Patent Application: US-201514615045-A

Abstract:
this invention relates to a screening method for the identification of agents which modulate the activity of a dna replication protein as a target for intervention in cancer therapy and includes agents which modulate said activity . the invention also relates to the use of the dna replication protein , and its rna transcripts in the prognosis and diagnosis of proliferative disease e . g ., cancer .

Description:
we have exploited a polyclonal antibody ( antibody v1 ) that was raised against recombinant human cdc6 ( coverley et al ., 2000 ; stoeber et al ., 1998 ; williams et al ., 1998 ) to identify and study an unknown antigen whose behavior correlates with initiation of dna replication in vitro . the antigen has an apparent mr of 100 kda ( called p85 ) and is readily detectable in extracts from 3t3 cells ( fig1 a ). dna synthesis can be activated in cell - free replication experiments using ‘ replication competent ’ late g1 phase nuclei , g1 extracts , and recombinant cyclin a - cdk2 . under these conditions nuclei will incorporate labelled nucleotides into nascent dna , in a manner strictly dependent on the concentration of active protein kinase ( fig1 b ). above and below the optimum concentration no initiation of dna replication takes place . however , other events occur which inversely correlate with initiation ( coverley et al ., 2002 ). here we use activation of dna synthesis ( fig1 b ), and mcm2 phosphorylation ( which results in increased mobility , fig1 c ), to calibrate the effects of recombinant cyclin a - cdk2 in cell - free replication experiments , and correlate the behavior of p85 with activation of dna synthesis . in g1 nuclei that are re - isolated from reactions containing initiation - inducing concentrations of cyclin a - cdk2 , p85 antigen is more prevalent compared to nuclei exposed to lower or higher concentrations of kinase ( fig1 d ). this suggests that p85 is regulated at some level by cyclin a - cdk2 , in a manner that is co - incident with activation of dna synthesis . no other antigens correlate so closely with this stage in the cell - free initiation process , therefore we used antibody v1 to clone the gene for mouse p85 . when applied to a cdna expression library derived from 11 - day mouse embryos antibody v1 picked out two clones that survived multiple rounds of screening ( see methods ). one encoded mouse cdc6 , while the other encoded 716 amino acids of the murine homologue of human ciz1 ( mitsui et al ., 1999 ). full - length human and mouse ciz1 have approximately 70 % overall homology at the amino - acid level , with greatest (& gt ; 80 %) homology in the n and c terminal regions . ciz1 is conserved among vertebrates as homologues exist in rat and fugu , but no proteins with a high degree of homology or similar domain structure could be identified in lower eukaryotes , raising the possibility that ciz1 evolved to perform a specialized role in vertebrate development . a previous publication on human ciz1 ( mitsui et al 1999 ) demonstrated interaction with the cell - cycle protein p21 - cip1 , leading to investigation of a proposed role as a transcription factor , not a dna replication factor . a second paper ( warder and keherly 2003 ) published after the priority date of this patent application suggests a role for ciz1 in tumorigenesis , but does not demonstrate a role in dna replication or recognize the importance of ciz1 splice variant expression . the predicted mouse ciz1 open reading frame and a cdna derived from a mouse mammary tumor library ( bc018483 ) contain three regions that are not present in our embryonic clone ( aj575057 ), hereafter referred to as eciz1 ( fig2 a ; seq id no : 27 ). the three variable regions in eciz1 appear to be the result of alternative splicing of exons 2 / 3 , 6 and 8 ( fig2 b ). mouse melanoma clone ak089986 lacks two of the same three regions as eciz1 ( fig2 a ), while the third encodes an n - terminal polyglutamine stretch that is also absent from human medulloblastoma derived clones . a fourth sequence block derived from exons 3 / 4 is absent from ciz1 transcripts derived from mouse es cells , and from exon 4 in mouse primordial germ cells ( fig7 ). human ciz1 is also alternatively spliced at the rna level to yield transcripts that exclude combinations of the same four sequence blocks as mouse ciz1 ( see below ). in fact , all known variations in mouse ciz1 cdnas have close human parallels , some of which are identical at the amino - acid level . this suggests that the different ciz1 isoforms have functional significance . a fifth variable region ( not yet observed in the mouse ) is alternatively spliced in human ciz1 transcripts derived mainly from carcinomas . the data suggest that shorter forms of ciz1 ( lacking the alternatively spliced exons ) are most prevalent early in development and in cell lineages that give rise to the germ line . in the analysis shown in fig7 , only ciz1 from fully developed neonatal heart shows no alternative splicing , while all embryonic cell types contain alternatively spliced forms . furthermore , the only complete ciz1 cdnas in public databases ( human or mouse ) are derived from non - embryonic cell types , and the only ones derived from embryonic sources are alternatively spliced . therefore , ciz1 splice variant expression appears to occur preferentially in cell types that are not yet fully differentiated . notably , ciz1 cdnas from pediatric cancers are also alternatively spliced ( see below ). this lead us to the hypothesis that failure to express the appropriate ciz1 isoform at the right point in development leads to inappropriately regulated ciz1 activity . this could contribute to unscheduled proliferation and cellular transformation . upon exposure to cytosolic extract from s phase cells , late g1 phase nuclei initiate dna replication and begin synthesizing nascent dna ( krude et al ., 1997 ). we used this cell - free assay to test the effect of eciz1 , and derived recombinant fragments , on dna synthesis ( fig3 ). full - length eciz1 protein consistently increased the number of nuclei that replicated in vitro , from 30 % (+/− 0 . 9 %) to 46 % (+/− 5 . 5 %), which suggests that ciz1 is limiting for initiation in s phase extracts ( fig3 a ). only two other classes of protein ( cyclin - dependent kinases , coverley et al ., 2002 ; krude et al ., 1997 ; laman et al ., 2001 , and the cdc6 protein , coverley et al ., 2002 ; stoeber et al ., 1998 ) have been previously found to stimulate cell - free initiation . thus , eciz1 is the first protein to have this property that was not already known to be involved in the replication process . the positive effect of recombinant eciz1 on cell - free initiation argues that endogenous ciz1 plays a positive role in dna replication in mammalian cells . stimulation of cell - free initiation is concentration - dependent with peak activity in s phase extract at around 1 nm eciz1 ( fig3 b ). this echoes previous cell - free analyses with other recombinant proteins ( coverley et al ., 2002 ; krude et al ., 1997 ), where stimulation of initiation typically peaks and then falls back to the un - stimulated level at high concentrations . for eciz1 , the reason for the drop in activity at high concentrations is not yet clear . however , mutagenesis studies ( see below ) suggest that the restraining mechanism is likely to be active and specific rather than due to a general imbalance in the composition of higher order protein complexes . down regulation of eciz1 involves threonines 191 / 192 ciz1 is likely to be a phospho - protein in vivo since it contains numerous putative phosphorylation sites , and it displays altered mobility when 3t3 cell extracts are treated with lambda phosphatase ( not shown ). murine ciz1 contains two rxl cyclin binding motifs and five putative cdk - phosphorylation sites , which are present in all known variants . four of these are located in the n - terminal fragment of eciz1 that contains in vitro replication activity ( see below ), and one is adjacent to the site at which exon 6 is alternatively spliced to exclude a short dsssq ( seq id no : 1 ) sequence motif ( fig2 a , c ). as this motif is 100 % identical and alternatively spliced in both mouse and man we reasoned that conditional inclusion might serve to regulate ciz1 activity , identifying this region of the protein as potentially important . we therefore chose to focus on the cdk site that is four residues upstream and which is also conserved in mouse and man , by combining a genetic approach with cell - free replication assays . starting with eciz1 , two threonines at 191 and 192 were changed to two alanines , generating eciz1t ( 191 / 2 ) a ( fig2 d ). when tested in vitro for dna replication activity , eciz1 t ( 191 / 2 ) a stimulated initiation in late g1 nuclei to a similar extent as eciz1 ( fig3 c ). however unlike eciz1 , stimulation of initiation was maintained over a broad range of concentrations that extended over at least three orders of magnitude . therefore , a mechanism to restrict the activity of excess eciz1 exists and operates in a cell - free environment . in a separate construct , the threonine at position 293 was also changed to alanine generating eciz1 t ( 293 ) a ( fig2 d ), but this alteration had little effect on eciz1 activity assayed in vitro ( fig3 d ). these results demonstrate that down - regulation of eciz1 activity involves threonine 191 / 2 , and is probably caused by cyclin - dependent kinase mediated phosphorylation at this site . this links ciz1 activity to the cdk - dependent pathways that control all major cell - cycle events , including initiation of dna replication . most pre - replication complex proteins and many replication fork proteins are phosphorylated in vivo , often by cyclin - dependent kinases ( bell and dutta , 2002 ; fujita , 1999 ). our data suggests that nuclear accumulation of p85 - ciz1 antigen is regulated ( directly or indirectly ) by cyclin a - cdk2 , and it shows that a specific consensus cdk phosphorylation site at threonine 191 / 192 is involved in controlling ciz1 activity . when this site is made unphosphorylatable ciz1 activity is maintained over a broader range of concentrations in cell - free assays . therefore , ciz1 activity is normally down regulated by modification at this site . the functions of the other conserved cdk phosphorylation sites , and the effect of conditional inclusion of an rxl cyclin - binding motif in the alternatively spliced n - terminal portion of ciz1 , remain to be determined . thus , the simple negative relationship between ciz1 activity and cdk - dependent phosphorylation that has been uncovered here , is unlikely to be the whole story . however , our analysis so far links ciz1 with the cdk - dependent pathways that control all major cell - cycle transitions , and is therefore consistent with our main conclusion that ciz1 is involved in initiation of dna replication . ciz1 possesses several c - terminal features that may anchor the protein within the nucleus . the matrin 3 domain suggests interaction with the nuclear matrix and the three zinc - fingers imply interaction with nucleic acids . indeed , recent evidence suggests that human ciz1 binds dna in a weakly sequence specific manner ( warder and keherley , 2003 ). to determine whether c - terminal domains are important for eciz1 replication activity we divided the protein into two fragments ( fig2 d ). nterm442 ( which contains the nls , two conserved cdk sites , one zinc finger and all known sites where variable splicing has been observed ) stimulates initiation to a similar extent and at the same concentration as eciz1 ( fig3 e ). in contrast , the c - terminal portion ( cterm274 ) contains no residual replication activity ( fig3 f ). therefore , the matrin 3 domain , one of the cyclin - dependent kinase phosphorylation sites and two of the zinc - fingers are not required for the dna replication activity of eciz1 , when assayed in vitro . it should be noted however that this analysis measures eciz1 activity in trans under conditions where the consequences of mis - localisation are unlikely to be detected . therefore , it remains possible that the matrin 3 domain and zinc fingers act in vivo to direct ciz1 activity to specific sites in the nucleus and thus limit the scope of ciz1 activity . endogenous ciz1 antibody v1 recognizes cdc6 as well as p85 - ciz1 ( fig1 a ), so it is not suitable for immuno - fluorescence experiments aimed at visualizing the sub - cellular localization of endogenous ciz1 . we therefore generated two new rabbit polyclonal anti - sera against recombinant eciz1 fragment nterm442 , designated anti - ciz1 1793 and 1794 . as expected , purified nterm442 is recognized by anti - ciz1 antibodies 1793 and 1794 in western blots , but it is also recognized by antibody v1 ( fig4 a ), supporting the conclusion that p85 ( p100 ) is indeed ciz1 . when applied to protein extracts derived from growing 3t3 cells , anti - ciz1 1793 recognized two antigens , with mr of 125 and 100 kda ( fig4 b ), whose relative proportions vary from preparation to preparation . the 100 kda band co - migrates with the cyclin - a responsive antigen that is recognized by antibody v1 ( fig1 and 4b ), which suggests that both antibodies recognize the same protein in vivo . we confirmed that the p100 - ciz1 bands recognized by antibody v1 and 1793 are the same protein by immuno - precipitation ( fig4 c ). antibody v1 precipitated a 100 kda band that was recognized in western blots by 1793 , and vice versa . furthermore , in the same experiment 1793 , and to a lesser extent antibody v1 , precipitated a 125 kda antigen , that was recognized in western blots by 1793 . taken together our observations show that the 100 kda band is indeed ciz1 ( previously known as p85 ), and they suggest that ciz1 protein exists in at least two forms in cycling cells . in addition to the immuno - precipitation evidence described above , several other observations lead to the conclusion that p125 is also a form of ciz1 . first , both of our anti - ciz1 antibodies ( 1793 and 1794 ) have this band in common . both antibodies produce the same pattern of nuclear staining in immuno - fluorescence experiments , and this is disrupted in cells treated with ciz1 sirna ( see below ). second , the relative proportions of p100 and p125 vary from preparation to preparation , and could therefore be the result of proteolytic cleavage . thirdly , our results are strikingly similar to those of mitsui et al ( 1999 ) whose anti - human ciz1 monoclonal antibody detected two antigens with apparent mr of 120 and 95 kda in hek293 cells . they proposed that the 120 kda form of human ciz1 protein is processed to produce the 95 kda form and our results are consistent with this proposal . the 125 kda band recognized by antibody 1793 in mouse and human cells resolves into three ciz1 - related bands during high - resolution electrophoresis of material derived from non - transformed human cells ( wi38 - see later ), and mouse cells ( nih3t3 — not shown ). this may be the result of post - translational modification of the ciz1 protein or of alternative splicing of the ciz1 transcript . sub - cellular distribution of ciz1 anti - ciz1 1793 was used to visualize the sub - cellular distribution of ciz1 protein ( p85 and p125 ) in 3t3 cells ( fig5 a ), and in hela cells ( not shown ). in both cell types 1793 reacted with a nuclear - specific antigen , and this was blocked by inclusion of recombinant nterm442 fragment ( fig5 b ). unlike cdc6 , which is shown for comparison ( fig5 a ), ciz1 is clearly detectable in all 3t3 cells in this cycling population . therefore ciz1 is present in the nucleus throughout interphase , although minor variations in quantity , or isoform would not be detected by this method . after detergent treatment overall nuclear ciz1 staining was reduced in all nuclei , which suggests that ciz1 is present in the nucleus as both a soluble fraction and also bound to insoluble nuclear structures . when soluble protein is washed away , the insoluble , immobilized antigen resolves into a punctate sub - nuclear speckled pattern at high magnification ( fig5 c , d ). ciz1 speckles show a similar size range and distribution as replication ‘ foci ’ or ‘ factories ’, the sites at which dna synthesis takes place in s phase . to ask whether ciz1 is coincident with sites of replication factories , we compared the position of ciz1 speckles to the position of pcna , a component of replication complexes in s phase cells ( fig5 c ). in confocal section , pcna foci are less abundant than ciz1 foci , but they are almost all co - incident with ciz1 ( fig5 d , e , f ). this is particularly striking for foci in the medium size range . in merged images , overlap between the positions of pcna and ciz1 foci results in yellow spots , while the remaining ciz1 foci that are not co - incident with pcna are red . green ( pcna alone ) foci are virtually absent , which suggests that ciz1 is present at all sites where dna replication factories have formed . ciz1 is also present at sites that don &# 39 ; t contain pcna ( fig5 d ), and unlike pcna , ciz1 foci persist throughout interphase ( fig5 a ). one interpretation of these observations is that ciz1 marks the positions in the nucleus at which pcna - containing replication factories are able to form in s phase , but that not all of these sites are used at the same time . it remains to be determined whether different ciz1 foci become active sites of dna replication at different times in s phase , or whether other nuclear activities also occur at sites where ciz1 is bound . indeed , at this stage it also remains possible that the 100 kda form and the 125 kda variants of ciz1 have different activities , and that they reside at nuclear sites with different functions . so far we have shown that the behavior of p85 ( p100 )- ciz1 correlates with initiation of dna replication in cell - free assays , that recombinant ciz1 stimulates the frequency of initiation , and that ciz1 resides at the same nuclear sites as the dna replication machinery . however , these data do not show that ciz1 has an essential function in proliferating cells . in order to test this we used rna interference ( rnai ) to selectively reduce ciz1 transcript levels in nih3t3 cells . four target sequences within ciz1 were chosen ( see fig2 a ) and short interfering ( si ) rna molecules were produced in vitro . when applied to cells , all four ciz1 sirna &# 39 ; s restricted growth ( fig6 a ) and caused a visible reduction in the level of ciz1 protein after 48 hours ( fig6 b ). the effect of ciz1 depletion on proliferation becomes apparent between 23 and 40 hours post - transfection , which suggests that the first cell cycle without ciz1 rna is relatively unaffected . by 40 hours , controls and ciz1 sirna treated cells diverged significantly with no further proliferation in the ciz1 depleted population . to verify the specificity of ciz1 depletion , transcript levels were monitored at 24 hours , before proliferation is significantly inhibited ( fig6 c ). at this point ciz1 transcripts were reduced to 42 % of the level in control cells treated with gapdh sirna . these experiments show that ciz1 is required for cell proliferation and are consistent with a primary function in dna replication . to test this further , cells were pulse - labelled with brdu 48 hours after sirna treatment to determine the fraction of cells engaged in dna synthesis ( fig6 d ). when ciz1 levels were reduced the brdu labelled fraction was also reduced , suggesting that dna synthesis is inhibited under these conditions . furthermore , cells in the ciz1 depleted population that did incorporate brdu ( approximately 15 % of the population ) were less intensely labelled . therefore , in some ciz1 sirna treated cells s phase is slowed down rather than inhibited completely , possibly due to incomplete depletion . inhibition of dna synthesis by ciz1 sirnas could be a secondary consequence of a general disruption of nuclear function . therefore , we looked in more detail at a range of other replication proteins whose levels are regulated in a cell cycle dependant manner , to ask whether depleted cells arrest randomly , or accumulate at a particular point . during initiation of eukaryotic dna replication mcm complex proteins assemble at replication origins in late g1 , in a cdc6 - dependent manner . sometime later , dna polymerases and their accessory factors ( including pcna ) become bound to chromatin and origins are activated . this is associated with nuclear export and proteolysis of the majority of cdc6 and , as dna synthesis proceeds , gradual displacement of the mcm complex from chromatin ( bell and dutta , 2002 ). in order to identify the point of action of ciz1 we used immuno - fluorescence to monitor mcm3 and pcna . in ciz1 depleted cells ( fig6 e , f ) both proteins were detectable within the nucleus bound to detergent resistant nuclear structures . therefore , these factors are unlikely to bind directly to ciz1 , or to be dependent upon ciz1 for their assembly . in fact , in four independent experiments the average number of cells with detergent - resistant chromatin - bound mcm3 actually increased from 31 % (+/− 6 %) to 51 % (+/− 5 %) ( fig6 e ). increased mcm3 indicates that the ciz1 dependent step occurs after pre - replication complex assembly ( but before completion of s phase ). in the same cell populations the pcna positive fraction also increased , from 32 % (+/− 5 %) to 49 % (+/− 6 %) ( fig6 f ), narrowing the point of ciz1 action to after pcna assembly . thus , ciz1 most likely acts to facilitate dna replication during a late stage in the initiation process , while failure to act inhibits progression through s phase , leaving mcm3 and pcna in place . taken together , our cell - free and cell - based investigations paint a consistent picture about the primary function of ciz1 . they suggest that ciz1 is a novel component of dna replication factories , and they show that ciz1 plays a positive role in the mammalian cell - cycle , acting to promote initiation of dna replication . three of our lines of investigation suggest that ciz1 is required during a late stage in the initiation process after pre - replication complex formation . first , p85 ( p100 )- ciz1 antigen accumulates in nuclei exposed to cyclin a - cdk2 concentrations that activate dna synthesis , implying that ciz1 functions during this step rather than during earlier replication complex assembly steps ( coverley et al ., 2002 ). second , functional studies with late g1 nuclei show that recombinant eciz1 increases the number of nuclei that incorporate labeled nucleotides in vitro . therefore , ciz1 must be active in a step that converts nuclei that are poised to begin dna synthesis into ones that are actively synthesizing dna . third , rna interference studies point to a ciz1 - dependent step after mcm complex formation and after pcna has become assembled onto dna , but before these proteins are displaced . these distinct lines of investigation lead to strikingly similar conclusions about the point of action of ciz1 placing it in the later stages of initiation . our analysis shows that ciz1 is essential for cell proliferation , and that targeting ciz1 is a viable strategy to restrain proliferation . the alternatively spliced forms of ciz1 that we observe in various cancers ( see below ) means that ciz1 could be targeted in a selective way to restrain proliferation in a subset of cells within a population . by way of example , this could be done by targeting sirna &# 39 ; s to the junction sequence created in ciz1 transcripts when the c - terminal sequence gttgaggaggaactctgcaagcag ( seq id no : 2 ) is missing , in small cell lung carcinoma cells , or by using ciz1 protein lacking the corresponding veeelckq ( seq id no : 3 ) sequence to select specific chemical inhibitors . accordingly the present invention also provides for the use of junction sequences created in ciz1 transcripts and proteins when alternatively spliced sequences are not present , as a diagnostic marker , prognostic indicator or therapeutic target . embryonic form ciz1 is localized to the nucleus rt - pcr analysis across potentially variable exons suggest that 3t3 cells predominantly express full - length ciz1 , so our immuno - localization work on endogenous ciz1 ( fig5 ) does not necessarily reflect the behavior of eciz1 , which lacks several sequence blocks and possibly therefore information that is used to localize the protein . to directly compare the localization of eciz1 and full - length ciz1 , enhanced gfp tagged constructs were transfected into 3t3 cells ( fig8 a ), and microinjected into mouse pro - nuclei ( fig8 b ). in all cases tagged ciz1 and eciz1 were exclusively nuclear , while a control construct expressing gfp alone was present in the nucleus and the cytoplasm . gfp - ciz1 and gfp - eciz1 were both visible in live cells as sub - nuclear foci , similar to replication foci seen in fixed cells by immuno - fluorescence . thus , the three sequence blocks that are absent from eciz1 do not appear to contribute to the nuclear localization of ciz1 . over the three day period following transfection no cell division was observed in the gfp - ciz1 and gfp - eciz1 transfected cells . these data suggest that overexpression of functional ciz1 has an inhibitory effect on the cell cycle ( in cells that have their regulatory pathways intact ). when gfp - tagged constructs in which the c - terminal one third of ciz1 had been removed were transfected into 3t3 cells , differences between eciz1 and full length ciz1 were observed ( fig8 c ). by 48 hours fl ciz1 n - term ( 442 equivalent ) had coalesced into large intra - nuclear blobs which only became apparent in the eciz1 n - term442 transfected population by day 3 or later . before this time eciz1 n - term442 was localized as a nuclear specific but diffuse pattern . thus ability to coalesce is quantifiably different between ciz1 and eciz1 , and is therefore affected by one of the three alternatively spliced exons ( 2 / 3 , 6 or 8 ). like cells transfected with full length ciz1 and eciz1 , cells transfected with constructs in which the c terminal one third was removed were not seen to multiply during the three day monitoring period . as described above , the difference between ciz1 and eciz1 n - term is masked when c - terminal domains are also present ( fig8 a ). furthermore the c - terminal fragment alone directs gfp tag to chromatin , forming an irregular pattern that is not as spotty ( focal ) as ciz1 or eciz1 , but which remains attached to chromosomes during mitosis ( fig8 d ). this suggests that c - terminal domains are involved in immobilizing ciz1 on a structural framework in the nucleus . notably , cells transiently transfected with c - terminal fragment continued to divide resulting in gradual dilution of green fluorescence . we looked at events occurring during the first day after transfection . the s phase fraction in transfected cells ( green ) was compared to the s phase fraction in untransfected cells , by labelling with brdu at various intervals . during long labelling windows including 0 - 22 hours ( fig8 e ), 0 - 12 hours and 0 - 7 hours ( not shown ), consistently more of the ciz1 and eciz1 transfected cells were engaged in dna synthesis , compared to untransfected cells . this suggests that ciz1 and eciz1 have a positive effect on the g1 - s transition , promoting unscheduled entry to s phase . similar results were obtained with 3t3 cell populations that were densely plated before transfection . this was done in order to minimize the fraction in the untransfected population that was engaged in s phase as part of the normal cell cycle . under these conditions the difference between the transfected and untransfected population was maximized , clearly demonstrating the effect of ectopic ciz1 on initiation of dna replication . conversely , when cells were labelled with brdu during a short pulse administered at 22 hours ( fig8 e ), or at 10 hours or 12 hours post - transfection ( not shown ), the labelled fraction was consistently reduced in the ciz1 and eciz1 transfected populations . this suggests that the s phase that is induced by ectopic ciz1 or eciz1 is abnormal , with slow or aborted dna synthesis that is not sufficient to label cells during short windows of exposure to brdu . therefore , ectopic ciz1 and eciz1 have two effects on s phase in cultured cells . they promote dna replication , but this results in slow or aborted dna synthesis . we also monitored transfected populations of 3t3 cells over a three week time period . in cells transfected with the gfp - nterm442 or the non - alternatively spliced equivalent and maintained under selection with g418 , large foci containing hundreds of cells were observed ( fig9 a ). these clusters contained large numbers of gfp expressing cells , demonstrating that over - expression of the n - terminal portion of eciz1 ( in which replication activity resides ) is not lethal , and suggesting that over - expression leads to altered proliferation phenotype , compared to untransfected cells , including loss of contact inhibition and failure to form a monolayer . this ciz1 - dependent altered behavior could contribute to tumor formation . a similar truncated version of mouse ciz1 , lacking putative chromatin interaction domains was previously isolated from a mouse melanoma ( fig2 ). as mentioned above human ciz1 is alternatively spliced at the rna level to yield transcripts that lack three of the same exons as mouse embryonic ciz1 . seven human ciz1 cdnas have been recorded in public databases ( fig1 ), submitted by mitsui et al ( 1999 ), warder and keherly ( 2003 ) and large - scale genome analysis projects ( nih - mgc project , nedo human cdna sequencing project ). only one is derived from normal adult tissue , and this contains all predicted exons ( ab030835 ). the rest are derived from embryonic cells ( ak027287 ), or notably from four different types of pediatric cancer ( medulloblastoma , af159025 , af0234161 , retinoblastoma , ak023978 , neuroblastoma , bc004119 and burkitt lymphoma , bc021163 ). the embryonic form and the cancer derived forms lack sequence blocks from the same three regions as our embryonic mouse clone , and from a fourth region which corresponds to exon 4 . therefore , the limited data suggests that alternatively spliced forms are more prevalent early in development . this correlation has not previously been noted in the scientific literature . the presence of alternatively spliced ciz1 in pediatric cancers raises the possibility that ciz1 mis - splicing might be linked to inappropriate cell proliferation . for example , one of the variable exons encodes a short conserved dsssq ( seq id no : 1 ) sequence motif that is absent in mouse eciz1 and in a human medulloblastoma . this is directly adjacent to the consensus cdk phosphorylation site that we have shown to be involved in regulation of eciz1 function . conditional inclusion of the dsssq ( seq id no : 1 ) sequence might make ciz1 the subject of regulation by the atm / atr family of protein kinases , which phosphorylate proteins at sq sequences , thereby restraining ciz1 initiation function in response to dna damage . the presence of alternatively spliced ciz1 in pediatric cancers prompted a detailed analysis of ciz1 ests . there are 567 expressed sequence tags ( ests ) included in ncbi unigene cluster hs . 23476 ( human ciz1 ). these are derived from a wide range of normal and diseased tissues and cell lines . sequences have been translated and mapped against the predicted full - length amino - acid sequence of human ciz1 . sequence alterations that give rise to amino - acid substitutions , deletions , frame - shifts and premature termination of translation have been recorded . alternatively spliced ciz1 variants were also seen in this est data set and are recorded here . the four sequence blocks that we previously reported to be alternatively spliced in human and mouse ciz1 ( fig2 ) were observed in the est sequences , as well as a previously undetected variant that lacks the exon 14 derived sequence veeelckq ( seq id no : 3 ). all of these recurrently variant sequence blocks are bounded by appropriate splice sites . a sixth variable sequence block was identified in one carcinoma derived library , caused by inclusion of gccacccacaccacgaagagatgtgttgcccacgttccagtgcaggggtggagca cagcccggcttgttacagatat ( seq id no : 4 ). ests are grouped according to the cell type from which they were derived with the primary divisions occurring between neoplastic cells of adult , childhood or embryonic origin . ests from normal tissue of embryonic or adult origin are included for comparison . est - derived ciz1 protein maps are shown in fig1 a - e and the alternatively spliced exons summarized in fig1 f . three sequence blocks in the n - terminal end of human ciz1 are absent in transcripts from medulloblastomas and neuroblastoma ( fig1 a ), and occasionally absent from ciz1 transcripts from other cancers . we also found similar alternative splicing in a third pediatric cancer , ewings sarcoma ( see below ). pediatric cancer - associated alternatively spliced sequences are from exons 2 / 3 ( at least two versions ), exon 4 and exon 6 . exon 8 variants in which one or more copies of a q - rich degenerate repeat are absent have been noted in transcripts derived from normal cells ( of embryonic or adult neural origin ) and from various cancers . alternative splicing in this region could produce ciz1 with inappropriate activity , therefore exon 8 variant expression , or occurrence of point mutations which influence splicing in this region , might be useful as diagnostic or prognostic markers in cancer . the alternatively spliced degenerate repeats in exon 8 are detailed below and summarized in fig1 f . in the c - terminal half of the human ciz1 protein two sequence blocks are variably spliced . one of these is missing from transcripts derived from three out of five lung carcinoma and lung carcinoid libraries , and from three other carcinoma libraries ( but very rarely from transcripts from other cell types ). the second variant sequence block is due to inappropriate inclusion of extra sequence in transcripts from the epidermoid carcinoma library ( mgc102 ). these sequences and the junction sequences formed in ciz1 proteins , and ciz1 transcripts when these segments are excluded or included , are potential targets for selective inhibition of cell proliferation in a wide range of different cancers . the remaining non - variant sequences are potential targets for non - selective inhibition of cell proliferation . in addition to splicing variations , other non - typical ciz1 transcripts were found to preferentially occur in some cancers . in rhabdomyosarcomas ciz1 is prematurely terminated leading to a predicted protein that lacks c - terminal nuclear binding domains . this could lead to inappropriate dna replication and might therefore be a therapeutic target or marker in this type of cancer . several transcripts contain point mutations that lead to amino - acid substitutions in putative cyclin - dependent kinase ( cdk ) phosphorylation sites . in the cervical carcinoma library mgc12 , this occurs twice . we have shown that two cdk phosphorylation sites are involved in restraining ciz1 activity ( fig3 c and d ), implicating these mutations in the deregulation of proliferation in cancer cells . one of these is the same as the carcinoma - derived mutant mentioned above ( fig1 e ). cancer - derived transcripts with point mutations in ciz1 could also be targeted by rna interference , or have value as diagnostic or prognostic indicators . ciz1 variant expression was investigated in 6 ewings sarcoma family tumor cell lines ( esfts ) and two neuroblastoma cell lines , using rtpcr with primer sets that span three regions of known ciz1 variability ( fig1 a ). this analysis showed that the pattern of ciz1 variant expression is different in esft cells compared to neuroblastoma cells compared to non - transformed cells , but apparently very similar within sets of cell lines from the same tumor . therefore , ciz1 variant expression could have prognostic or diagnostic potential for these cancers . minor variations within a set of lines from the same tumor type could have prognostic value . by subcloning and sequencing amplified transcripts we found that all six esft lines tested express an exon 4 minus form of ciz1 . as ciz1 is essential for cell proliferation ( see below ), this offers a possible route for selective restraint of esft cells . transcripts from the two neuroblastoma cell lines tested rarely lack exon 4 but frequently lack sequences the dsssq ( seq id no : 1 ) motif encoded by exon 6 ( fig1 b ). this experimental analysis confirms that pediatric cancers express forms of ciz1 with variable inclusion of exons 4 , 6 and probably exons 2 / 3 . two versions of the sequence encompassing exon 8 and one form of the sequence encompassing the veeelckq - coding sequence were detected in esfts , neuroblastomas and control suggesting that these regions do not contribute to deregulation of ciz1 in these paediatric cancers . in all cases , ciz1 rt - pcr products were most abundant in reactions carried out with rna samples from cancer cell lines , compared to controls ( wi38 , hek293 , nih3t3 cells , and primary human osteoblasts ). this is consistent with increased expression of ciz1 variants in tumors . normal , non - transformed human lung fibroblasts ( and mouse nih3t3 cells ) express two major forms of ciz1 that are detected by anti - ciz1 polyclonal antibody 1793 in western blots ( fig1 a ). the larger ( approximately 125 kda ) band resolves into three distinct bands that are present in equal proportions in wi38 cells , but grossly uneven proportions in prostate cancer cell lines pc3 and lncap ( and esft cell lines — not shown ). we postulate that these protein isoforms are generated by expression of variably spliced exons . both tumor cell lines also contain more ciz1 antigen than wi38 cells , consistent with over - expression of ciz1 in these cancer cell lines . taken together , our results ( experimental and bioinformatics analysis of genome data ) support the conclusion that ciz1 is mis - regulated in a wide range of human cancers . we have shown that the ciz1 protein plays a positive role in the dna replication process , therefore mutant ciz1 could contribute to cellular transformation , rather than be a consequence of it . if deregulation of ciz1 is a common step in this process it represents a very attractive target for development of therapeutic agents . we have also associated particular changes with specific cancers , making it a real possibility that ciz1 could be useful as a diagnostic or prognostic marker . alternative splicing in the n - terminal part of the protein ( that contains replication activity in vitro ) in pediatric cancers . point mutations in cyclin - dependent kinase phosphorylation sites known to be involved in restraining ciz1 replication activity . non - typical expression and nuclear binding properties of ciz1 - p125 forms in prostate carcinoma cell lines , possibly due to mis - regulated splicing of the degenerate repeats in exon 8 , or other exons . conditional exclusion of a discrete motif ( veeelckq ) in the c - terminal end of ciz1 ( probably involved in localization of ciz1 protein within the nucleus ) in small cell carcinoma of the lung and other carcinomas . increased levels of ciz1 protein and rna ( detected by western blot and by rt - pcr ) in all cancer derived cells lines tested so far , compared to wi38 normal embryonic lung fibroblast , human osteoblast rna and mouse nih3t3 fibroblasts . the sequences shown in fig1 to 21 are of use for the development of therapeutic , diagnostic , or prognostic reagents . a lamba triplex 5 ′- stretch , full length enriched cdna expression library derived from 11 day old mouse embryos ( clontech ml5015t ) was used to infect e . coli xl1blue according to the recommended protocol ( clontech ). plaques were lifted onto 0 . 45 micron nitrocellulose filters pre - soaked in 10 mm iptg ( sigma ). affinity purified antibody v1 was applied to approximately 3 × 10 6 plaques at 1 / 1000 dilution in pbs , 10 % non - fat milk powder , 0 . 4 % tween20 , after blocking for 30 minutes in the absence of antibody . after two hours filters were washed three times with the same buffer and reactive plaques were visualized with anti - rabbit secondary antibody conjugated to horse - radish peroxidase ( sigma ), and enhanced chemi - luminescence ( ecl , amersham ) according to standard procedures . 43 independent plaques were picked but only two strains of phage survived a further three rounds of screening . these were converted to ptriplex by transforming into bm25 . 8 and sequenced . one codes for mouse cdc6 ( clone p ) and the other ( clone l ) for an unknown mouse protein that is homologous to human ciz1 . we refer to this as embryonic ciz1 ( eciz1 ) and it was submitted to embl under the accession number aj575057 . bacterial expression pgex based bacterial expression constructs ( amersham ) were used to produce eciz1 proteins for in vitro analysis . pgex - eciz1 was generated by inserting a 2 . 3 kb smai - xbai ( blunt ended ) fragment from clone l into the smai site of pgex - 6p - 3 . pgex - nterm442 was generated by inserting the 1 . 35 kb xmai - xhoi fragment into xmai - xhoi digested pgex - 6p - 3 , and pgex - cterm274 by inserting the 0 . 95 kb xhoi fragment into xhoi digested pgex - 6p - 3 . pgex - t ( 191 / 2 ) a was generated from pgex - eciz1 by site directed mutagenesis ( stratagene quikchange ) using primers aaccccctcttccgccgcccccaatcgcaaga ( seq id no : 5 ) and tcttgcgattgggggcggcggaagagggggtt ( seq id no : 6 ). pgex - t ( 293 ) a was generated from pgex - eciz1 using primers aagcagacacaggccccggatcggctgcct ( seq id no : 7 ) and aggcagccgatccggggcctgtgtctgctt ( seq id no : 8 ). integrity and reading frame of all clones were sequence verified . recombinant ciz1 , ciz1 fragments and point mutants were produced in bl21 - plyss ( stratagene ) as glutathione s - transferase - tagged protein . this was purified from sonicated and cleared bacterial lysates by binding to glutathione sepharose 4b ( amersham ). recombinant protein was eluted by cleavage from the gst tag using precision protease ( as recommended by the manufacturer , amersham ), into buffer ( 50 mm tris - hc ph 7 . 0 , 150 mm nacl , 1 mm dtt ). this yielded protein preparations between 0 . 2 and 2 . 0 mg / ml . for replication assays serial dilutions were made in 100 mm hepes ph 7 . 8 , 1 mm dtt , 50 % glycerol so that not more than 1 ml of protein solution was added to 10 ml replication assays , yielding the concentrations shown . consistent with previous observations ( mitsui et al ., 1999 ; warder and keherly , 2003 ) recombinant ciz1 , and derived fragment n - term442 migrated through sds - page with anomalously high molecular weight . cyclin a - cdk2 was produced in bacteria as previously described ( coverley et al ., 2002 ). rabbit polyclonal antibody v1 ( coverley et al ., 2000 ; stoeber et al ., 1998 ; williams et al ., 1998 ) was raised against an internal fragment of bacterially expressed human cdc6 corresponding to amino - acids 145 - 360 , and affinity purified by standard procedures ( harlow and lane , 1988 ). this antibody reacts strongly with endogenous p100 - ciz1 and also with eciz1 nterm442 fragment . alignment of nterm442 with cdc6 amino - acids 145 - 360 suggest that the shared epitope could be at 294 - 298 or 304 - 312 in mouse ciz1 . recombinant nterm442 was used to generate two ciz1 - specific polyclonal anti - sera designated 1793 and 1794 ( abcam ). 1793 has been used routinely in the experiments described here . its specificity was verified by reciprocal immuno - precipitation and western blot analysis with antibody v , by inclusion of nterm 442 ( 25 μg / ml in antibody buffer , 10 mg / ml bsa , 0 . 02 % sds , 0 . 1 % triton x100 in pbs ), which blocked reactivity with endogenous epitopes , and by sirna - mediated depletion of ciz1 that specifically reduced 1793 nuclear staining . asynchronousy growing 3t3 cells were washed in pbs , rinsed in extraction buffer ( 20 mm hepes ph7 . 8 , 5 mm potassium acetate , 0 . 5 mm magnesium chloride ) supplemented with edta - free protease inhibitor cocktail ( roche ) and scrape harvested as for replication extracts . cells were lysed with 0 . 1 % triton x 100 and the detergent resistant pellet fraction extracted with 0 . 3m nacl in extraction buffer . 5 μl of 1793 or 2 μl of antibody v were used per 100 μl of extract and incubated for 1 hour at 4 ° c . antigen - antibody complexes were extracted with 100 μl of protein g - sepharose ( sigma ) and beads were washed five times with 50 mm tris ph 7 . 8 , 1 mm edta , 0 . 1 % np40 , 150 mm nacl . complexes were boiled in loading buffer ( 100 mm dtt , 2 % sds , 60 mm tris ph6 . 8 , 0 . 001 % bromophenol blue ) and resolved by 6 . 5 % sds - polyacrylamide gel electrophoresis . cells were grown on coverslips and fixed in 4 % paraformaldehyde , with or without brief pre - exposure to 0 . 05 % triton x100 in pbs . endogenous ciz1 was detected with 1793 serum diluted 1 / 2000 in antibody buffer following standard procedures . mcm3 was detected with monoclonal antibody sc9850 ( 1 / 1000 ), cdc6 with monoclonal sc9964 ( 1 / 100 ) and pcna with monoclonal antibody pc10 ( 1 / 100 , all santa cruz biotechnology ). co - localization analysis of dual stained fluorescent confocal images was carried out as described ( rubbi and milner , 2000 ; van steensel et al ., 1996 ). mouse 3t3 cells were synchronized by release from quiescence as previously described ( coverley et al ., 2002 ). nuclei prepared from cells harvested 17 hours after release ( referred to as ‘ late - g1 ’) were used in all cell - free replication experiments described here . this yielded populations containing s phase nuclei , replication competent late g1 nuclei and unresponsive early g1 / g0 nuclei , in varying proportions . recipient , mid - g1 3t3 extracts were prepared at 15 hours ( these typically contain approximately 5 % s phase cells ). the series of cell - free replication experiments described here required large amounts of standardized extract , therefore hela cells were used because they are easily synchronized in bulk . s phase hela extracts were prepared from cells released for two hours from two sequential thymidine - induced s phase blocks , as described ( krude et al ., 1997 ). dna replication assays were performed as described ( coverley et al ., 2002 ; krude et al ., 1997 ). briefly , 10 μl of mid g1 or s phase extract ( supplemented with energy regenerating system , nucleotides and biotinylated dutp ), and 5 × 10 4 late g1 phase nuclei were incubated for 60 mins at 37 ° c . reactions were supplemented with baculovirus lysate containing cyclin a - cdk2 ( fig1 b and c ), where 0 . 1 μl of lysate has the same specific activity as 1 nm purified kinase ( coverley et al ., 2002 ). all recombinant proteins were serially diluted in 100 mm hepes ph 7 . 8 , 1 mm dtt , 50 % glycerol , so that not more than 1 μl was added to 10 μl replication assays , generating the concentrations indicated . reactions were stopped with 50 μl of 0 . 5 % triton x100 and fixed by the addition of 50 μl of 8 % paraformaldehyde , for 5 minutes . after transfer to coverslips , nuclei were stained with streptavidin - fitc ( amersham ) and counterstained with toto - 3 - iodide ( molecular probes ). the proportion of labelled nuclei was quantified by inspection at 1000 × magnification , and all nuclei with fluorescent foci or intense uniform labelling were scored positive . images of in vitro replicating nuclei were generated by confocal microscopy at 600 × magnifications , of samples counterstained with propidium iodide . for analysis of nuclear proteins , nuclei were re - isolated after 15 minutes exposure to initiating conditions , by diluting reactions two fold with cold pbs and gentle centrifugation . prior to use in initiation assays each preparation of synchronized g1 phase nuclei is tested so that the proportion of nuclei that are already in s phase is established (‘% s ’). to do this nuclei are incubated in an extract that is incapable of inducing initiation of dna synthesis ( from mid - g1 phase cells harvested 15 hours after release from quiescence ), but that will efficiently support elongation dna synthesis from origins that were initiated in vivo . the elongating fraction of nuclei incorporates labeled nucleotides efficiently during in vitro initiation assays but is uninformative . routinely this fraction is pre - established and subtracted from the raw data . synchronized populations in which 20 % or less are in s phase are used for initiation assays . when 3t3 cells are released from quiescence by the protocol used here no more than 70 % of the total population enters s phase ( coverley et al ., 2002 ). however , the highest observed replication frequency in vitro is nearer 50 %; usually obtained by incubation with eciz1 . for the g1 population of 3t3 nuclei used here 17 % were in s phase (% s ) and the maximum number that replicated in any assay in vitro was 51 % (% replication ). therefore , 34 % of this population is competent to initiate replication in vitro (% c ). thus , for each data point in fig3 b - f , % initiation =(% replication −% s )/% c × 100 . endogenous ciz1 was targeted in proliferating nih3t3 cells using in vitro transcribed sirnas ( ambion silencer kit ), directed against four regions of mouse ciz1 . oligonucleotide sequences that were used to generate sirnas are aagcacagtcacaggagcagacctgt ( seq id no : 9 ) ctc and aatctgctcctgtgactgtgccctgtctc ( seq id no : 10 ) for sirna 4 , aatctgtcacaagttctacgacctgtctc ( seq id no : 11 ) and aatcgtagaacttgtgacagacctgtctc ( seq id no : 12 ) for sirna 8 , aatcgcaaggatcttcttctcctgtctc ( seq id no : 13 ) and aaagaagaagaatcctgcgacctgtctc ( seq id no : 14 ) for sirna 9 , and aatctgcagcagttcttccccctgtctc ( seq id no : 15 ) and aagggaaagaactgctgcagacctgtctc ( seq id no : 16 ) for sirna 11 . target sequences that are distributed throughout the ciz1 transcript were chosen based on low secondary structure predictions and on location within exons that are consistently expressed in all known forms of ciz1 ( sequences 4 , 8 , 11 ), with the exception of one ( sirna 9 ) that is known to be alternatively spliced . negative controls were untreated , mock treated ( transfection reagents but no sirna ) and cells treated with gapdh sirna ( ambion ). cy3 labelled sirnas ( ambion ) were used to estimate transfection efficiency , which was found to be greater than 95 %. rna interference experiments were performed in 24 well format starting with 2 × 10 4 cells per well in 500 μl of medium ( dmem with glutamax supplemented with 4 % fcs ). sirna &# 39 ; s were added 12 hours after plating using oligofectamine reagent for delivery ( invitrogen ). unless stated otherwise , sirnas were used in pairs ( at 2 nm total concentration in medium ), as two doses with the second dose delivered in fresh medium 24 hours after the first . results were assessed at 48 hours after first exposure , by counting cell number , s phase labelling , and immuno - staining . northern blots were performed on rnas isolated from cells treated for 24 hours with a single dose of sirna , in reactions that were scaled up 5 fold . rna was prepared using trizol reagent ( invitrogen ) and samples were electrophoresed through 1 % agarose , transferred onto hybond n + nylon membrane ( amersham ), and sequentially hybridized at 50 ° c . with cdna probes using northernmax kit reagents ( ambion ), following manufacturers instructions . the membrane was stripped between each hybridization using 0 . 5 % sds solution at 90 ° c ., allowed to cool slowly to room temperature . probes were [ 32 p ]- dctp labelled using random primers dna labelling system ( gibco brl ), and used in the following order : i . a 1 . 35 kb xmal - xhol fragment derived from eciz1 . ii . human β - actin cdna ( clontech ) and iii . mouse gapdh cdna ( rnway laboratories ). the membrane was washed twice in 2 × ssc 0 . 2 % sds for 30 - 60 mins each , followed by one wash in 0 . 2 × ssc 0 . 2 % sds for 30 mins , at 55 - 65 ° c ., depending on probe used . hybridization signals were quantified using an amersham biosciences typhoon 9410 variable mode imager , and image quant tl software ( v2002 ). band intensities are expressed in arbitrary units ( in parentheses ), and results for ciz1 and gapdh were normalized against those for β - actin , and expressed as a %. the fraction of nuclei undergoing dna synthesis in vivo was monitored by supplementing culture medium with 20 μm bromodeoxyuridine ( brdu , sigma ) for 20 minutes . incorporated brdu was visualized after acid treatment with fitc - conjugated anti - brdu monoclonal antibody ( alexis biochemicals ) according to manufacturers instructions . nuclei were counterstained with hoescht 33258 and scored under high ( 1000 ×) magnification . full - length mouse ciz1 cdna was obtained from uk hgmp resource centre ( mgc clone 27988 ) and the sequence fully verified . a 2 . 8 kb smai - xbai ( blunt ended ) full length ciz1 fragment from this clone , and a 2 . 3 kb smai - xbai ( blunt ended ) eciz1 fragment from ptriplex - clone l were ligated in frame with enhanced green fluorescent protein ( egfp ) into the smai site of pegfp - c3 ( clontech ). pegfp - c3 with no insert was used as a control . constructs were transfected into nih3t3 cells using transit - 293 ( mirus ), following manufacturers instructions or microinjected into the male pro - nucleus of fertilized mouse eggs at the one cell stage . growing 3t3 cells transfected with full length egfp - ciz1 , or egfp - eciz1 were analysed by live cell fluorescent microscopy up to three days after transfection . dna synthesis was monitored during the first 24 hours after transfection , by including the nucleotide analogue brdu in cell culture medium for various time periods as indicated in figure legends . as described above any cells undergoing dna synthesis while exposed to brdu stain with anti - brdu monoclonal antibody generating red nuclei . ciz1 transfected cells were also maintained under selection with 50 μg / ml g418 , in standard culture medium ( dmem glutamax plus 10 % fetal calf serum ) for up to a month , yielding cell populations with altered morphology . individual expressed sequence tags ( ests ) mapping to ncbi unigene cluster hs . 23476 ( human ciz1 ) were translated using genejockey and the predicted amino - acid sequence compared to the predicted sequence for full length ciz1 , with the aim of identifying recurrent changes in cancer cells . in order to exclude errors that reflect poor quality dna sequence such as that which occurs at the end of long sequencing runs , only those changes positioned more than 8 amino - acids from the end of uninterrupted sequence are included in this analysis . frame - shifts that are restored by a second alteration later in the read , and frame - shifts that are followed by a stop codon are only included if followed by uninterrupted sequence . thus the majority of sequencing errors are excluded from this analysis . however , it is expected that many of the point mutations that remain ( including frame - shifts and stops ) reflect errors introduced during sequencing . therefore , this analysis is aimed at uncovering trends , with weight being given to point mutations only if they appear more than once . of 567 sequences that map to ciz1 unigene cluster , we have analyzed most ( all paediatric cancers , prostate and lung carcinomas , leukemias and lymphomas and a wide range of non - diseased tissues ). some were not mapped because they are extremely short reads or yielded very short amino - acid sequences upon translation , and for a small number we detected no homology to the ciz1 coding sequence . a small number of ests were excluded from the analysis because of multiple frameshifts that produced stretches of homology in all three frames , with no indication of the reading frame used in vivo . these were all from cancer derived material , usually adenocarcinomas . rna was isolated using trizol reagent following recommended procedures , dnase treated and reverse transcribed using random hexamers and superscript ii , then amplified with ciz1 specific primers : h / m5 ( seq id no : 17 ) cagtccccaccacaggcc , h / m2 ( seq id no : 18 ) ggcttcctcagacccctctg . h / m3 ( seq id no : 19 ) acacagacctctccagagcacttag h / m4 ( seq id no : 20 ) atggtgaccttcagggagc h4 ( seq id no : 21 ) tccttggcga tgtcctctgg gcagg h3 ( seq id no : 22 ) tccctcctca acggctccat gctgc h6 ( seq id no : 23 ) cg tgggggcgac ttgagcgttg agg h1 ( seq id no : 24 ) gatgccaggggt atggggcgcc ggg h2 ( seq id no : 25 ) tccgagccct tccactcctc tctgg . cells were grown in dmem with 10 % fcs until sub - 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