BACKGROUND
one of the common obstacles encountered in gene therapy trials is the potential deleterious effect of the integration of the ectopic gene to the cellular genome.
as an example a serious adverse event after successful gene therapy for x-linked severe combined immunodeficiency has been described with a lmo2-associated clonal t cell proliferation in two patients  <cit> .
a way to eradicate this negative effect is to induce the death of the modified cells upon request including a suicide gene in the gene transfer vector.
previous approaches used gancyclovir-induced cell death post transduction with a viral vector containing a herplex simplex virus-thymidine kinase expression cassette  <cit> .
however the effectiveness of this strategy may be blunted because of their more limited effect on quiescent or slowly dividing cells that require prolonged expression of the therapeutic gene and long term administration of the prodrugs.
another way to induce the death of gene modified cells is to promote expression of a pro-apoptotic protein, a cytotoxic protein or a drug sensitive inducer protein such as cd <dig> as suggested recently  <cit>  via a pharmacological control of the transgene transcription  <cit> .
transcription regulation is usually achieve by means of cell-permeant-inducing agents such as tetracycline, macrolides, oestrogen, progesterone, isopropyl-b-d-thiogalactoside and ectysone  <cit> .
here we proposed a post translational control of a protein.
we studied a way to pharmacologically induce protein function upon request by reversible sub-cellular localization of the protein.
protein prenylation is required for the biological functions of several proteins by permitting association with the cell membranes and encouraging protein-protein interactions with other regulatory molecules.
protein isoprenylation is a post translational isoprenoid lipid modification of substrate proteins by isoprenic lipids  <cit> .
<dig>  to  <dig> % of cellular proteins are isoprenylated , including members of the rasgtpase superfamily, several protein kinases and phosphatases, and a variety of proteins involved in nuclear integrity and centromere function  <cit> .
two type of enzymes catalyse protein isoprenylation, the caax prenyl transferase, farnesyl transferase  and geranylgeranyl transferase i  that recognize caax  c terminus peptide motif and rabggtase or ggtaseii that recognizes ccx or cxc c terminus motifs.
ftase or ggtasei catalyse the covalent attachment of the  <dig> carbon farnesyl or the  <dig> carbon geranylgeranyl respectively to the cysteine of the caax motif.
the terminal x residue of the caax motif determines whether farnesylation or geranylgeranylation occurs: ftase prefers x to be methionine, serine, alanine or glutamine, as for ras proteins  <cit>  while ggtasei prefers leucine or isoleucine.
there are exceptions to this general rule since rhob can be farnesylated or geranylgeranylated in vivo by ftase and ggtasei respectively  <cit>  and since n-ras or k-ras but not h-ras can be geranylgeranylated by ggtasei when the ftase is inhibited.
protein prenylation is the first step of a complex protein processing including proteolytic cleavage of the aax peptide, carboxymethylation of the prenylated cysteine residue, and lastly for some proteins, attachment of a palmitate residue near the prenylated cysteine.
the development of ftase inhibitors  has raised the possibility of specifically inhibiting the function of proteins involved in oncogenesis such as ras oncoproteins.
by preventing ras farnesylation, ftis severely impair ras functions because of the inability of the non farnesylated protein to anchor to the membranes  <cit> .
ftis are currently under evaluation in phase ii/iii clinical trials for the treatment of cancer  <cit>  and show relatively low-toxic effects  <cit> .
we recently showed that a artificial three-component chimaera consisting of the ribosome recruitment core of the eif4g <dig> eukaryotic translation factor, the rna-binding domain of the r <dig> bacteriophage coat protein and the plasma membrane localization caax motif of farnesylated h-ras can have its translational activity inhibited through protein farnesylation that could be restored by fti treatment.
this result put on light the possibility of a pharmacological switche that control gene expression at the translational level  <cit> .
here, we hypothezize that this concept could be applied to control the proteins that exert their functions in specific cellular compartment such as nuclear transcriptional factor as p <dig>
the gene encoding p <dig> mediates a major tumor suppression pathway that is frequently altered in human cancers  <cit> .
p <dig> is inhibited during normal cell growth by mdm <dig> a proto-oncogene through either ubiquitin-dependent p <dig> degradation in the cytoplasm or repression of the transcriptional activity of p <dig> in the nucleus  <cit> .
p <dig> is activated following cellular stresses leading to its phosphorylation and translocation to the nucleus.
nuclear activated p <dig> binds to specific dna sequences and triggers the transcription of target genes thus functionning, at least in part, as a transcriptional regulator.
p <dig> contributes to tumor suppression through at least two mechanisms, arrest of cell proliferation  <cit>  and induction of cell death through apoptosis  <cit> .
the aim of this study was to find a way to induce the death  of targeted cells upon request.
the way that has been chosen is to control the function of a protein  by acting on its localization.
to inactivate p <dig> function we targeted it to the cell membranes by post translational modifications.
we have generated a chimeric p <dig> protein with the  <dig> cooh term amino acids of h-ras  fused to the cooh term of p <dig>  we have demonstrated that an inactive chimeric p53hrcaax gains its cellular functions in saos- <dig> cells  under fti treatment.
these data highlight the fact that the artificial prenylation of proteins provide a novel system for controlling the function of a transactivating proteins.
RESULTS
the saos- <dig> human osteosarcoma is a poorly differentiated, growth factor-insensitive cell line.
this p53-/- cell line was selected because an ectopic expression of p <dig> induces their apoptosis  <cit> .
several ad vectors encoding different mutated forms of the pro-apoptotic gene p <dig> were constructed to transduce these saos- <dig> cells.
in this study, our aim was to compare the efficiency of mutated form of p <dig> versus wtp <dig> in inducing cellular responses, such as apoptosis only in the presence of fti.
adenoviral vectors have been constructed to efficiently transduce saos- <dig> cells.
the proapoptotic genes were under the transcriptional control of the strong cmv promoter.
we generated adp53wt, adp53hrcaax in which the tga codon of the p <dig> coding gene has been replaced by the sequence encoding  <dig> cooh term amino acids of h-ras which correspond to the membrane binding domain of h-ras allowing the whole post translational processing of the protein, and adp53hrsaax as a control.
in this control vector, the tga codon of the p <dig> coding gene has been replaced by the sequence encoding  <dig> cooh term amino acids of h-ras with a single base mutation allowing the translation of a serine instead of a cysteine in the caax motif of the chimeric protein.
this p53hrassaax is unable to be processed.
brand et al  <cit>  showed that in vitro Δe <dig> ad vector could lead to growth retardation, prolongation of the g2/m phase and induction of apoptosis if applied at a high moi.
to distinguish between the cytotoxicity of the vectors and the effect of the transgene we compared the vectors mentioned above with either adegfp  or adluc .
using adegfp, we confirmed that more than 95% of the cells were gfp positive when using  <dig> physical particles/cell and no significant cytotoxicity was seen 7-days post transduction .
these data demonstrated, under our conditions, that cell responses observed bellow were due to transgene expression and that modest doses of vectors led to efficient transduction.
ad transduced saos- <dig> cells could express the farnesylated form of p53
forty eight hr post transduction of saos- <dig> cells by the different ad vectors, cells extracts were analyzed by western blot .
as expected, adluc transduced cells did not express p <dig> protein.
following transduction with adp53wt, adp53hrcaax or adp53hrsaax, p <dig> protein expression was observed.
these results are consistent with expression of the ectopic p <dig> gene.
all p <dig> mutants were expressed roughtly at the same level.
moreover, treatment of the cells with fti did not induce any variation of the p <dig> expression irrespective of the form tested.
several proteins, aside from the ras family are known to be farnesylated.
these farnesylated proteins may be useful surrogate markers for determining whether a specific fti is actually inhibiting farnesylation, by providing an indirect measure of ftase inhibition.
example of these proteins include hdj- <dig>  a chaperone protein.
the inhibition of farnesylation of hdj- <dig> can be monitored by immunoblotting, since its unfarnesylated form displays a reduced mobility in sds-page relative to its farnesylated versions  <cit> .
when saos- <dig> transduced cells were exposed to  <dig> μm fti- <dig>  a mobility shift on sds-page was observed in hdj- <dig>  reflecting inhibition of ftase.
moreover, a slight mobility shift in the p53hrcaax band was noted, suggesting that fti- <dig> prevents the protein farnesylation of this mutant .
hras membrane binding domain induced p <dig> membranes localization
the sub-cellular localization of the different p <dig> mutants was analyzed  <dig> hr post transduction of saos- <dig> cells by the different ad vectors .
transducing the cells with adp53hrcaax resulted in a distinctive, punctuate staining pattern in the cytosol with a perinuclear concentration  excluding the nucleus.
confocal imaging confirmed that p53hrcaax was perinuclear and cytoplasmic .
in the presence of  <dig> μm fti, adp53hrcaax transduced saos- <dig> cells expressed p <dig> exclusively in the nucleus suggesting that inhibition of farnesylation of p <dig> abolished its membranes sequestration.
mihara et al  <cit>  recently reported that a fraction of stess-induced wild-type p <dig> translocates to mitochondria during p <dig> dependent apoptosis after dna damage and under hypoxia.
we have shown here that the farnesylated form of p <dig> did not localize preferentially to the cell plasma membrane as expected.
therefore we checked by confocal microscopy whether p53hrcaax could localize to the mitochondria in the absence of fti by colocalization with mitochondrial markers .
just as wtp <dig>  p53hrcaax did not translocate to the mitochondria in the transduced saos- <dig> cells either  <dig> or  <dig> hr post transduction .
as a control, when adp53wt or adp53hrsaax transduced cells were stained, a nuclear localization of p <dig> was observed irrespective of whether the cells are untreated or treated or not with fti .
culture with fti had no effect on p <dig> localization for these p <dig> forms.
the farnesylated form of p <dig>  had reduced transactivation activity
the tumor suppressor activity of p <dig> is related to its ability to interact with both dna and proteins.
as a result of complex patterns of both dna-protein and protein-protein interactions, expression of the down-stream genes that participate in the control of dna replication, repair, cell cycle and apoptosis can be either elevated or decreased by p <dig>  we have used a reporter gene assay to analyse the transcriptional activity of the different p <dig> mutants using the reporter vector pgl3-in which the luc reporter gene is driven by a promoter that is sensitive to induction by p <dig>  it is anticipated that the expression of the luciferase gene will be enhanced in the presence of a functional p <dig>  using the polyethylenimine  method, we transiently transfected the p53-/- cells saos- <dig> with this luc gene as reporter, plus a renilia luciferase expression plasmid to control transfection efficiency.
then, these cells were transduced with the different ad vectors in the presence or absence of  <dig> μm fti.
both lucf and lucr activities were measured.
as shown in figure 3a, p53-mediated transcriptional transactivation was effective in cells transduced with either adp53wt or adp53hrsaax in the presence or absence of fti.
in contrast, p53-mediated transcriptional transactivation was effectively lowered in p53hrcaax transduced cells, although fti- <dig> treatment restored the transactivation efficiency of the p53hrcaax mutant.
we then analysed the induction of the p <dig> endogenous target genes, p21waf1/cip <dig> and bax.
western blotting was performed for p21waf1/cip <dig> and bax to detect functional p <dig> activity in cells with and without exposure to the different vectors in the presence or absence of the fti.
as expected p53wt, but not p53hrcaax transduced cells expressed p21waf1/cip <dig> within  <dig> hr of transduction .
both cells expressed bax at different levels .
fti treatment induced p21waf1/cip <dig> expression or a higher level of bax expression in p53hrcaax cells, while no modification of protein levels was observed in adp53wt transduced cells.
.
overall these data demonstrated that the hrcaax domain fused with the p <dig> protein impaired the transcriptional transactivation function of the protein, although this could be reinduced by farnesyl transferase inhibition.
fti triggered cell growth inhibition and apoptosis of the p53hrcaax mutant
the p <dig> protein is a potent inhibitor of cell growth, arresting the cell cycle at several points and, under certain circumstances, activating the apoptotic machinery leading to cell death  <cit> .
to compare the antiproliferative activity of wtp <dig>  p53hrcaax and p53hrsaax, cell viability was determined using an mtt assay at  <dig>   <dig>   <dig> and  <dig> hr post-transduction by the different ad vectors.
saos- <dig> cells transduced with adp53wt, adp53hrsaax in the presence or absence of fti or those transduced with adp53hrcaax plus the fti all showed a similar level of cell death.
in contrast saos- <dig> cells transduced with adp53hrcaax in the absence of the fti were more resistant to cell death .
to quantify the apoptosic effects, annexin v-positive populations were estimated by fluorescence microscopy at various times after transduction .
transdution of cells with adp53hrcaax in the absence of fti resulted in only background levels of apoptotic cells, similar to those of mock tranduction.
in contrast, adp53hrcaax in the presence of fti induced the apoptosis of more than  <dig> % of saos- <dig> cells, as seen with adp53wt.
transduction of cells with adluc resulted in only a a very few apoptotic cells, such as in the non transduced cells.
therefore, we have shown that the farnesylated form of p <dig> elicits no apoptosis of saos- <dig> in the absence of the fti.
yet the ability to elicit apoptosis could be restored in the presence of the fti.
discussion
we have shown that the artificial prenylation of proteins could provide a novel system for controlling the function of a protein such as here a transactivating factor.
the farnesylation of a protein changed its cellular localization therefore its function was inhibited.
this post translational modification could be prevented by inhibition of farnesyl transferase leading to protein activation.
these new properties of an ectopic protein could be used to develop a 'gain of function' approach designed to allow the death by apoptosis of targeted gene modified cells upon request.
to prove the concept we have chosen to use p <dig> as it is a tumor suppressor gene involved in cell cycle arrest and programmed cell death .
the properties of p <dig> are mainly linked to its ability to induce the transcription of genes, such as the cyclin-dependent kinase inhibitor p21waf1/cip <dig> or bax  <cit> .
we constructed a p <dig> chimeric protein which is a substrate for farnesyl transferase by fusing the membrane binding domain of h-ras that includes the  <dig> cooh term amino acids of the protein to the cooh end of p <dig>  we used this domain because, in contrast to n-ras and k-ras, h-ras membrane binding domain is not a substrate of ggtasei when ftase is inhibited.
this eliminates the possibility of protein geranylgeranylation under fti treatment.
we have demonstrated that it is the c terminus membrane binding domain of h-ras, but not that of the caax box permitting only farnesylation which avoids nuclear localization of the p <dig> transcriptional factor .
this highlight the fact that only farnesylation is not sufficient to induce membrane attachment of a chimeric protein and that complete post translational processing is necessary for this purpose.
the post translational modification of the chimeric p <dig> protein is illustrated by a slight shift of the farnesylated form of the p <dig> protein, as observed in the figure  <dig>  we have verified that the non processed form of the h-ras binding domain p <dig> chimeric protein does not impair its transcriptional activity.
indeed, p53hrsaax, like p53hrcaax in the presence of the fti, was able to induce transcription of responder genes as efficiently in vitro as in vivo and was also able to induce apoptosis of the saos- <dig> cells.
p <dig> shuttles within the cell between the nucleus and the cytoplasm, possessing nuclear localization signals  and a nuclear export signal   <cit> .
it was a challenge to impede the nuclear translocation of a protein containing an nls domain and this was achieved by lipid modification which avoids nuclear localization of the p <dig> transcriptional factor.
this highlights the possibility of developing this strategy so as to control the protein function of other nuclear proteins.
we have shown here that the lipid processed form of p <dig> whilst located in part in the plasma membrane, was found preferentially in the cytoplasm.
this observation is not surprising since chiu et al  <cit>  have shown that farnesylation of the caax motif could also target ras proteins to the endoplasmic reticulum and golgi membranes.
in these locations they encounter rce protease and prenylcysteine-directed carboxymethyltransferase.
thus nascent ras proteins are present, at least transiently, on the endoplasmic reticulum and golgi.
the fact that farnesylation could control cellular localization of different chimeric protein  has also been described previously  <cit> .
we have used farnesylation of p <dig> to control its localization and therefore it's function.
we have shown here that bypassing the nucleus by targeting p <dig> to other cell compartments is sufficient to significantly reduce the marked apoptosis of p53-deficient tumor cells in the absence of an fti.
this therefore establishes the proof of principle that a chimeric p <dig> can initiate apoptosis only upon treatment of cells with the fti,.
ftis have previously demonstrated low toxicity in normal cells.
several ftis are currently being evaluated in phase ii and phase iii clinical trials  <cit> .
ftis can exert dramatic effects on cancer cells, including morphological changes, inhibition of anchorage-independent growth and alteration of cell cycle progression.
although ftis clearly inhibit ras farnesylation it is unclear whether their antiproliferative effects result exclusively from their inhibition of ras functioning  <cit> .
ftis have effects on several other prenylated proteins involved in crucial cellular signal transduction pathways, such as the centromere binding protein -e and cenp-f, peroxysomal membrane and nuclear membrane  associated proteins, or members of the rho proteins family.
ftis affect the pi3-k/akt cell survival pathway.
they also inhibit soft agar growth of several breast cancer cells lines independent of their ras mutant status, probably through an alternative target such as the protein rhob which regulates receptor trafficking and cell adhesion/motility.
in total more than  <dig> polypeptides possess a caax sequence that potentially can be farnesylated and such ftis may have multiple targets that may be inhibited to produce a net antiproliferative effect or apoptosis  <cit> .
as expected in the transformed saos- <dig> cellular model used in these data, few apoptotic cells have been noted following fti treatment.
however, a high percentage of apoptosis has been noted in our experiments only if fti treated cells were previously transduced by p53hrcaax.
our results confirmed the data reported by nielsen et al.
that combination therapy with a replication-deficient recombinant adenovirus, which expresses the human p <dig>  and an fti have synergistic or additive antiproliferative effects on a panel of tumor cells in vitro  <cit> .
CONCLUSIONS
this work has been performed to find application in gene therapy protocols including, amongst others, i) the potential for inducing the activation so that the function of an ectopic protein at a defined moment and ii) the safety of therapeutic gene expression for the cells being treated.
for this second purpose, integrating vectors could be constructed as a polycistronic vector.
it would contain the gene of interest and the gene encoding a chimeric pro-apoptotic protein such as bax, trail or p <dig> such as what is currently done with the hsv-tk strategy.
upon request, if deleterious effect appears on gene modified cells such as transformation of the gene modified cells, we could apply fti and so induce the death of the modified cells.
the combination of fti which have antitumoral effect plus the apoptosis of the newly transformed cells could allow the destruction of the unwanted transformed cells.
the synergestic effect of the fti and p <dig> induced apoptosis could be of greater interest than the hsv-tk strategy.
undoubtedly, the challenge now is to tease apart the intricate circuitry that controls the cellular location of pro-apoptotic-proteins.

 one of the common obstacles encountered in gene therapy trials is the potential deleterious effect of the integration of the ectopic gene to the cellular genome. as an example a serious adverse event after successful gene therapy for x-linked severe combined immunodeficiency has been described with a lmo2-associated clonal t cell proliferation in two patients. a way to eradicate this negative effect is to induce the death of the modified cells upon request including a suicide gene in the gene transfer vector. previous approaches used gancyclovir-induced cell death post transduction with a viral vector containing a herplex simplex virus-thymidine kinase expression cassette. however the effectiveness of this strategy may be blunted because of their more limited effect on quiescent or slowly dividing cells that require prolonged expression of the therapeutic gene and long term administration of the prodrugs. another way to induce the death of gene modified cells is to promote expression of a pro-apoptotic protein, a cytotoxic protein or a drug sensitive inducer protein such as cd as suggested recently via a pharmacological control of the transgene transcription. transcription regulation is usually achieve by means of cell-permeant-inducing agents such as tetracycline, macrolides, oestrogen, progesterone, isopropyl-b-d-thiogalactoside and ectysone. here we proposed a post translational control of a protein. we studied a way to pharmacologically induce protein function upon request by reversible sub-cellular localization of the protein. protein prenylation is required for the biological functions of several proteins by permitting association with the cell membranes and encouraging protein-protein interactions with other regulatory molecules. protein isoprenylation is a post translational isoprenoid lipid modification of substrate proteins by isoprenic lipids. to % of cellular proteins are isoprenylated, including members of the rasgtpase superfamily, several protein kinases and phosphatases, and a variety of proteins involved in nuclear integrity and centromere function. two type of enzymes catalyse protein isoprenylation, the caax prenyl transferase, farnesyl transferase and geranylgeranyl transferase i that recognize caax c terminus peptide motif and rabggtase or ggtaseii that recognizes ccx or cxc c terminus motifs. ftase or ggtasei catalyse the covalent attachment of the carbon farnesyl or the carbon geranylgeranyl respectively to the cysteine of the caax motif. the terminal x residue of the caax motif determines whether farnesylation or geranylgeranylation occurs: ftase prefers x to be methionine, serine, alanine or glutamine, as for ras proteins while ggtasei prefers leucine or isoleucine. there are exceptions to this general rule since rhob can be farnesylated or geranylgeranylated in vivo by ftase and ggtasei respectively and since n-ras or k-ras but not h-ras can be geranylgeranylated by ggtasei when the ftase is inhibited. protein prenylation is the first step of a complex protein processing including proteolytic cleavage of the aax peptide, carboxymethylation of the prenylated cysteine residue, and lastly for some proteins, attachment of a palmitate residue near the prenylated cysteine. the development of ftase inhibitors has raised the possibility of specifically inhibiting the function of proteins involved in oncogenesis such as ras oncoproteins. by preventing ras farnesylation, ftis severely impair ras functions because of the inability of the non farnesylated protein to anchor to the membranes. ftis are currently under evaluation in phase ii/iii clinical trials for the treatment of cancer and show relatively low-toxic effects. we recently showed that a artificial three-component chimaera consisting of the ribosome recruitment core of the eif4g eukaryotic translation factor, the rna-binding domain of the r bacteriophage coat protein and the plasma membrane localization caax motif of farnesylated h-ras can have its translational activity inhibited through protein farnesylation that could be restored by fti treatment. this result put on light the possibility of a pharmacological switche that control gene expression at the translational level. here, we hypothezize that this concept could be applied to control the proteins that exert their functions in specific cellular compartment such as nuclear transcriptional factor as p the gene encoding p mediates a major tumor suppression pathway that is frequently altered in human cancers. p is inhibited during normal cell growth by mdm a proto-oncogene through either ubiquitin-dependent p degradation in the cytoplasm or repression of the transcriptional activity of p in the nucleus. p is activated following cellular stresses leading to its phosphorylation and translocation to the nucleus. nuclear activated p binds to specific dna sequences and triggers the transcription of target genes thus functionning, at least in part, as a transcriptional regulator. p contributes to tumor suppression through at least two mechanisms, arrest of cell proliferation and induction of cell death through apoptosis. the aim of this study was to find a way to induce the death of targeted cells upon request. the way that has been chosen is to control the function of a protein by acting on its localization. to inactivate p function we targeted it to the cell membranes by post translational modifications. we have generated a chimeric p protein with the cooh term amino acids of h-ras fused to the cooh term of p we have demonstrated that an inactive chimeric p53hrcaax gains its cellular functions in saos- cells under fti treatment. these data highlight the fact that the artificial prenylation of proteins provide a novel system for controlling the function of a transactivating proteins.  the saos- human osteosarcoma is a poorly differentiated, growth factor-insensitive cell line. this p53-/- cell line was selected because an ectopic expression of p induces their apoptosis. several ad vectors encoding different mutated forms of the pro-apoptotic gene p were constructed to transduce these saos- cells. in this study, our aim was to compare the efficiency of mutated form of p versus wtp in inducing cellular responses, such as apoptosis only in the presence of fti. adenoviral vectors have been constructed to efficiently transduce saos- cells. the proapoptotic genes were under the transcriptional control of the strong cmv promoter. we generated adp53wt, adp53hrcaax in which the tga codon of the p coding gene has been replaced by the sequence encoding cooh term amino acids of h-ras which correspond to the membrane binding domain of h-ras allowing the whole post translational processing of the protein, and adp53hrsaax as a control. in this control vector, the tga codon of the p coding gene has been replaced by the sequence encoding cooh term amino acids of h-ras with a single base mutation allowing the translation of a serine instead of a cysteine in the caax motif of the chimeric protein. this p53hrassaax is unable to be processed. brand et al showed that in vitro Δe ad vector could lead to growth retardation, prolongation of the g2/m phase and induction of apoptosis if applied at a high moi. to distinguish between the cytotoxicity of the vectors and the effect of the transgene we compared the vectors mentioned above with either adegfp or adluc. using adegfp, we confirmed that more than 95% of the cells were gfp positive when using physical particles/cell and no significant cytotoxicity was seen 7-days post transduction. these data demonstrated, under our conditions, that cell responses observed bellow were due to transgene expression and that modest doses of vectors led to efficient transduction. ad transduced saos- cells could express the farnesylated form of p53 forty eight hr post transduction of saos- cells by the different ad vectors, cells extracts were analyzed by western blot. as expected, adluc transduced cells did not express p protein. following transduction with adp53wt, adp53hrcaax or adp53hrsaax, p protein expression was observed. these results are consistent with expression of the ectopic p gene. all p mutants were expressed roughtly at the same level. moreover, treatment of the cells with fti did not induce any variation of the p expression irrespective of the form tested. several proteins, aside from the ras family are known to be farnesylated. these farnesylated proteins may be useful surrogate markers for determining whether a specific fti is actually inhibiting farnesylation, by providing an indirect measure of ftase inhibition. example of these proteins include hdj- a chaperone protein. the inhibition of farnesylation of hdj- can be monitored by immunoblotting, since its unfarnesylated form displays a reduced mobility in sds-page relative to its farnesylated versions. when saos- transduced cells were exposed to μm fti- a mobility shift on sds-page was observed in hdj- reflecting inhibition of ftase. moreover, a slight mobility shift in the p53hrcaax band was noted, suggesting that fti- prevents the protein farnesylation of this mutant. hras membrane binding domain induced p membranes localization the sub-cellular localization of the different p mutants was analyzed hr post transduction of saos- cells by the different ad vectors. transducing the cells with adp53hrcaax resulted in a distinctive, punctuate staining pattern in the cytosol with a perinuclear concentration excluding the nucleus. confocal imaging confirmed that p53hrcaax was perinuclear and cytoplasmic. in the presence of μm fti, adp53hrcaax transduced saos- cells expressed p exclusively in the nucleus suggesting that inhibition of farnesylation of p abolished its membranes sequestration. mihara et al recently reported that a fraction of stess-induced wild-type p translocates to mitochondria during p dependent apoptosis after dna damage and under hypoxia. we have shown here that the farnesylated form of p did not localize preferentially to the cell plasma membrane as expected. therefore we checked by confocal microscopy whether p53hrcaax could localize to the mitochondria in the absence of fti by colocalization with mitochondrial markers. just as wtp p53hrcaax did not translocate to the mitochondria in the transduced saos- cells either or hr post transduction. as a control, when adp53wt or adp53hrsaax transduced cells were stained, a nuclear localization of p was observed irrespective of whether the cells are untreated or treated or not with fti. culture with fti had no effect on p localization for these p forms. the farnesylated form of p had reduced transactivation activity the tumor suppressor activity of p is related to its ability to interact with both dna and proteins. as a result of complex patterns of both dna-protein and protein-protein interactions, expression of the down-stream genes that participate in the control of dna replication, repair, cell cycle and apoptosis can be either elevated or decreased by p we have used a reporter gene assay to analyse the transcriptional activity of the different p mutants using the reporter vector pgl3-in which the luc reporter gene is driven by a promoter that is sensitive to induction by p it is anticipated that the expression of the luciferase gene will be enhanced in the presence of a functional p using the polyethylenimine method, we transiently transfected the p53-/- cells saos- with this luc gene as reporter, plus a renilia luciferase expression plasmid to control transfection efficiency. then, these cells were transduced with the different ad vectors in the presence or absence of μm fti. both lucf and lucr activities were measured. as shown in figure 3a, p53-mediated transcriptional transactivation was effective in cells transduced with either adp53wt or adp53hrsaax in the presence or absence of fti. in contrast, p53-mediated transcriptional transactivation was effectively lowered in p53hrcaax transduced cells, although fti- treatment restored the transactivation efficiency of the p53hrcaax mutant. we then analysed the induction of the p endogenous target genes, p21waf1/cip and bax. western blotting was performed for p21waf1/cip and bax to detect functional p activity in cells with and without exposure to the different vectors in the presence or absence of the fti. as expected p53wt, but not p53hrcaax transduced cells expressed p21waf1/cip within hr of transduction. both cells expressed bax at different levels. fti treatment induced p21waf1/cip expression or a higher level of bax expression in p53hrcaax cells, while no modification of protein levels was observed in adp53wt transduced cells.. overall these data demonstrated that the hrcaax domain fused with the p protein impaired the transcriptional transactivation function of the protein, although this could be reinduced by farnesyl transferase inhibition. fti triggered cell growth inhibition and apoptosis of the p53hrcaax mutant the p protein is a potent inhibitor of cell growth, arresting the cell cycle at several points and, under certain circumstances, activating the apoptotic machinery leading to cell death. to compare the antiproliferative activity of wtp p53hrcaax and p53hrsaax, cell viability was determined using an mtt assay at and hr post-transduction by the different ad vectors. saos- cells transduced with adp53wt, adp53hrsaax in the presence or absence of fti or those transduced with adp53hrcaax plus the fti all showed a similar level of cell death. in contrast saos- cells transduced with adp53hrcaax in the absence of the fti were more resistant to cell death. to quantify the apoptosic effects, annexin v-positive populations were estimated by fluorescence microscopy at various times after transduction. transdution of cells with adp53hrcaax in the absence of fti resulted in only background levels of apoptotic cells, similar to those of mock tranduction. in contrast, adp53hrcaax in the presence of fti induced the apoptosis of more than % of saos- cells, as seen with adp53wt. transduction of cells with adluc resulted in only a a very few apoptotic cells, such as in the non transduced cells. therefore, we have shown that the farnesylated form of p elicits no apoptosis of saos- in the absence of the fti. yet the ability to elicit apoptosis could be restored in the presence of the fti. discussion we have shown that the artificial prenylation of proteins could provide a novel system for controlling the function of a protein such as here a transactivating factor. the farnesylation of a protein changed its cellular localization therefore its function was inhibited. this post translational modification could be prevented by inhibition of farnesyl transferase leading to protein activation. these new properties of an ectopic protein could be used to develop a'gain of function' approach designed to allow the death by apoptosis of targeted gene modified cells upon request. to prove the concept we have chosen to use p as it is a tumor suppressor gene involved in cell cycle arrest and programmed cell death. the properties of p are mainly linked to its ability to induce the transcription of genes, such as the cyclin-dependent kinase inhibitor p21waf1/cip or bax. we constructed a p chimeric protein which is a substrate for farnesyl transferase by fusing the membrane binding domain of h-ras that includes the cooh term amino acids of the protein to the cooh end of p we used this domain because, in contrast to n-ras and k-ras, h-ras membrane binding domain is not a substrate of ggtasei when ftase is inhibited. this eliminates the possibility of protein geranylgeranylation under fti treatment. we have demonstrated that it is the c terminus membrane binding domain of h-ras, but not that of the caax box permitting only farnesylation which avoids nuclear localization of the p transcriptional factor. this highlight the fact that only farnesylation is not sufficient to induce membrane attachment of a chimeric protein and that complete post translational processing is necessary for this purpose. the post translational modification of the chimeric p protein is illustrated by a slight shift of the farnesylated form of the p protein, as observed in the figure we have verified that the non processed form of the h-ras binding domain p chimeric protein does not impair its transcriptional activity. indeed, p53hrsaax, like p53hrcaax in the presence of the fti, was able to induce transcription of responder genes as efficiently in vitro as in vivo and was also able to induce apoptosis of the saos- cells. p shuttles within the cell between the nucleus and the cytoplasm, possessing nuclear localization signals and a nuclear export signal. it was a challenge to impede the nuclear translocation of a protein containing an nls domain and this was achieved by lipid modification which avoids nuclear localization of the p transcriptional factor. this highlights the possibility of developing this strategy so as to control the protein function of other nuclear proteins. we have shown here that the lipid processed form of p whilst located in part in the plasma membrane, was found preferentially in the cytoplasm. this observation is not surprising since chiu et al have shown that farnesylation of the caax motif could also target ras proteins to the endoplasmic reticulum and golgi membranes. in these locations they encounter rce protease and prenylcysteine-directed carboxymethyltransferase. thus nascent ras proteins are present, at least transiently, on the endoplasmic reticulum and golgi. the fact that farnesylation could control cellular localization of different chimeric protein has also been described previously. we have used farnesylation of p to control its localization and therefore it's function. we have shown here that bypassing the nucleus by targeting p to other cell compartments is sufficient to significantly reduce the marked apoptosis of p53-deficient tumor cells in the absence of an fti. this therefore establishes the proof of principle that a chimeric p can initiate apoptosis only upon treatment of cells with the fti,. ftis have previously demonstrated low toxicity in normal cells. several ftis are currently being evaluated in phase ii and phase iii clinical trials. ftis can exert dramatic effects on cancer cells, including morphological changes, inhibition of anchorage-independent growth and alteration of cell cycle progression. although ftis clearly inhibit ras farnesylation it is unclear whether their antiproliferative effects result exclusively from their inhibition of ras functioning. ftis have effects on several other prenylated proteins involved in crucial cellular signal transduction pathways, such as the centromere binding protein -e and cenp-f, peroxysomal membrane and nuclear membrane associated proteins, or members of the rho proteins family. ftis affect the pi3-k/akt cell survival pathway. they also inhibit soft agar growth of several breast cancer cells lines independent of their ras mutant status, probably through an alternative target such as the protein rhob which regulates receptor trafficking and cell adhesion/motility. in total more than polypeptides possess a caax sequence that potentially can be farnesylated and such ftis may have multiple targets that may be inhibited to produce a net antiproliferative effect or apoptosis. as expected in the transformed saos- cellular model used in these data, few apoptotic cells have been noted following fti treatment. however, a high percentage of apoptosis has been noted in our experiments only if fti treated cells were previously transduced by p53hrcaax. our results confirmed the data reported by nielsen et al. that combination therapy with a replication-deficient recombinant adenovirus, which expresses the human p and an fti have synergistic or additive antiproliferative effects on a panel of tumor cells in vitro.  this work has been performed to find application in gene therapy protocols including, amongst others, i) the potential for inducing the activation so that the function of an ectopic protein at a defined moment and ii) the safety of therapeutic gene expression for the cells being treated. for this second purpose, integrating vectors could be constructed as a polycistronic vector. it would contain the gene of interest and the gene encoding a chimeric pro-apoptotic protein such as bax, trail or p such as what is currently done with the hsv-tk strategy. upon request, if deleterious effect appears on gene modified cells such as transformation of the gene modified cells, we could apply fti and so induce the death of the modified cells. the combination of fti which have antitumoral effect plus the apoptosis of the newly transformed cells could allow the destruction of the unwanted transformed cells. the synergestic effect of the fti and p induced apoptosis could be of greater interest than the hsv-tk strategy. undoubtedly, the challenge now is to tease apart the intricate circuitry that controls the cellular location of pro-apoptotic-proteins. 
artificial protein farnesylation impaired the transcriptional activity of p this could be prevented by farnesyl transferase inhibition. indeed, the translation products of ras genes are cytosolic proteins that become secondarily associated with membranes through a series of post-translational modifications initiated by a caax motif present at the c terminus of ras proteins. these data highlight the fact that the artificial prenylation of proteins provides a novel system for controlling the function of a transactivating factor. the use of integrating viral vectors in gene therapy clinical trials has pointed out the problem of the deleterous effect of the integration of the ectopic gene to the cellular genome and the safety of this strategy.
 the use of integrating viral vectors in gene therapy clinical trials has pointed out the problem of the deleterous effect of the integration of the ectopic gene to the cellular genome and the safety of this strategy. we proposed here a way to induce the death of gene modified cells upon request by acting on a pro-apoptotic protein cellular localization and on the activation of its apoptotic function.  we constructed an adenoviral vector coding a chimeric p protein by fusing p sequence with the cooh term amino acids sequence of h-ras. indeed, the translation products of ras genes are cytosolic proteins that become secondarily associated with membranes through a series of post-translational modifications initiated by a caax motif present at the c terminus of ras proteins. the chimeric p53hrcaax protein was farnesylated efficiently in transduced human osteosarcoma p53-/- cell line. the farnesylated form of p resided mainly in the cytosol, where it is non-functional. farnesyl transferase inhibitors specifically inhibited farnesyl isoprenoid lipid modification of proteins. following treatment of the cells with an fti, p53hrcaax underwent translocation into the nucleus where it retained transcription factor activity. shifting p into the nucleus resulted in the induction of p21waf1/cip and bax transcription, cell growth arrest, caspase activation and apoptosis.  artificial protein farnesylation impaired the transcriptional activity of p this could be prevented by farnesyl transferase inhibition. these data highlight the fact that the artificial prenylation of proteins provides a novel system for controlling the function of a transactivating factor. 