Patent Abstract:
method of blocking aberrant ras signaling in a mammal while avoiding excessive cell toxicity by administration of lovastatin and geranylgeraniol .

Detailed Description:
lovastatin was obtained from merck . geranylgeraniol was obtained as mixed isomers from sigma chemical co . or the all - trans isomer from american radiolabelled chemicals , inc . both agents were prepared as stock solutions in dimethylsulfoxide ( dmso ) with 10 mm dithiothreitol ( dtt ). dtt is not expected to be necessary in the formulation , since neither lovastatin nor ggoh possesses a thiol functional group . for convenience , the following abbreviations are used in the specification : ftase , farnesyltransferase ; ggtase , geranylgeranyltransferase ; fpp , farnesylpyrophosphate ; ggpp , geranylgeranylpyrophosphate ; ggoh , geranylgeraniol ; hmg - coa , hydroxymethylglutaryl - coenzyme a ; sds - page , sodium dodecyl sulfate polyacrylamide gel electrophoresis ; mapk , mitogen - activated protein kinase ; dtt , dithiothreitol ; dmso , dimethylsulfoxide ; pmsf , phenylmethylsulfonyl fluoride ; mva , mevalonic acid ; pbs , phosphate - buffered saline ; caax , c = cysteine , a = aliphatic , x = any amino acid ; mtt , 3 -[ 4 , 5 - dimethylthiazol - 2 - yl ]- 2 , 5 - diphenyltetrazolium bromide . nih - 3t3 mouse fibroblasts ( atcc ) were maintained in dulbecco &# 39 ; s modified eagles medium ( dmem ) supplemented with 10 % fetal bovine serum and 1 % pen - strep ( life technologies inc .). nih - 3t3 cells transfected with the oncogene encoding h - ras - cvls ( 12r ) or h - ras - cvls ( 61l ) or with the empty vector ( pzipneo ) were kind gifts from dr . channing der and dr . adrienne cox ( university of north carolina , chapel hill ) and were maintained in dmem supplemented with 10 % calf serum , 1 % pen - strep , and 400 μg / ml g418 . nih - 3t3 cells transfected with either the h - ras - cvls ( 61l ) oncogene or the empty vector were seeded into 96 - well plates ( 10 , 000 / well ) and the following day treated ( at 50 - 70 % confluency ) with increasing concentrations of lovastatin ( 0 - 300 μm ) in the presence or the absence of 15 μm ggoh . after 4 days , the medium was replaced with 100 μl of mtt ( 1 mg / ml ) in dmem . after 3 h incubation at 37 ° c . the tetrazolium / formazan reaction was stopped and the uptake of mtt assessed by replacing the medium with 100 μl dmso , shaking the plate for 5 min to solubilize all the dye , and measuring the absorbance at 492 nm with a titertek multiskan spectrophotometer ( flow laboratories , mcclean va .). cells were seeded into 100 mm plates ( 8 . 0 × 10 5 - 1 . 0 × 10 6 / plate ) on day 0 such that the following day they were 50 - 70 % confluent . cells were treated twice ( days 1 and 2 ) with vehicle ( 10 mm dtt in dmso ) or concentrations of lovastatin and / or ggoh as indicated in the figure legends . on day 3 , cells were harvested in ice cold pbs , ph 7 . 5 , pelleted , and then lysed in 50 mm hepes , ph 7 . 5 , 10 mm nacl , 1 % triton x - 100 , 10 % glycerol , 5 mm mgcl 2 , 1 mm egta , 25 μg / ml leupeptin , 2 mm na 3 vo 4 , 1 mg / ml soybean trypsin inhibitor , 10 μg / ml aprotinin and 6 . 4 mg / ml phosphatase substrate . after clearing the lysates ( 14 , 000 rpm , 4 ° c ., 10 min ), equivalent amounts of protein were applied to sds - polyacrylamide gels ( 12 . 5 % for protein processing ; 15 % for map kinase ), separated by electrophoresis , and subsequently transferred to nitrocellulose filters . filters were blocked with 5 % non - fat dry milk in pbs , 0 . 1 % tween - 20 ( pbs - t ) and then probed with either anti - ras ( y13 - 238 or y13 - 259 , atcc ), anti - rapla ( santa cruz biotechnology , santa cruz , calif .) anti - rhob ( santa cruz biotechnology , santa cruz , calif . ), anti - rab5 ( transduction laboratories , lexington , ky .) or anti - map kinase ( erk2 , ubi , lake placid , n . y .) antibodies in 3 % non - fat dry milk in pbs - t . positive antibody reactions were detected using appropriate horseradish peroxidase - conjugated antibodies ( oncogene science and jackson immunoresearch laboratories inc ., west grove , pa .) and an enhanced chemiluminescence detection system ( ecl , amersham corp .). nih - 3t3 cells transfected with h - ras - cvls ( 61l ) were seeded into 12 - well plates ( 200 , 000 cells / well ) and incubated the following day ( at 90 - 100 % confluency ) with tritiated ggoh in a manner similar to that described by crick et al . ( 1994 ). cells were incubated with 500 μl dmem medium containing 30 μci / ml 3 h - ggoh ( 50 - 60 ci / mmole ; 0 . 6 μm ggoh , final ), 3 - 5 % calf serum and 20 μm lovastatin . as noted in the legend to fig6 some cells were treated with additional unlabeled ggoh which increased the final ggoh concentration to 5 μm but decreased the specific activity by 8 . 7 - fold . in addition , some cells were treated overnight with 20 μm lovastatin prior to incubation with the labeled ggoh . after 21 - 22 h at 37 ° c ., the labeling medium was removed and the cells were washed once with 1 . 5 ml ice - cold pbs , ph 7 . 5 and harvested using two 1 . 0 - ml aliquots of ice - cold pbs , ph 7 . 5 . the cells were spun down and the pellets were disrupted in ice - cold ch 3 oh . the delipidated protein pellets were extracted twice with chcl 3 / ch 3 oh ( 2 : 1 ), air dried and subsequently dissolved in 95 μl sds - page sample buffer . an aliquot ( 5 μl ) was used to determine the amount of tritium incorporated into protein and the remainder loaded onto a 12 . 5 % sds - polyacrylamide gel . after electrophoretically separating the labeled proteins , the gels were fixed with ch 3 oh / ch 3 co 2 h / h 2 o ( 9 : 2 : 9 ), treated with entensify ( dupont nen , boston , mass . ), dried down on whatman paper , and exposed to x - ray film for 2 - 16 days at - 80 ° c . nih - 3t3 cells were seeded into 100 mm plates ( 3 . 8 × 10 6 cells / plate ) and were treated the following day ( about 70 % confluency ) with either vehicle , 25 μm ggoh , 50 μm lovastatin , or 50 μm lovastatin and 25 μm ggoh . after 40 h , the cells were washed , harvested in ice - cold pbs , ph 7 . 5 , and the cell pellet volume ( pv ) estimated . cells were disrupted by sonication in 1 . 4 × pv of 50 mm tris , ph 7 . 5 , 1 mm egta , 1 mm dtt , 1 mm pmsf , 25 μg / ml leupeptin , and 10 μg / ml aprotinin and the cytosolic ( 60s ) fraction prepared by centrifugation at 25 , 000 rpm for 1 h at 4 ° c . using a beckman swt155 swinging bucket rotor . equivalent amounts of cytosolic protein ( 160 μg ) were incubated in 50 mm tris , ph7 . 5 , 50 μm zncl 2 , 20 mm kcl , 3 mm mgcl 2 , 1 mm dtt with exogenously added human ggtase i ( mono q - purified from human burkitt lymphoma ( daudi ) cells ). prenylation of 60s proteins was started by addition of 4 . 8 μci 3 h - ggpp and the reaction allowed to proceed for 45 - 60 min at 37 ° c . total reaction volume was 55 . 4 μl . the reaction was stopped by briefly placing tubes on ice and then adding 25 μl sds - page sample buffer ( 2 ×). samples were electrophoresed on 12 . 5 % sds - polyacrylamide gels and the gels processed for fluorography as described above for 3 h - ggoh metabolic labeling . the effects of ggoh on lovastatin inhibition of protein farnesylation were investigated in nih - 3t3 cells transformed with an oncogenic , gtp - locked mutant of h - ras - cvls ( an ftase substrate ). cells were treated with lovastatin and ggoh alone or in combination , and the extent of oncogenic ras processing , as well as its ability to stimulate mapk , were assessed . nih - 3t3 cells transformed with oncogenic h - ras - cvll ( mutated at its caax box to become a ggtase i substrate ) were treated in parallel with those transformed by oncogenic h - ras - cvls and served as a control exhibiting processing and signaling that is geranylgeranylation - dependent . after 2 days of treatment , the cells were harvested and lysed and the lysate proteins subsequently separated by sds - page and immunoblotted with anti - ras or anti - mapk antibodies as described in example 3 . cells treated with vehicle or ggoh alone exhibited only the processed form of ras and both the active ( hyperphosphorylated ) and inactive ( hypophosphorylated ) forms of mapk ( fig1 a , lanes 1 and 5 ). lovastatin partially inhibited the processing of both h - ras - cvls and h - ras - cvll and inhibited the activation of mapk slightly in h - ras - cvls - transformed cells and completely in h - ras - cvll - transformed cells ( fig1 a , lanes 2 and 6 ). while co - treatment with ggoh completely prevented the inhibitory effect of lovastatin in h - ras - cvll - transformed cells , h - ras - cvls processing and mapk activation were inhibited to an extent that was greater than that observed for lovastatin treatment alone ( fig1 a , lanes 4 and 8 ). to assess the sensitivity of protein farnesylation to inhibition by lovastatin / ggoh co - treatment , cells were treated with various concentrations of both compounds , and the extent of h - ras processing assessed . as shown in fig1 b , ggoh treatment alone ( 0 - 30 μm ; lanes 1 - 4 ) as well as 1 μm lovastatin alone ( lane 5 ) did not inhibit h - ras processing . however , co - treatment of cells with 1 μm lovastatin and 7 . 5 μm ggoh ( fig1 b , lane 6 ) achieved detectable inhibition of ras processing ; significant inhibition was observed with the same concentration of lovastatin and 30 μm ggoh ( lane 8 ). while treatment of cells with 5 μm and 15 μm lovastatin produced a partial inhibition of ras processing , co - treatment with 7 . 5 μm ggoh resulted in a much greater inhibition ( fig1 b , lanes 9 - 16 ). co - treatment of cells with lovastatin / ggoh ( 15 μm / 30 μm ) was observed to completely inhibit ras processing ( fig1 b , lane 16 ). when the same nitrocellulose membrane was reprobed with an antibody against rapla , the processing of this endogenous geranylgeranylated protein was observed to be significantly inhibited at 5 μm and 15 μm lovastatin ( fig1 b , lanes 9 and 13 ), but was restored upon co - treatment with ggoh at even 7 . 5 μm ( lanes 10 and 14 ). these dramatic and divergent effects of ggoh on lovastatin inhibition of oncogenic ras and rapla processing are depicted graphically in fig2 . based on three independent experiments ( table i ), cells treated with 15 μm lovastatin demonstrated an inhibition of h - ras processing of 54 %. and of rapla processing of 93 %. co - treatment with 15 μm lovastatin and 15 μm ggoh further reduced levels of processed oncogenic ras to 20 % while those of rapla were completely restored . analysis of the data in table i using a student &# 39 ; s two - tailed paired t - test demonstrates that the inhibition of oncogenic ras processing achieved by lovastatin / ggoh co - treatment is statistically different ( p & lt ; 0 . 01 ) from that achieved by lovastatin treatment alone . hence , by employing this treatment with cells that express constitutively activated ras , the processing and signaling of this oncoprotein can be potently inhibited without affecting the processing / function of geranylgeranylated proteins . table i . data from three independent experiments illustrating the divergent effects of ggoh on ras and rapla processing in lovastatin - treated cells . ______________________________________ % of processed ras % of processed rap1a lova + lova + expt . control lova ggoh control lova______________________________________ ggoh1 100 27 5 100 9 1002 100 36 9 100 9 1003 100 75 45 100 4 100______________________________________ the processing of ras and rapla was assessed after treating cells 2 days with either vehicle ( control ), 15 μm lovastatin , or both 15 μm ggoh and 15 μm lovastatin . statistical analysis of the data obtained from these experiments demonstrated that the inhibition of ras processing effected by lovastatin / ggoh cotreatment is statistically different from that achieved by using lovastatin alone ( p & lt ; 0 . 01 using a student &# 39 ; s two - tailed paired t - test ). lovastatin / ggoh co - treatment inhibits the processing of ras but not that of rapla in human lung carcinoma cells in an effort to demonstrate that lovastatin / ggoh co - treatment is an effective means of inhibiting the processing of ras in human cancer cells , human lung carcinoma ( a549 ) cells were treated 2 days with lovastatin and ggoh alone and in combination and the extent of processing for ras and rapla assessed . when cell lysates were electrophoresed and immunoblotted with anti - ras antibodies , two distinct bands of ras were detected in control cells ( fig3 lane 1 ). as shown in fig3 the processing of the ras protein corresponding to the faster - migrating ( lower ) band , as well as that of the endogenous rapla in the same cells , was observed to be sensitive to treatment with lovastatin alone ( lane 3 ). however , in cells co - treated with lovastatin and ggoh , inhibition of processing of the lower ras band was still observed but that of rapla was restored ( lane 4 ). these results , therefore , show the efficacy of the lovastatin / ggoh co - treatment toward inhibition of processing of ras , but not rapla , in a human cancer line . geranylgeraniol ( ggoh ) rescues lovastatin - induced cell rounding , cytoxicity , and inhibition of processing of geranylgeranylated proteins ras - transformed nih - 3t3 cells were treated with lovastatin , ggoh , or a combination of both . fig4 a - 4d illustrate the characteristic rounded cell morphology produced by treatment of cells with lovastatin alone , as well as the complete prevention of this change by co - treatment with ggoh . since lovastatin is known to be cytotoxic at concentrations that cause cell rounding , we investigated whether ggoh could attenuate lovastatin cytoxicity . cells were plated at high density in 96 - well plates and treated with various concentrations of lovastatin alone or in combination with ggoh . fig4 e shows that while lovastatin inhibited ras - transformed 3t3 cell growth with an ic 50 of 8 μm , co - treatment with ggoh ( 15 μm ) significantly attenuated lovastatin cytotoxicity , increasing the ic 50 by 15 - fold . these findings were similar for all cell lines tested , both non - transformed and transformed . the rounding of cells induced by lovastatin has been suggested to be due to its inhibitory effects on protein geranylgeranylation ( fenton et al ., 1992 ). rho and rac proteins , both of which are geranylgeranylated , have been demonstrated to regulate polymerization of actin and to play a pivotal role in maintaining cellular shape ( nobes and hall 1995 ). thus the ability of ggoh to prevent lovastatin - induced rounding suggested that it might be serving to preserve protein geranylgeranylation by replenishing the depleted pool of geranylgeranyl in lovastatin - treated cells . in order to address whether the processing of geranylgeranylated proteins is indeed rescued by co - treatment with ggoh , cells were treated with either lovastatin alone , ggoh alone , or a combination of both , and the processing of rapla and rhob ( substrates for ggtase i ) and rab5 ( a ggtase ii substrate ) was assessed . fig5 shows that while the processing of all of these proteins was inhibited by lovastatin , no detectable inhibition of processing was observed when cells were co - treated with both lovastatin and ggoh . these findings strongly suggest that ggoh is converted to a metabolite that can be utilized by ggtases for protein geranylgeranylation . in order to establish that ggoh was being used as metabolic source for protein prenylation in mouse fibroblasts , ras - transformed nih - 3t3 cells were treated with 3 h - ggoh and 20 μm lovastatin for 20 h and the delipidated proteins were separated by sds - page and visualized by fluorography . as shown in fig6 ( lane 3 ) a pattern of labeled proteins ( mw range of 21 - 29 kda ) resembling that obtained for small g proteins was observed . although treatment of cells with lovastatin at the time of 3 h - ggoh labeling did not significantly enhance tritium incorporation into protein ( data not shown ), pre - treatment of cells with 20 μm lovastatin for 24 h before co - treatment with lovastatin and 3 h - ggoh did significantly increase protein labeling ( fig6 lane 4 ). in addition , optimal metabolic labeling of protein was found to occur when unlabeled ggoh was added such that the final concentration was increased from 0 . 6 μm ( 3 h - ggoh alone ) to 5 . 0 μm ( fig6 lanes 1 and 2 ). hence although the specific activity of 3 h - ggoh was decreased approximately 8 - fold by addition of unlabeled ggoh , increased tritium incorporation was observed . presumably , this is due to kinetic factors involved in the conversion of ggoh to an activated form and / or the use of this activated form for protein prenylation . further addition of unlabeled ggoh ( 10 μm , final ) did not result in a further increase in protein labeling ( data not shown ). these results suggest that ggoh is being metabolically converted by mouse fibroblasts to an activated form ( possibly ggpp ) and used for protein geranylgeranylation . this form replenishes the ggpp pool that is depleted in lovastatin - treated cells . ggoh co - treatment significantly reduces the levels of all unprocessed ggtase i protein substrates in the cytosolic fraction of lovastatin - treated nih - 3t3 cells the effect of ggoh co - treatment on the processing of all ggtase i protein substrates in nih - 3t3 cells was assessed using a novel approach . cells were treated with lovastatin ( 50 μm ) and ggoh ( 25 μm ) alone or in combination , harvested , and cytosolic fractions prepared and used as a source of protein substrates in an in vitro geranylgeranylation assay . as shown in fig7 the cytosolic fractions obtained from cells treated with vehicle or ggoh alone contained no unprocessed proteins capable of serving as substrates in the in vitro assay ( lanes 1 and 2 ). however , the cytosolic fractions from lovastatin - treated nih - 3t3 cells contained several protein substrates for ggtase i , as demonstrated by the profile of tritium - labeled geranylgeranylated proteins ranging from 21 - 29 kda that was detected using this assay ( fig7 lane 3 ). when the assay was performed on the cytosolic fractions from nih - 3t3 cells co - treated with lovastatin and ggoh , all of the protein substrates for ggtase i ( detected in the cytosolic fractions from lovastatin - treated cells ) were observed to be significantly reduced ( fig7 lane 4 ). in longer exposures , a few other bands of higher and lower molecular weight appeared in the lane containing cytosol from lovastatin - treated cells but these also were not detected in the cytosol from cells co - treated with lovastatin and geranylgeraniol . thus , this novel approach demonstrates that ggoh is converted by the cells to a form capable of rescuing the processing of virtually all proteins that are geranylgeranylated by ggtase i in the cell . the examples presented herein clearly demonstrate that simultaneous lovastatin and ggoh treatment of cells preserves the processing of geranylgeranylated proteins while that of farnesylated ras is further inhibited ( fig1 and 5 ) above that which occurs after lovastatin treatment alone . the fact that ggoh has no apparent effect on protein prenylation in the absence of lovastatin ( fig1 and 7 ) indicates that it acts to replenish the pool of ggpp or to provide an acceptable substitute . this is consistent with data from crick et al . ( 1994 ) who previously demonstrated that when 3 h - ggoh metabolically - labeled proteins were subjected to pronase e digestion , a labeled product that was chromatographically identical to geranylgeranylcysteine was released . by achieving the selective restoration of protein geranylgeranylation , not only can cell morphology be maintained at the same phenotype as control cells ( fig4 a - 4d ), but much of the cytotoxic effects of lovastatin can be alleviated ( fig4 e ). the maintenance of cell shape as well as cell cycle progression through g 1 might be expected since these cellular processes have recently been shown to be regulated by geranylgeranylated proteins ( rho and rac ; nobes and hall , 1995 ; olson et al 1995 ). thus , ggoh enhances the inhibitory effect of lovastatin on the uncontrolled signalling caused by farnesylated oncogenic ras while preserving normal cellular functions that are dependent on geranylgeranylated proteins . based on the results presented here , we have proposed a model depicting the mechanism by which ggoh confers to lovastatin a selective and enhanced ability to inhibit protein farnesylation ( fig8 ). as discussed above , ggoh is suggested to be taken up and converted by cells to an activated form ( designated ggx in fig8 ) which serves as the geranylgeranyl donor for protein prenylation catalyzed by ggtases i and ii ( only ggtase i is depicted in fig8 ). moreover , ggx is proposed to competitively inhibit fpp from the active site of ftase . previous work shows that ggpp can bind to the active site of ftase with a similar affinity to that of fpp but cannot be used as a substrate for protein prenylation ( reiss et al ., 1992 ). moreover , it has been demonstrated in vitro that ggpp inhibits ftase with an ic 50 of 5 μm ( unpublished data ). thus , in the presence of lovastatin endogenous pools of fpp are low and ggx can act to potently inhibit ftase ( fig8 ): in the absence of lovastatin , no inhibition of ras processing is observed since the levels of ggx formed are not high enough to compete out endogenous fpp from the ftase active site ( fig1 and 5 ). the examples presented herein demonstrate that lovastatin - induced cell rounding and cytotoxicity are not due to inhibition of protein farnesylation . more importantly , the data indicate that inhibition of protein geranylgeranylation by lovastatin is solely responsible for these untoward effects . therefore , the novel approach of the present invention is a powerful tool for selectively inhibiting oncogenic ras signaling while sparing cells from lovastatin cytotoxicity . it should be a safe and effective treatment for cancers and other pathological conditions which are related to h - ras signaling as well as any pathological condition which depends on a protein that requires farnesylation for its function / activity ( e . g . hdv large antigen ). for convenience , references cited herein are set forth below in their entirety and are hereby incorporated by reference . adamson , p ., marshall , c . j . hall , a ., and tilbrook , p . a . 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