featherstone & amp ; russell , 1991 ; parker et al ., 1991 ; parker et al ., 1992 ). while p107 wee1 appears to control p34 cdc2 activity to ensure completion of s - phase , other studies suggest that p107 wee is not required for the dna - damage - dependent mitotic checkpoint . in this context , normal mitotic arrest has been observed after irradiation of schizosaccharomyces pombe cells with a defective or missing wee1 gene ( barbet & amp ; carr , 1993 ). other studies have shown that p34 cdc2 is phosphorylated on tyrosine in yeast wee1 minus mutants ( gould et al ., 1990 ). the present results in mammalian cells suggest that regulation of p34 cdc2 following exposure to alkylating agents involves activation of p56 / p53 lyn . the association of p56 / p53 lyn and p34 cdc2 in mmc - treated cells , as well as the finding that p56 / p53 lyn can phosphorylate the tyr - 15 site of p34 cdc2 in vitro , support the possibility that p56 / p53 lyn contributes to signaling from the mitotic checkpoint that monitors for alkylating agent - induced damage . ionizing radiation activates the lyn tyrosine kinase and promotes tyrosine phosphorylation of p34 cdc2 treatment of human hl - 60 myeloid leukemia cells with ionizing radiation is associated with activation of the lyn tyrosine kinase . the lyn gene encodes two forms of this kinase , p56 lyn and p53 lyn , as a result of alternate splicing ( yamanashi et al ., 1987 ; 1989 ). both p56 / p53 lyn , but not certain other src - related kinases , are activated in irradiated hl - 60 cells . activation of p56 / p53 lyn represents a signaling pathway distinct from those involved in x - ray - induced early response gene expression . hl - 60 myeloid leukemia cells were grown in rpmi - 1640 medium containing 15 % heat - inactivated fetal bovine serum ( fbs ) supplemented with 100 units / ml penicillin , 100 mg / ml streptomycin , 2 mm l - glutamine , 1 mm sodium pyruvate and 1 mm non - essential amino acids . cells in logarithmic growth phase were suspended in complete rpmi - 1640 medium with 0 . 5 % fbs 18 hours prior to irradiation . irradiation was performed at room temperature using a gammacell 1000 ( atomic energy of canada , ottawa ) under aerobic conditions with a 137 cs source emitting at a fixed dose rate of 13 . 3 gy / min as determined by dosimetry . hl - 60 cells were also treated with 50 mm h 2 o 2 ( sigma chemical co ., st . louis , mo . ), 30 mm n - acetyl cysteine ( nac ; sigma ), 10 μm genistein ( gibco / brl , gaithersburg , md .) or 10 μm herbimycin a ( gibco / brl ). cells ( 2 − 3 × 10 7 ) were washed twice with ice cold phosphate buffered saline ( pbs ) and lysed in 2 ml of lysis buffer ( 20 mm tris , ph 7 . 4 , 150 mm nacl , 1 % np - 40 , 1 mm sodium vanadate , 1 mm phenylmethylsulfonyl fluoride , 1 mm dtt , and 10 mg / ml of leupeptin and aprotinin ). after incubation on ice for 30 min , insoluble material was removed by centrifugation at 1400 rpm for 10 min at 4 ° c . soluble proteins were precleared by incubation with 5 mg / ml rabbit anti - mouse igg for 1 hour at 4 ° c . and then addition of protein a sepharose for 30 min . the supernatants were incubated with 2 . 5 μl of anti - human fyn , 2 μl of anti - human lyn , 3 μl of anti - human lyk ( n - terminal ) or 3 μl of anti - src antibody ( ubi , lake placid , n . y .) for 1 hour at 4 ° c . followed by 30 min with protein a - sepharose . the immune complexes were washed three times with lysis buffer , once with kinase buffer ( 20 mm hepes , ph 7 . 0 , 10 mm mncl 2 and 10 mm mgcl 2 ) and resuspended in 30 μl of kinase buffer containing 1 mci / ml [ γ - 32 p ] atp ( 3000 ci / mmol ; nen , boston , mass .). the reaction was incubated for 10 min at 30 ° c . and terminated by the addition of 2 × sds sample buffer . the proteins were resolved in 10 % sds - polyacrylamide gels , dried and analyzed by autoradiography . immune complexes as prepared for autophosphorylation assays were washed three times with lysis buffer and once with kinase buffer . the beads were resuspended in 30 μl of kinase buffer containing 1 mci / ml [ γ - 32 p ] atp and 3 - 5 mg of acid treated enolase ( sigma ). the reaction was incubated for 10 min at 30 ° c . and terminated by the addition of 2 × sds sample buffer . the proteins were resolved by 10 % sds - page . equal loading of the enolase was determined by staining with coomassie blue . the gels were then destained and analyzed by autoradiography . radioactive bands were also excised from the gel and quantitated by scintillation counting . previous studies have demonstrated that p59 fyn and p56 / p53 lyn are expressed in hl - 60 cells ( katagiri et al ., 1991 ). using autophosphorylation assays , the present inventors herein show that irradiation of hl - 60 cells with 200 cgy was associated with little if any change in p59 fyn activity at 15 min and 12 hours ( fig9 a ). a more detailed analysis between those time points revealed similar findings . in contrast , p56 / p53 lyn activity was increased at both 15 min and 12 hours after irradiation as compared to that in untreated cells ( fig9 b ). studies of p56 lck demonstrated little detectable activity in hl - 60 cells before or after exposure to ionizing radiation ( fig9 c ). these findings show that p56 / p53 lyn is selectively activated in hl - 60 cells by ionizing radiation . this conclusion is further supported by the absence of an increase in c - src activity following irradiation . hl - 60 cells were also irradiated with 200 cgy and immunoprecipitates assayed for both p56 / p53 lyn autophosphorylation and enolase ( a substrate protein ) phosphorylation . irradiation was associated with an increase in p56 / p53 lyn autophosphorylation at 5 min that persisted through 12 hours ( fig1 a ). however , assays at 24 hours after x - ray treatment revealed declines in p56 / p53 lyn signals ( fig1 a ). similar findings were obtained when using enolase as the substrate . while stimulation of p56 / p53 lyn autophosphorylation was less apparent under these conditions , increases in enolase phosphorylation were clearly detectable when comparing anti - lyn immunoprecipitates from control and irradiated hl - 60 cells ( fig1 b ). this increase in activity was rapid and sustained for at least 12 hours ( fig1 b ). quantitation of 32 p - incorporation into enolase by scintillation counting demonstrated x - ray - induced increases in p56 / p53 lyn activity of approximately 3 - fold at 15 min to 12 hours ( fig1 b ). as observed in autophosphorylation studies , enolase phosphorylation was also decreased at 24 hours ( fig1 b ). similar studies were performed at different doses of ionizing radiation ( fig1 ). treatment with 25 cgy had little if any effect on phosphorylation of p56 / p53 lyn or enolase . doses of 50 cgy , however , were associated with increases in p56 / p53 lyn activity ( fig1 ). moreover , on the basis of enolase phosphorylation there was an apparent dose - dependent stimulation of this kinase ( fig1 ). the cellular effects of ionizing radiation are believed to be related to direct interaction of x - rays with dna or through the formation of reactive oxygen intermediates ( rois ) which damage dna and cell membranes ( hall , 1988 ). while the role of different classes of rois in activation of the src - like kinases is unclear , recent studies have demonstrated that h 2 o 2 and diamide , which oxidize free sulfhydryl groups in cells , activate p56 lck in t cells ( nakamura et al ., 1993 ). hl - 60 cells were either treated with h 2 o 2 for the indicated times or pretreated with 30 mm nac for 1 hour , irradiated ( 200 cgy ) and harvested at 12 hours . irradiated hl - 60 cells treated with h 2 o 2 did not show a detectable increase in phosphorylation of p56 / p53 lyn or enolase ( fig1 a ). cells were also treated with the antioxidant nac ( roederer et al ., 1990 ; staal et al ., 1990 ), an agent that abrogates oxidative stress by scavenging certain rois and increasing intracellular glutathione levels ( aruoma et al ., 1989 ; burgunder et al ., 1989 ). nac had little effect on x - ray - induced p56 / p53 lyn activity ( fig1 a ), while this agent completely blocks induction of c - jun and egr - 1 gene expression in irradiated hl - 60 cells ( datta et al ., 1992b ; 1993 ). hl - 60 cells were treated with 10 μm herbimycin ( h ) or 10 μm genistein ( g ) for 1 hour , irradiated ( 200 cgy ) and then harvested at 12 hours . cell lysates were immunoprecipitated with anti - lyn and the immunoprecipitates were analyzed for phosphorylation of p56 / p53 lyn and enolase . in marked contrast , the tyrosine kinase inhibitors , herbimycin and genistein inhibited x - ray - induced p56 / p53 lyn activity ( fig1 b ). previous work has demonstrated that both ionizing radiation and h 2 o 2 are potent inducers of c - jun gene transcription ( datta et al ., 1992b ). these two agents have also been used to support the role of rois in targeting cc ( a / t ) 6 gg sequences to mediate activation of the egr - 1 gene ( datta et al ., 1993 ). the finding that such induction of early response gene transcription is inhibited by nac further supports the role of some of these intermediates in x - ray - induced nuclear signaling mechanisms . the present invention provides for the activation of p56 / p53 lyn as a distinct cellular response to ionizing radiation and not to h 2 o 2 - induced oxidative stress . these findings contrast work by others which suggested that src - like tyrosine kinases , including p56 / p53 lyn , are not responsible for signaling in irradiated b cells ( uckun et al ., 1992a ). the demonstration that ionizing radiation , and not h 2 o 2 , induces p56 / p53 lyn activity by an nac - insensitive mechanism therefore indicates that activation of this tyrosine kinase is independent from those signals responsible for x - ray - induced early response gene expression . the finding in b cells that p56 / p53 lyn is functionally associated with the cell surface ( yamanashi et al ., 1992 ) suggests that activation of this kinase by ionizing radiation may be generated near the plasma membrane rather than in the nucleus . indeed , the available evidence supports the involvement of receptor - mediated signaling in the activation of p56 / p53 lyn ( yamanashi et al ., 1992 ; pleiman et al ., 1993 ). src - like proteins may be activated through dephosphorylation by tyrosine phosphatases ( mustalin & amp ; altman , 1990 ; cantley et al ., 1991 ; hartwell & amp ; weinart , 1989 ) and potentially other mechanisms ( cantley et al ., 1991 ; hartwell & amp ; weinart , 1989 ). in regard to the effect of ionizing radiation on the phosphorylation of p34 cdc2 on tyrosine , hl - 60 cells were grown in rpmi 1640 medium containing 15 % heat - inactivated total bovine serum supplemented with 100 units / ml penicillin , 100 μg / ml streptomycin and 2 mm l - glutamine . exponentially growing cells were suspended in serum free media 18 h prior to irradiation . irradiation was performed at room temperature using a gammacell 1000 ( atomic energy of canada , ottawa ) with a 137 cs source emitting at a fixed dose rate of 13 . 3 gy / min as determined by dosimetry . cells were washed twice with ice cold phosphate buffered saline and lysed in buffer a ( 10 mm tris , ph 7 . 4 , 1 mm egta , 1 mm edta , 50 mm nacl , 5 mm β - glycerophosphate , 1 % triton x - 100 , 0 . 5 % np - 40 , 1 mm sodium vanadate , 1 mm dtt , 1 mm phenylmethylsulfonyl fluoride and 10 μg / ml of leupeptin and aprotinin ). insoluble material was removed by centrifugation at 14000 rpm for 5 min at 4 ° c . protein concentration was determined by coomassie blue staining using bsa as standard . soluble proteins ( 50 μg ) were separated by electrophoresis in 10 % sds - polyacrylamide gels and then transferred to nitrocellulose paper . the residual binding sites were blocked by incubating the filter in 5 % dry milk in pest ( pbs / 0 . 05 % tween 20 ) for 1 h at room temperature . the filters were then incubated for 1 h with either mouse anti - phosphotyrosine ( anti - p - tyr ; 4g10 ) monoclonal antibody ( 4g10 , ubi , lake placid , n . y .) or a mouse anti - p34 cdc2 monoclonal antibody which is unreactive with other cyclin - dependent kinases ( sc - 54 ; santa cruz biotechnology , santa cruz , calif .). after washing twice with pest , the blots were incubated with anti - mouse or anti - rabbit igg peroxidase conjugate ( sigma chemical co ., st . louis , mo .). the antigen - antibody complexes were visualized by chemiluminescence ( ecl detection system , amersham , arlington heights , ill .). immunoprecipitations were performed with anti - p - tyr or anti - p34 cdc2 at 5 μg / ml cell lysate . immune complexes were collected with protein a - sepharose ( pharmacia ) and immunoprecipitates were analyzed by 10 % sds - page . after transfer to nitrocellulose and blocking , immunoblot analysis was performed with either anti - p34 cdc2 or anti - p - tyr and detected with the appropriate hrp - conjugated second antibody using the ecl system . hl - 60 cells were exposed to 200 cgy ionizing radiation and monitored for proteins with increased levels of phosphotyrosine . using an anti - p - tyr antibody in immunoblot analyses , reactivity with a protein of approximately 34 kd was increased at 1 min after ionizing radiation treatment ( fig1 a ). similar findings were obtained at 5 and 10 min , while reactivity was decreased at 15 min ( fig1 a ). the filters were washed and reprobed with an anti - p34 cdc2 antibody . the anti - p - tyr and anti - p34 cdc2 signals were superimposable . moreover , there was little detectable change in p34 cdc2 protein levels following exposure to ionizing radiation ( fig1 b ). similar findings were obtained with doses of ionizing radiation from 50 to 500 cgy ( fig1 a ). the finding that the signals obtained with the anti - p34 cdc2 antibody ( fig1 b ) were also superimposable over those found with anti - p - tyr suggested that p34 cdc2 may undergo phosphorylation on tyrosine following ionizing radiation treatment . extracts of irradiated cells were subjected to immunoprecipitation with anti - p34 cdc2 . the immunoprecipitates were then monitored by immunoblotting with anti - p - tyr . the signal for p34 cdc2 was increased in irradiated as compared to control cells ( fig1 a ). while this result further supported increased tyrosine phosphorylation of p34 cdc2 , the filter was washed and reprobed with anti - p34 cdc2 to assay for levels of p34 cdc2 protein . the finding that the anti - p34 cdc2 signals were similar in control and irradiated cells ( fig1 b ) indicated that p34 cdc2 undergoes increased phosphorylation on tyrosine following ionizing radiation exposure . activation of p34 cdc2 requires association with cyclin b ( pines & amp ; hunter , 1989 ; russel & amp ; nurse , 1987 ) and certain posttranslational modifications . in schizosaccharomyces pombe , the p34 cdc2 / cyclin b complex is inactivated by phosphorylation of p34 cdc2 on tyrosine 15 by weel ( featherstone & amp ; russell , 1991 ; parker et al ., 1991 ; 1992 ; gould & amp ; nurse , 1989 ). dephosphorylation of p34 cdc2 on tyr - 15 by the cdc25 gene product is necessary for activation of p34 cdc2 and entry into mitosis ( gould et al ., 1989 ; enoch & amp ; nurse , 1990 ). the weel and cdc25 gene products thus determine the timing of entry into mitosis by a series of phosphorylations and dephosphorylations of p34 cdc2 . other work in s . pombe has demonstrated that mitotic checkpoints monitor dna synthesis and the presence of dna damage ( al - khodairy & amp ; carr , 1992 ; rowley et al ., 1992 ; lock & amp ; ross , 1990 ). the dna damage checkpoint evidently regulates p34 cdc2 by mechanisms distinct from those induced by the replication checkpoint ( rowley et al ., 1992 ; lock & amp ; ross , 1990 ). other studies have demonstrated that p34 cdc2 kinase activity is decreased when cho cells are exposed to 8 gy ionizing radiation ( uckun et al ., 1992b ). the present invention discloses activation of src - like tyrosine kinases and phosphorylation of tyrosine kinase substrates , such as p34 cdc2 , as a rapid response to ionizing radiation . inhibition of the radiation - induced activation of those tyrosine kinases prevents or inhibits substrate phosphorylation . because the function of those substrates depends on their state of phosphorylation , inhibition of phosphorylation alters the function of those substrates . to the extent that substrate function is responsible for all or part of the cascade of changes associated with radiation , altering substrate function by inhibition of phosphorylation alters the cells response to radiation . thus , the present invention contemplates a process to alter the response of cell to radiation , the process comprising inhibiting tyrosine kinase activity . in a preferred embodiment , the tyrosine kinase in a src - like tyrosine kinase of the lyn family . following radiation exposure , many single strand breaks are produced in dna , but these are readily repaired using the opposite strand of dna as a template . x - ray energy deposition on dna may lead not only to strand breakage but to base damage . the breakage may result in incorrect rejoining in pre - replication chromosomes in the g 1 phase , leading to chromosomal aberrations , or if the radiation is given late in s or g 2 , chromatid aberrations will result . the skilled artisan in directed to “ remington &# 39 ; s pharmaceutical sciences ” 15th edition , chapter 33 , in particular pages 624 - 652 . some variation in dosage will necessarily occur depending on the condition of the subject being treated . the person responsible for administration will , in any event , determine the appropriate dose for the individual subject . moreover , for human administration , preparations should meet sterility , pyrogenicity , general safety and purity standards as required by fda office of biologics standards . a variety of other dna damaging agents may be used with the tyrosine kinase inhibitors , as provided by this invention . this includes agents that directly crosslink dna , agents that intercalate into dna , and agents that lead to chromosomal and mitotic aberrations by affecting nucleic acid synthesis . agents that induce dna alkylation , such as mitomycin c , may be used . mitomycin c is an extremely toxic antitumor antibiotic that is cell cycle phase - nonspecific . it is almost always given intravenously , at a dose of 20 mg / meter 2 , either in a single dose or given in 10 separate doses of 2 mg / meter 2 each given over 12 days . it has been used clinically against a variety of adenocarcinomas ( stomach , pancreas , colon , breast ) as well as certain head and neck tumors . another option is to employ cisplatin , which has also been widely used to treat cancer , with efficacious doses used in clinical applications of 20 mg / m 2 for 5 days every three weeks for a total of three courses . cisplatin is not absorbed orally and must therefore be delivered via injection intravenously , subcutaneously , intratumorally or intraperitoneally . agents that damage dna also include compounds that interfere with dna replication , mitosis , and chromosomal segregation . examples of these compounds include adriamycin , also known as doxorubicin , etoposide , verapamil , podophyllotoxin , and the like . widely used in clinical setting for the treatment of neoplasms these compounds are administered through bolus injections intravenously at doses ranging from 25 - 75 mg / m2 at 21 day intervals for adriamycin , to 35 - 50 mg / m2 for etoposide , intravenously or double the intravenous dose orally . agents that disrupt the synthesis and fidelity of nucleic acid precursors , and subunits also lead to dna damage . as such a number of nucleic acid precursors have been developed . particularly useful are agents that have undergone extensive testing and are readily available . as such , agents such as 5 - fluorouracil ( 5 - fu ), are preferentially used by neoplastic tissue , making this agent particularly useful for targeting to neoplastic cells . although quite toxic , 5 - fu , is applicable in a wide range of carriers , including topical , however intravenous administration with doses ranging from 3 to 15 mg / kg / day being commonly used . tyrosine protein kinase activities are known to be associated with oncogene products of the retroviral src gene family , and also with several cellular growth factor receptors such as that for epidermal growth factor ( egf ). activation of protein tyrosine phosphorylation by p56 / p53 lyn in the present studies demonstrates that the lyn protein is associated with the cell cycle regulatory protein p34 cdc2 , contributing to mitotic arrest . if this association is blocked , such as by use of protein tyrosine kinase inhibitors such as genistein or herbimycin a , the cells are unable to arrest in the g 2 phase , forcing cell cycle traverse and expression of potentially lethal damage . thus , the combined use of dna damaging agents such as ionizing radiation or alkylating agents with tyrosine kinase inhibitors is a novel approach to enhancing cell killing . genistein , a natural isoflavonoid phytoestrogen , has been reported to exhibit specific inhibitory activity against tyrosine kinases of egf receptor , pp60 v - src and pp110 gag - fes . it has been generally shown to block a number of egf dependent phenomena , including both receptor autophosphorylation and histone phosphorylation . herbimycin a has also been shown to inhibit the autophosphorylation of egf - stimulated receptors in intact cells in a time and dose dependent manner . herbimycin a both decreases the receptor quantity and the egf - stimulated receptor kinase activity . other tyrosine kinase inhibitors may also be used , for example , those isolated from natural sources . one such compound is erbstatin ( umezawa and imoto m , 1991 ; sugata et al ., 1993 ) and its analogues , e . g ., rg 14921 ( hsu et al ., 1992 ). lavendustin a from streptomyces griseolavendus ( onoda et al ., 1989 ), which is about 50 times more inhibitory than erbstatin , and analogues thereof , are also contemplated for use as protein - tyrosine kinase inhibitors ( smyth et al ., 1993b ). piceatannol ( 3 , 4 , 3 ′, 5 ′- tetrahydroxy - trans - stilbene ; geahlen and mclaughlin , 1989 ) and polyhydroxylated stilbene analogues thereof ( thakkar et al ., 1993 ) may also be used . further natural tyrosine kinase inhibitors that may be used are emodin ( 3 - methyl - 1 , 6 , 8 - trihydroxyanthraquinone ), an inhibitor from the chinese medicinal plant polygonum cuspidatum ( jayasuriya et al ., 1992 ; chan et al ., 1993 ); desmal ( 8 - formyl - 2 , 5 , 7 - trihydroxy - 6 - methylflavanone ), isolated from the plant desmos chinensis ( kakeya et al ., 1993 ); the chlorosulfolipid , malhamensilpin a , isolated from the cultured chrysophyte poterioochromonas malhamensis ( chen et al ., 1994 ); flavonoids obtained from koelreuteria henryi ( abou - shoer et al ., 1991 ); fetuin , a natural tyrosine kinase inhibitor of the insulin receptor ( rauth et al ., 1992 ). another group of compounds known to be tyrosine kinase inhibitors are the tyrphostins , which are low molecular weight synthetic inhibitors ( gazit et al ., 1989 ). the tyrphostins ag17 , ag18 , t23 and t47 have been shown to inhibit pancreatic cancer cell growth in vitro ( gillespie et al ., 1993 ). tyrphostins have also been shown to have antiproliferative effects on human squamous cell carcinoma in vitro and in vivo ( yoneda et al ., 1991 ). rg - 13022 and rg - 14620 were found to suppress cancer cell proliferation in vitro and tumor growth in nude mice . another active tyrphostin is ag879 ( ohmichi et al ., 1993 ). various chemical compounds may also be used in combination with dna damaging agents , such as ionizing radiation , as have been described in the literature for use alone . one example is rg50864 ( merkel et al ., 1993 ). further examples are the indole substituted 2 , 2 ′- dithiobis ( 1 - methyl - n - phenyl - 1h - indole - 3 - carboxamides , especially the 5 - substituted derivative , as described by rewcastle et al . ( 1994 ). ( z )- alpha -[( 3 , 5 - dichlorophenyl ) methylene ]- 3 - pyridylacetonitrile ( rg 14620 ) is another active tyrosine kinase inhibitor that may be used in a topical or intravenous form ( khetarpal et al ., 1994 ). be - 23372m , ( e )- 3 -( 3 , 4 - dihydroxybenzylidene )- 5 -( 3 , 4 - dihydroxyphenyl )- 2 ( 3h )- furanone , is also a tyrosine kinase inhibitor ( tanaka et al ., 1994a ). this may be synthesized from 3 -( 3 , 4 - dimethoxybenzoyl ) propionic acid and veratraldehyde or 3 , 4 - diacetoxy - benzaldehyde , as described by tanaka et al . ( 1994b ). be - 23372m may also be isolated from the culture broth of a rhizoctonia solani fungus ( strain f23372 ) using acetone and then purified by solvent extraction and column chromatography ( okabe et al ., 1994 ). further tyrosine kinase inhibitors that may be used include 4 , 5 - dianilinophthalimide , which has , alone , been shown to have in vivo antitumor activity ( buchdunger et al ., 1994 ). hydroxylated 2 -( 5 ′- salicyl ) naphthalenes form another group of inhibitors that could be used in the present invention , and may be prepared as described by smyth et al . ( 1993a ). 1 ) patients exhibiting neoplastic disease are treated with a protein kinase inhibitor , for example genistein , at a concentration of between 1 and 100 μm , or herbimycin a at a concentration of between about 1 and 100 μm , for 6 hours prior to exposure to a dna damaging agent . 2 ) patients are exposed to ionizing radiation ( 2 gy / day for up to 35 days ), or an approximate a total dosage of 700 gy . 3 ) as an alternative to ionizing radiation exposure , patients are treated with a single intravenous dose of mitomycin c at a dose of 20 mg / m 2 . it is contemplated that mitomycin c treatment in combination with tyrosine protein kinase inhibitors will be effective against cancer of the stomach , pancreas , oral cavity , breast and head / neck . all of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure . while the compositions and methods of this invention have been described in terms of preferred embodiments , it will be apparent to those of skill in the art that variations may be applied to the composition , methods and in the steps or in the sequence of steps of the method described herein without departing from the concept , spirit and scope of the invention . more specifically , it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved . all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit , scope and concept of the invention as defined by the appended claims . the following references , to the extent that they provide exemplary procedural or other details supplementary to those set forth herein , are specifically incorporated herein by reference . abou - shoer , m ., ma , g e ., et al . 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