the idea of a constitutively expressed death machinery in each cell has given way to the notion that survival factors repress this machinery and , if such factors are unavailable , cells default into death [ 1 , 2 , 3 ] . this theory is supported by findings showing that many forms of programmed cell death do not require mrna or protein synthesis .
in fact , mrna and protein synthesis inhibitors can induce apoptosis , suggesting that in some cases transcriptional activity might actually impede cell death [ 4 , 5 ] . to identify genes that are transcriptionally regulated in cells undergoing apoptosis by survival factor deprivation
gene trapping involves introduction of a reporter gene into a random collection of chromosomal sites , including transcriptionally active regions . by selecting for gene expression ,
recombinants are obtained in which the reporter gene is fused to the regulatory elements of an endogenous gene .
transcripts generated by these fusions faithfully reflect the activity of a disrupted cellular gene and serve as a molecular tag to clone any gene linked to a specific function [ 6 , 7 ] . to identify genes that are transiently expressed during a biological process
, we developed a strategy , which makes use of the site - specific recombination system cre / loxp . by combining gene trap mutagenesis with site - specific recombination
, it is possible to uncouple a trapped cellular promoter from a transduced reporter gene .
this enables the recovery of recombinants even in the absence of an active cellular promoter and thus allows selection for integrations into transiently expressed genes [ 8 , 9 ] .
we employed this strategy to isolate genes induced in hematopoietic cells ( fdcp1 ) undergoing apoptosis by growth factor withdrawal .
briefly , the interleukin-3 ( il-3)-dependent hematopoietic cells ( floxil3 ) expressing a reporter plasmid encoding hsv - thymidine - kinase , neomycin - phosphotransferase and murine il-3 , were transduced with a retroviral gene trap vector carrying coding sequences for cre recombinase ( cre ) in the u3 region .
activation of cre expression from integrations into active genes resulted in a permanent switching between the selectable marker genes that converted the floxil3 cells to factor independence .
selection for autonomous growth yielded recombinants in which cre sequences in the u3 region were expressed from upstream cellular promoters . as
the expression of the marker genes is independent of the trapped cellular promoter , genes could be identified that were transiently induced by il-3 withdrawal ( figure 1 ) .
( a ) u3cre gene trap activation from integrations in genes induced by il-3 withdrawal excises the tkneo fusion gene , which is flanked by loxp sites from the reporter plasmid pgklxtkneoil3 .
this places the il-3 cdna immediately downstream of the pgk promoter and enables its expression .
cre , cre recombinase ; pol ii , rna polymerase ii ; pgk , phosphoglycerate kinase promoter ; lx , loxp target sequence for cre recombinase ; tkneo , hsv - thymidine - kinase / neomycin - phosphotransferase fusion gene ; pa , bovine growth hormone polyadenylation sequence ; il-3 , murine interleukin-3 cdna .
u3cre - infected floxil3 cells were first selected in g418 to eliminate integrations into constitutively expressed genes .
the g418-resistant cells were then plated into semisolid ( agar ) cultures in the absence of il-3 .
analysis of several recombinants obtained by this method indicated that cells upregulate survival genes before committing to cell death . as a result , cells receiving a transient apoptotic stimulus exhibit improved survival in a growth factor deficient environment .
from a floxil3 gene trap integration library consisting of approximately 2  10 unique proviral integrations , we have isolated 125 individual clones that converted to factor independence upon il-3 withdrawal .
cellular sequences adjacent to u3cre integration sites ( gene trap sequence tags , gtsts ) were obtained from 102 clones by inverse pcr or 5'race using cre - specific primers .
ninety clones yielded informative gtsts ( > 30 nucleotides ) , of which 15 ( 17% ) belonged to known genes , 10 ( 11% ) matched single ests or full cdnas with unknown function and 65 ( 72% ) had no match within the public databases ( figure 2 ) .
interestingly , the frequency of unknown genes recovered with this method was significantly higher than that obtained with a standard gene trap protocol and a similar gene trap vector .
for example , only 782 ( 46% ) of 1,687 gtsts recovered from embryonic stem ( es ) cells expressing a u3 geo gene trap virus were novel .
this difference is consistent with the ability of the cre / loxp approach to enrich for transiently expressed genes that are difficult to isolate by standard methodology and therefore underrepresented in the nucleotide databases .
figure 3a shows the structure of the proviral integration sites in the 15 previously characterized genes . like earlier studies involving retroviral gene trap vectors with a reporter gene in the u3 region of the long terminal repeat ( ltr ) , most integrations occurred in or near 5 ' exons , although integrations further downstream were observed occasionally . to confirm transcriptional induction by il-3 withdrawal
, polyadenylated rnas were isolated from fdcp1 cells after various intervals of factor deprivation and hybridized on northern blots to gene - specific probes .
( a ) gene trap position in previously characterized genes . gtsts were either obtained by 5'race or inverse genomic pcr .
exon / intron boundaries were either available from the databases or were determined by rt - pcr reactions using mrna from fdcp1 cells and exon - specific primers .
polyadenylated rnas ( 5 g per lane ) extracted from fdcp1 cells at various intervals following il-3 withdrawal were fractionated on formaldehyde - agarose gels , blotted onto nylon filters and hybridized to p -labeled gene- or exon ( net1)-specific probes .
as summarized in table 1 , a significant number of the trapped known genes encode proteins involved in cell growth and survival .
for example , the guanine nucleotide exchange factors ( gefs ) for the rho - family gtpases net1 and h - pem2 ( kiaa0424 ) [ 10 , 11 ] belong to a large family of proteins that regulate cell growth and can transform cells in culture .
transformation is dependent on the highly conserved dbl homology ( dh ) domain and in most cases requires amino - terminal truncation of the protein [ 13 , 14 ] .
interestingly , a novel splice variant of net1 , net1a , which was isolated in these experiments , encodes an amino - terminally truncated protein that is transforming in its native form ( table 1 ) .
thus , when overexpressed in nih3t3 fibroblasts , net1a but not net1 produces transformed foci with typical rho morphology ( f.w . and h.v.m .
in addition to the dh domain , rho - gefs have a pleckstrin homology domain that connects the rho signal transduction pathway to the phosphatidylinositol-3-phosphate kinase ( pi3 kinase ) signal transduction pathway .
the latter is responsible for mediating survival signals , particularly in hematopoietic cells , and is directly activated by il-3 .
surprisingly , two members of this pathway , the phosphatidylinositol 4/5 phosphate kinases ( pip5 kinases ) type i ( which corresponds to human pip5 kinase type i ) and type i , were induced by il-3 withdrawal ( table 1 ) .
pip5 kinase converts the lipid phosphatidylinositol-4-phosphate to phosphatidylinositol-4,5-phosphate ( pip2 ) , which enhances cell survival by at least three mechanisms .
moreover , overexpression of pip5 kinase type i in cultured cells prevents apoptosis induced by caspase 9 or tumor necrosis factor- ( tnf- ) .
second , pip2 is phosphorylated by pl3 kinase to give phosphatidylinositol 3,4,5 phosphate ( pip3 ) , which activates the survival kinase akt .
akt prevents cell death by phosphorylating and inactivating the proapoptotic proteins bad , caspase 9 and the forkhead family transcription factor fkhrl1 , which induces the expression of fas ligand [ 17 , 18 , 19 , 20 ] .
third , pip5 kinase activates atp - dependent potassium channels and thereby helps to maintain the polarity of the cell membrane .
as shown previously , apoptotic signals can cause membrane depolarization , which leads to cell death by directly inhibiting potassium channels .
summary of previously characterized genes disrupted by gene trap mutagenesis another gene with a putative survival function encodes the cell - cycle - regulated protein p38 - 2g4 ( ebp1 ) .
although less well characterized than the above proteins , p38-g4 is absent from go cells and may stimulate cell - cycle progression [ 23 , 24 ] .
it includes the il-12 receptor , which inhibits fas - mediated apoptosis in certain types of t lymphocytes   and the transcription factor yb-1 , which represses fas gene expression .
interestingly , yb-1 also activates the expression of the multidrug resistance gene ( mdr1 ) whose product , the p glycoprotein , is an efflux pump that eliminates a variety of toxins from the cell , including proapoptotic anticancer drugs .
similarly cytoprotective is vmat2 , the protein product of the vesicular monoamine transporter gene , which moves cytoplasmic monoamine transmitters into secretory vesicles and prevents cell death by sequestering proapoptotic molecules such as mpp and histamine [ 28 , 29 ] . finally , il-3 withdrawal induced transcription of several dna repair genes .
these were the mouse homologs of the bacterial alkb gene and of the yeast rec8 and rad50 genes .
while alkb protects dna from damage by alkylating agents , rec8 and rad50 are dna double - strand break repair proteins [ 31 , 32 , 33 ] .
only a few genes upregulated by il-3 withdrawal ( 4 out of 15 ) were not directly related to cell death or survival and their induction may reflect some secondary events occurring during apoptosis ( table 1 ) . however , the low frequency with which such genes were recovered underscores the specificity of the gene trap approach . to further study transcriptional regulation during growth factor starvation , we analyzed the expression patterns of a large number of previously characterized genes in fdcp1 cells undergoing apoptosis by il-3 withdrawal using atlas mouse cdna arrays ( clontech ) .
atlas arrays contain cdna probes for 588 genes involved in development , oncogenesis , dna repair , tumor suppression and apoptosis .
figure 4a shows that of the 588 genes displayed on the arrays , only 12 were differentially expressed after factor deprivation .
differential expression of these genes was reproducible in independent array experiments and could be confirmed by northern blotting ( figure 4c ) .
( a ) comparative atlas array hybridization with cdna probes derived from fdcp1 cells after 0 h ( left ) and 2 h ( right ) of il-3 deprivation .
filters were scanned with a phosphoimager ( molecular dynamics ) and analyzed with the atlasimage 1.5 software .
( b ) relative changes in gene expression on a log scale . 1 , no regulation ; >
1 , upregulation ; < 1 , downregulation . note that the apparent downregulation of bcl-2 could not be confirmed on northern blots .
induction of c - kit and chop10 ( asterisked ) was observed only on arrays hybridized to cdna recovered from cells after 8 h of factor deprivation .
( c ) northern blot analysis of transcripts identified as differentially expressed by the atlasimage 1.5 software .
poly(a ) rna ( 2.5 g ) prepared after 0 , 2 , 4 , 6 and 8 h of il-3 deprivation was fractionated on formaldehyde gels , blotted onto nylon filters and hybridized with p - labeled gene - specific probes .
similar to the trapping results , several genes with survival functions were upregulated in the absence of il-3 . among these
were the survival kinase akt , the c - kit receptor tyrosine kinase , the potent inhibitor of apoptosis flipl , and the il-3-receptor itself [ 34 , 35 , 36 ] .
moreover , survival functions seemed to be strengthened by the transient downregulation of the proapoptotic activin receptor [ 37 , 38 ] . in confirmation of previous results , however , the expression of several survival genes appeared il-3 dependent .
thus , transcripts for antiapoptotic pim-1 , c - myc and bcl - xl proteins were repressed in the absence of il-3 .
finally , only two genes with arguably proapoptotic functions ( that is , jund and chop10 ) were upregulated in these experiments ( figure 4c ) .
taken together , the gene trap and cdna array results suggested that the transcriptional changes at the onset of apoptosis should favor cell survival rather than cell death .
the activation of protective mechanisms during the early stages of programmed cell death suggested that a transient exposure to an apoptotic stimulus might be favorable for cell survival .
accordingly , a transient factor deprivation should , by upregulating anti - apoptotic gene expression , reduce the fdcp1 cell 's need for il-3 . to test this
, we evaluated the ability of il-3-deprived and non - deprived cells to form colonies in agar cultures that contained suboptimal amounts of il-3 .
figure 5a shows that cells pre - exposed to factor deprivation generated significantly more colonies than their non - exposed counterparts , indicating that survival mechanisms were induced by factor deprivation . whereas growth stimulation was highest in cultures containing il-3 at concentrations that normally fail to support colony formation , it gradually subsided with prolonged il-3 deprivation .
after incubation for 0 , 1 , 2 and 4 h , il-3 was added at the indicated concentrations .
1  10 cells were deprived of il-3 for 1 and 2 h , respectively .
after re - adding il-3 for 4 h , cells were washed and incubated without il-3 .
after 6 , 9 and 12 h factor deprivation , the cells were stained with annexin and the frequency of apoptotic cells was estimated by flow cytometry .
, we directly tested whether cells pre - exposed to factor deprivation develop increased apoptotic tolerance to subsequent factor starvation . to this end , il-3 was initially withdrawn from fdcp1 cells for 1 or 2 hours .
subsequently , il-3 was re - added for 4 hours only to be removed again for 6 , 9 and 12 hours . at these time points cells
figure 5b shows that pretreated cells died significantly more slowly that their non - pretreated counterparts , indicating that apoptotic prestimulation increases resistance to factor deprivation . like the clonal survival assays , the cell protective effect was highest after a prestimulation of 2 hours . on the basis of these results ,
we conclude that cell protective mechanisms are activated following growth factor withdrawal and transiently prevail prior to irreversible commitment to death .
from a floxil3 gene trap integration library consisting of approximately 2  10 unique proviral integrations , we have isolated 125 individual clones that converted to factor independence upon il-3 withdrawal .
cellular sequences adjacent to u3cre integration sites ( gene trap sequence tags , gtsts ) were obtained from 102 clones by inverse pcr or 5'race using cre - specific primers .
ninety clones yielded informative gtsts ( > 30 nucleotides ) , of which 15 ( 17% ) belonged to known genes , 10 ( 11% ) matched single ests or full cdnas with unknown function and 65 ( 72% ) had no match within the public databases ( figure 2 ) .
interestingly , the frequency of unknown genes recovered with this method was significantly higher than that obtained with a standard gene trap protocol and a similar gene trap vector .
for example , only 782 ( 46% ) of 1,687 gtsts recovered from embryonic stem ( es ) cells expressing a u3 geo gene trap virus were novel .
this difference is consistent with the ability of the cre / loxp approach to enrich for transiently expressed genes that are difficult to isolate by standard methodology and therefore underrepresented in the nucleotide databases .
figure 3a shows the structure of the proviral integration sites in the 15 previously characterized genes . like earlier studies involving retroviral gene trap vectors with a reporter gene in the u3 region of the long terminal repeat ( ltr )
, most integrations occurred in or near 5 ' exons , although integrations further downstream were observed occasionally . to confirm transcriptional induction by il-3 withdrawal
, polyadenylated rnas were isolated from fdcp1 cells after various intervals of factor deprivation and hybridized on northern blots to gene - specific probes .
( a ) gene trap position in previously characterized genes . gtsts were either obtained by 5'race or inverse genomic pcr .
exon / intron boundaries were either available from the databases or were determined by rt - pcr reactions using mrna from fdcp1 cells and exon - specific primers .
polyadenylated rnas ( 5 g per lane ) extracted from fdcp1 cells at various intervals following il-3 withdrawal were fractionated on formaldehyde - agarose gels , blotted onto nylon filters and hybridized to p -labeled gene- or exon ( net1)-specific probes .
as summarized in table 1 , a significant number of the trapped known genes encode proteins involved in cell growth and survival .
for example , the guanine nucleotide exchange factors ( gefs ) for the rho - family gtpases net1 and h - pem2 ( kiaa0424 ) [ 10 , 11 ] belong to a large family of proteins that regulate cell growth and can transform cells in culture .
transformation is dependent on the highly conserved dbl homology ( dh ) domain and in most cases requires amino - terminal truncation of the protein [ 13 , 14 ] .
interestingly , a novel splice variant of net1 , net1a , which was isolated in these experiments , encodes an amino - terminally truncated protein that is transforming in its native form ( table 1 ) .
thus , when overexpressed in nih3t3 fibroblasts , net1a but not net1 produces transformed foci with typical rho morphology ( f.w . and h.v.m . , unpublished data ) .
in addition to the dh domain , rho - gefs have a pleckstrin homology domain that connects the rho signal transduction pathway to the phosphatidylinositol-3-phosphate kinase ( pi3 kinase ) signal transduction pathway .
the latter is responsible for mediating survival signals , particularly in hematopoietic cells , and is directly activated by il-3 .
surprisingly , two members of this pathway , the phosphatidylinositol 4/5 phosphate kinases ( pip5 kinases ) type i ( which corresponds to human pip5 kinase type i ) and type i , were induced by il-3 withdrawal ( table 1 ) .
pip5 kinase converts the lipid phosphatidylinositol-4-phosphate to phosphatidylinositol-4,5-phosphate ( pip2 ) , which enhances cell survival by at least three mechanisms .
moreover , overexpression of pip5 kinase type i in cultured cells prevents apoptosis induced by caspase 9 or tumor necrosis factor- ( tnf- ) .
second , pip2 is phosphorylated by pl3 kinase to give phosphatidylinositol 3,4,5 phosphate ( pip3 ) , which activates the survival kinase akt .
akt prevents cell death by phosphorylating and inactivating the proapoptotic proteins bad , caspase 9 and the forkhead family transcription factor fkhrl1 , which induces the expression of fas ligand [ 17 , 18 , 19 , 20 ] .
third , pip5 kinase activates atp - dependent potassium channels and thereby helps to maintain the polarity of the cell membrane .
as shown previously , apoptotic signals can cause membrane depolarization , which leads to cell death by directly inhibiting potassium channels .
summary of previously characterized genes disrupted by gene trap mutagenesis another gene with a putative survival function encodes the cell - cycle - regulated protein p38 - 2g4 ( ebp1 ) .
although less well characterized than the above proteins , p38-g4 is absent from go cells and may stimulate cell - cycle progression [ 23 , 24 ] .
it includes the il-12 receptor , which inhibits fas - mediated apoptosis in certain types of t lymphocytes   and the transcription factor yb-1 , which represses fas gene expression .
interestingly , yb-1 also activates the expression of the multidrug resistance gene ( mdr1 ) whose product , the p glycoprotein , is an efflux pump that eliminates a variety of toxins from the cell , including proapoptotic anticancer drugs .
similarly cytoprotective is vmat2 , the protein product of the vesicular monoamine transporter gene , which moves cytoplasmic monoamine transmitters into secretory vesicles and prevents cell death by sequestering proapoptotic molecules such as mpp and histamine [ 28 , 29 ] . finally , il-3 withdrawal induced transcription of several dna repair genes .
these were the mouse homologs of the bacterial alkb gene and of the yeast rec8 and rad50 genes .
while alkb protects dna from damage by alkylating agents , rec8 and rad50 are dna double - strand break repair proteins [ 31 , 32 , 33 ] .
only a few genes upregulated by il-3 withdrawal ( 4 out of 15 ) were not directly related to cell death or survival and their induction may reflect some secondary events occurring during apoptosis ( table 1 ) . however , the low frequency with which such genes were recovered underscores the specificity of the gene trap approach .
to further study transcriptional regulation during growth factor starvation , we analyzed the expression patterns of a large number of previously characterized genes in fdcp1 cells undergoing apoptosis by il-3 withdrawal using atlas mouse cdna arrays ( clontech ) .
atlas arrays contain cdna probes for 588 genes involved in development , oncogenesis , dna repair , tumor suppression and apoptosis .
figure 4a shows that of the 588 genes displayed on the arrays , only 12 were differentially expressed after factor deprivation .
differential expression of these genes was reproducible in independent array experiments and could be confirmed by northern blotting ( figure 4c ) .
( a ) comparative atlas array hybridization with cdna probes derived from fdcp1 cells after 0 h ( left ) and 2 h ( right ) of il-3 deprivation .
filters were scanned with a phosphoimager ( molecular dynamics ) and analyzed with the atlasimage 1.5 software .
( b ) relative changes in gene expression on a log scale . 1 , no regulation ; > 1 , upregulation ; < 1 , downregulation . note that the apparent downregulation of bcl-2 could not be confirmed on northern blots .
induction of c - kit and chop10 ( asterisked ) was observed only on arrays hybridized to cdna recovered from cells after 8 h of factor deprivation .
( c ) northern blot analysis of transcripts identified as differentially expressed by the atlasimage 1.5 software .
poly(a ) rna ( 2.5 g ) prepared after 0 , 2 , 4 , 6 and 8 h of il-3 deprivation was fractionated on formaldehyde gels , blotted onto nylon filters and hybridized with p - labeled gene - specific probes .
similar to the trapping results , several genes with survival functions were upregulated in the absence of il-3 . among these
were the survival kinase akt , the c - kit receptor tyrosine kinase , the potent inhibitor of apoptosis flipl , and the il-3-receptor itself [ 34 , 35 , 36 ] .
moreover , survival functions seemed to be strengthened by the transient downregulation of the proapoptotic activin receptor [ 37 , 38 ] . in confirmation of previous results , however , the expression of several survival genes appeared il-3 dependent .
thus , transcripts for antiapoptotic pim-1 , c - myc and bcl - xl proteins were repressed in the absence of il-3 .
finally , only two genes with arguably proapoptotic functions ( that is , jund and chop10 ) were upregulated in these experiments ( figure 4c ) . taken together
, the gene trap and cdna array results suggested that the transcriptional changes at the onset of apoptosis should favor cell survival rather than cell death .
the activation of protective mechanisms during the early stages of programmed cell death suggested that a transient exposure to an apoptotic stimulus might be favorable for cell survival .
accordingly , a transient factor deprivation should , by upregulating anti - apoptotic gene expression , reduce the fdcp1 cell 's need for il-3 . to test this
, we evaluated the ability of il-3-deprived and non - deprived cells to form colonies in agar cultures that contained suboptimal amounts of il-3 .
figure 5a shows that cells pre - exposed to factor deprivation generated significantly more colonies than their non - exposed counterparts , indicating that survival mechanisms were induced by factor deprivation . whereas growth stimulation was highest in cultures containing il-3 at concentrations that normally fail to support colony formation , it gradually subsided with prolonged il-3 deprivation .
after incubation for 0 , 1 , 2 and 4 h , il-3 was added at the indicated concentrations .
1  10 cells were deprived of il-3 for 1 and 2 h , respectively .
after re - adding il-3 for 4 h , cells were washed and incubated without il-3 .
after 6 , 9 and 12 h factor deprivation , the cells were stained with annexin and the frequency of apoptotic cells was estimated by flow cytometry .
, we directly tested whether cells pre - exposed to factor deprivation develop increased apoptotic tolerance to subsequent factor starvation . to this end
subsequently , il-3 was re - added for 4 hours only to be removed again for 6 , 9 and 12 hours . at these time points cells were stained with annexin and apoptosis was quantified by flow cytometry .
figure 5b shows that pretreated cells died significantly more slowly that their non - pretreated counterparts , indicating that apoptotic prestimulation increases resistance to factor deprivation . like the clonal survival assays , the cell protective effect was highest after a prestimulation of 2 hours . on the basis of these results ,
we conclude that cell protective mechanisms are activated following growth factor withdrawal and transiently prevail prior to irreversible commitment to death .
a gene trap strategy was used to identify genes induced in hematopoietic cells undergoing apoptosis by growth factor withdrawal .
this approach enables identification of transiently expressed genes that are difficult to isolate by standard methodology .
a comparatively large proportion of unknown genes were recovered , which underscores the strategy 's potential for isolating novel genes .
in contrast to conventional gene trapping , which tags and disrupts random genes , the cre / loxp approach allows enrichment for genes induced by specific biological stimuli .
as most regulatory genes are expressed in a temporally and spatially restricted manner , we believe that the combination of gene trap mutagenesis and site - specific recombination provides a sound alternative for functional gene analysis in the post - genomic era . as judged by the small fraction of recombinants recovered after growth factor deprivation ( that is , 124 of 2  10 or 0.006% )   and by the identity of the trapped
known genes , the strategy seems highly specific for regulatory genes induced during programmed cell death .
accordingly , the majority of genes upregulated by il-3 withdrawal were associated with cell death and survival .
although this was similar on the cdna arrays , the differentially expressed genes recovered with the two methods were quite different .
thus , with the exception of yb1 and rad50 , none of the genes displayed on the arrays were tagged by gene trap mutagenesis .
moreover , yb1 and rad50 , despite being recovered in the gene trap approach as induced by il-3 withdrawal , appeared constitutively expressed or even downregulated on both arrays and northern blots , respectively ( figures 3,4 ) .
first , the integration library used here covered only 25% of the genome . as only seven genes were induced on the arrays ( those encoding akt , il-3 receptor , glutathione - s - transferase , flipl , c - kit , jund and chop10 ) their recovery from an unsaturated library was unlikely .
second , transient gene trapping as opposed to cdna hybridization has no bias towards highly expressed genes .
third , the gene trap strategy selects for real gene inductions and , unlike the arrays and northern blots , is independent of pre - existing steady - state mrna levels subjected to post - transcriptional regulation . taken together
, these considerations suggest that for the functional analysis of the mammalian genome , gene trapping effectively complements cdna - based strategies , including cdna arrays , which are unable to distinguish between transcriptional and/or post - transcriptional changes in gene expression . in terms of apoptosis
whereas in most cells conflicts between prosurvival and apoptotic signals are carried out post - translationally by well characterized proteins ( for example , bcl-2 family members , caspases ) , there is increasing evidence for transcriptional regulators of apoptosis capable of tilting the balance between the constitutively expressed pro- and anti - apoptotic proteins [ 20 , 41 ] .
these transcriptional regulators of apoptosis - still largely unknown - are likely to confer tissue specificity on the apoptotic process .
the identity of such regulators is of considerable interest as they could provide valuable targets for prospective anti - neoplastic and/or anti - degenerative drugs .
most genes identified in this study encode cell protective and/or pro - survival functions . in line with this ,
the biological experiments described here have shown such functions to materialize in cells exposed to apoptotic prestimulation .
accordingly , cells receiving stimulation exhibited improved survival and reduced apoptosis in a growth factor deprived environment .
interestingly , a similar cytoprotective phenomenon known as ischemic preconditioning has been observed in animal models . in this , a short , sublethal period of ischemia induces profound resistance to subsequent ischemic events .
thus , induction of anti - apoptotic gene expression prior to a lethal stimulus seems to raise the threshold required for that stimulus to be effective .
this provides an additional safety mechanism that can prevent unwanted loss of cells exposed only accidentally to apoptotic stimulation . in summary
, the present experiments have shown that hematopoietic cells undergoing apoptosis by il-3 withdrawal activate survival genes that do impede cell death .
this suggests that apoptosis in hematopoietic cells is the end result of a conflict between death and survival signals , rather than a simple death by default .
inverse pcr from genomic dnas was performed using the cre - specific primers described previously   and a combination of the blunt end restriction enzymes sspi and hincii .
5'race was performed with 1 g of total rna using the 5'race kit from gibco - brl and the manufacturer 's instructions .
amplification reactions were performed in a perkin elmer thermocycler and cloned into the p - gemt - vector ( promega ) as described previously .
fdcp1 cells were propagated at concentrations of 2  10 cells / ml in dulbecco 's modified eagle 's medium ( dmem ; gibco ) , supplemented with 10% ( v / v ) fetal bovine serum ( boehringer - mannheim ) and 5 ng / ml recombinant mouse il-3 ( peprotech ) unless indicated otherwise .
agar cultures were an equal volume mixture of double - strength dmem supplemented with 40% ( v / v ) fetal bovine serum and 0.6% ( w / v ) bacto - agar ( difco ) in double distilled water as previously described .
apoptosis was measured in a facscan flow cytometer after staining the cells with annexin using the annexin - v - fluos detection kit ( roche ) and the manufacturer 's instructions .
poly(a ) rna samples from fdcp-1 cells were reverse transcribed in the presence of p - labeled datp and hybridized to atlas mouse cdna expression array ( clontech ) according to the manufacturer 's instructions .
filters were scanned with a phosphoimager ( molecular dynamics ) and analyzed with the atlasimagetm 1.5 software ( clontech ) . for northern blots , 2.5 g of poly(a )
was fractionated in 1% formaldehyde - agarose gels , transferred onto hybond n membranes ( amersham ) and hybridized to specific p - dctp - labeled probes produced by random priming ( rediprime , amersham ) .
cellular sequences adjacent to proviral integrations ( gene trap sequence tags ; gtsts ) were searched in the ncbi / nih genomic databases   using the blastn algorithm .
inverse pcr from genomic dnas was performed using the cre - specific primers described previously   and a combination of the blunt end restriction enzymes sspi and hincii .
5'race was performed with 1 g of total rna using the 5'race kit from gibco - brl and the manufacturer 's instructions .
amplification reactions were performed in a perkin elmer thermocycler and cloned into the p - gemt - vector ( promega ) as described previously .
fdcp1 cells were propagated at concentrations of 2  10 cells / ml in dulbecco 's modified eagle 's medium ( dmem ; gibco ) , supplemented with 10% ( v / v ) fetal bovine serum ( boehringer - mannheim ) and 5 ng / ml recombinant mouse il-3 ( peprotech ) unless indicated otherwise .
agar cultures were an equal volume mixture of double - strength dmem supplemented with 40% ( v / v ) fetal bovine serum and 0.6% ( w / v ) bacto - agar ( difco ) in double distilled water as previously described .
apoptosis was measured in a facscan flow cytometer after staining the cells with annexin using the annexin - v - fluos detection kit ( roche ) and the manufacturer 's instructions .
poly(a ) rna samples from fdcp-1 cells were reverse transcribed in the presence of p - labeled datp and hybridized to atlas mouse cdna expression array ( clontech ) according to the manufacturer 's instructions .
filters were scanned with a phosphoimager ( molecular dynamics ) and analyzed with the atlasimagetm 1.5 software ( clontech ) . for northern blots , 2.5 g of poly(a )
was fractionated in 1% formaldehyde - agarose gels , transferred onto hybond n membranes ( amersham ) and hybridized to specific p - dctp - labeled probes produced by random priming ( rediprime , amersham ) .
cellular sequences adjacent to proviral integrations ( gene trap sequence tags ; gtsts ) were searched in the ncbi / nih genomic databases   using the blastn algorithm .
this work was supported in part by grants from deutsche krebshilfe and the deutsche forschungsgemeinschaft , bonn to h.v.m and by schering ag , berlin .
 backgroundthe existence of a constitutively expressed machinery for death in individual cells has led to the notion that survival factors repress this machinery and , if such factors are unavailable , cells die by default . in many cells , however , mrna and protein synthesis inhibitors induce apoptosis , suggesting that in some cases transcriptional activity might actually impede cell death . 
 to identify transcriptional mechanisms that interfere with cell death and survival , we combined gene trap mutagenesis with site - specific recombination ( cre / loxp system ) to isolate genes from cells undergoing apoptosis by growth factor deprivation.resultsfrom an integration library consisting of approximately 2  106 unique proviral integrations obtained by infecting the interleukin-3 ( il-3)-dependent hematopoietic cell line - floxil3 - with u3cre gene trap virus , we have isolated 125 individual clones that converted to factor independence upon il-3 withdrawal . 
 of 102 cellular sequences adjacent to u3cre integration sites , 17% belonged to known genes , 11% matched single expressed sequence tags ( ests ) or full cdnas with unknown function and 72% had no match within the public databases . 
 most of the known genes recovered in this analysis encoded proteins with survival functions.conclusionswe have shown that hematopoietic cells undergoing apoptosis after withdrawal of il-3 activate survival genes that impede cell death . 
 this results in reduced apoptosis and improved survival of cells treated with a transient apoptotic stimulus . 
 thus , apoptosis in hematopoietic cells is the end result of a conflict between death and survival signals , rather than a simple death by default . 
Background
Results
A majority of gene trap sequence tags recovered following IL-3 withdrawal correspond to unknown genes
Most genes induced by IL-3 withdrawal exhibit cell-protective functions
Mouse cDNA expression arrays reveal differential expression of cell death and survival genes following IL-3 withdrawal
Apoptotic prestimulation improves survival in the absence of IL-3
Discussion
Materials and methods
Amplification and cloning of upstream sequences
Cell cultures and apoptosis assays
Mouse cDNA expression arrays and northern blot hybridizations
Database searches
Acknowledgements