Patent Application: US-60126700-A

Abstract:
the present invention relates to the identification of the genomic promoter region of the human and mouse telomerase rna gene . telomerase activity is necessary for the unrestricted proliferative capacity of many human cancers . it is proposed that mutation or dysregulation of the telomerase repression pathway may cause reactivation or upregulation of telomerase expression in cancer . the invention provides details of elements important for the regulation of telomerase rna genes , including the sp family of transcription factors . there is further provided methods for screening elements having the ability for suppressing telomerase rna gene promoter activity and use of such elements in the treatment of cancers . in addition , evidence is also provided for the development of new transcription based therapies for cancer and for genetic approaches to targeting therapeutic genes to cancer cells . namely , transcriptional repression and the disruption of signal transduction pathways regulating telomerase activation . tumour specific gene expression for genetic therapy via telomerase rna gene promoters .

Description:
preparation of tissue sections for in situ hybridisation formalin fixed paraffin embedded tissue blocks were obtained from pathology department files . tissue sections were deparaffinised , rehydrated through graded concentrations of ethanol , ( 100 %, 90 %, 70 %, 50 %, 30 % etoh , 10 sec . each ), rinsed in 0 . 85 % sodium chloride for 5 minutes , followed by pbs for 5 minute . sections were fixed in 4 % paraformaldehyde / pbs for 20 minutes , rinsed in pbs , and treated with proteinase k ( 40 μg / ml ) in 50 mm tris - hcl ph 7 . 5 , 5 mm edta for 7 . 5 minutes at room temperature . after rinsing for 5 minutes in pbs , sections were post fixed in 4 % paraformaldehyde / pbs for 5 minutes , rinsed in water , and acetylated in freshly prepared 0 . 25 % acetic anhydride / 0 . 1m triethanolamine for 10 minutes . the slides were rinsed in 0 . 85 % saline , followed by pbs for 5 minutes each and dehydrated in gradually increasing concentrations of ethanol prior to hybridisation . the riboprobe plasmid containing telomerase rna sequences used for rna in situ hybridisation is as previously described ( soder et al ., 1997b ). control riboprobes were human histone h3 and glyceraldehyde 3 - phosphate dehydrogenase ( gapdh ), ( ambion , tex .). the probes were labelled with ( 35 s )- utp using a rna labelling kit ( amersham , uk ). transcripts were purified using a sephadex g - 50 column ( pharmacia ), phenol / chloroform extracted and precipitated in ethanol . the probes were resuspended in 50 mm dithiothreitol . northern blot analysis of normal human tissue confirmed the specificity and sensitivity of the htr probe to detect htr expression in normal testis , ( data not shown ). sections were hybridised overnight at 52 ° c . in 60 % formamide , 0 . 3m nacl , 10 mm tris - hcl ( ph 7 . 5 ), 5 mm edta , 10 % dextran sulphate , 1 × denhardts ( 0 . 02 % bsa , 0 . 02 % ficoll , 0 . 02 % polyvinylpyrrolidone ), 0 . 5 mg / ml yeast trna , 50 mm dtt ( freshly added ), and 50 000 cpm / ul 35 s - labelled crna probe . the tissue was washed stringently at 50 ° c . in 5 × ssc , 0 . 1 % β - mercaptoethanol for 25 minutes , at 65 ° c . in 50 % formamide , 2 × ssc , 1 % β - mercaptoethanol for 25 minutes , and washed twice at 37 ° c . in 0 . 5m nacl , 10 mm tris - hcl ph 7 . 5 , 5 mm edta for 15 minutes before treatment with 20 μg / ml rnasea at 37 ° c . for 30 minutes . rnase a only digests single stranded rna . this removes single stranded , and therefore unhybridised probe , leaving the rna : rna duplexes intact . thus this step helps reduce background probe signal . following washes in 50 % formamide , 2 × ssc , 1 % β - mercaptoethanol at 65 ° c . for 20 minutes , and twice in 2 × ssc at 50 ° c . for 15 , the slides were dehydrated and dipped in 0 . 1 % gelatine / 0 . 01 % chromealun , and then in hypercoat nuclear lm - 1 emulsion ( amersham ) and exposed for 2 weeks in light tight boxes with desiccant at 4 ° c . the microautradiographs were developed in 20 % phenisol for 2 . 5 minutes , washed in 1 % acetic and water each for 30 seconds , fixed in 30 % sodium thiosulphate for 5 minutes , rinsed in water 30 minutes , and counter - stained with haematoxylin . cloning of sequences encompassing the human and mouse telomerase rna genes the present inventors have previously reported the identification of genomic clones in p1 vectors containing htr and terc transcribed sequences ( soder et al ., 1997b ; soder et al ., 1997c ). the human p1 clone , 9913 , is derived fron a human foreskin fibroblast p1 library and the mouse p1 clone , 11792 , is derived from a mouse c127 fibroblast p1 library . briefly , in order to subclone the promoter regions , the p1 clones were digested with ecori and hindiii and ligated into pbluscript . colonies containing telomerase rna gene sequences were identified by hybridisation with pcr generated probes as previously described ( soder et al ., 1997b ; soder et al ., 1997c ). plasmid dna was prepared from positively hybridising colonies , and inserts sequenced on both strands by dideoxy chain termination using the abi prism dye terminator cycle sequencing kit ( pe applied biosystems , warrington , uk ) and 25 ng oligonucleotide primers , dye labelled products were resolved and detected using the applied biosystems dna sequencer abi373 . sequence was analysed using the sequencing analysis program 3 . 0 . homology searches were carried out using blast ( basic local alignment search tool ), national centre for biotechnology information ( ncbi ). sequence was analysed for potential transcription factor binding sites by tess : transcription element search software on the www , jonathan schug and g . christian overton , technical report cbil - tr - 1997 - 1001 - v0 . 0 , of the computational biology and informatics laboratory , school of medicine , university of pennsylvania . identification of cpg islands was carried out using grail : gene recognition and assembly internet link , computational biology section of the life science division , oak ridge national laboratory . the full sequences have been submitted to genbank . the structures of the telomerase rna gene - luciferase constructs used in the study are shown in fig5 and 4 . promoter - luciferase constructs were made by inserting pcr products into pgl3 - basic , ( promega ). orientation and sequence of each insert was checked by sequencing . details of the primers are given in fig6 . all transfections were carried out in duplicate in 6 - well plates , ( 35 mm diameter ). cells were seeded at 6 × 10 4 cell per well and cultured overnight . transfection was carried out using superfect transfection reagent , ( qiagen ), according to the manufacturers instructions . cells were exposed to the transfection mix for three hours and harvested for analysis after 48 hours . equivalent amounts of cellular protein as determined by bio - rad assay , ( biorad ), were used in the luciferase assay . luciferase assays were performed according to the manufacturers protocols , ( promega ). to ensure reproducibility in the assays , particular care was taken over the following : dna used for transfection was quantified by spectrophotometry and direct visualisation by gel electrophoreses . all transfections were carried out in duplicate wells and this was found to be a good measure of the reproducibility of transfection . in each experiment , all deletion constructs were analysed together with both the basic cloning vector , pgl3 - basic and the positive control vector , pgl3 - control , which contains sv40 promoter and enhancer sequences . each extract was measured for luciferase activity at least twice . all transfections were carried out at least three times . initial transfection conditions were determined by using promoter fragments liked to a green fluorescent protein reporter gene , ( clontech ), as this allowed direct visualisation of promoter activity in live cells , ( data not shown ). the present inventors found it important to transfect and analyse the cells at sub - confluence and that it was important to avoid harsh transfection protocols such as electroporation , resulting in poor cell viability . tumour specific regulation of telomerase rna gene expression visualized by in situ hybridization . the patterns of htr expression were examined in epithelial cancer of lung , ovary , breast and cervix , ( table 1 ). twenty six percent of non - small cell lung cancers , ( nsclc ), were htr positive . however , the nsclc group consists of squamous , adenocarcinoma and large - cell anaplastic variants . interestingly however , expression was almost exclusively limited to the squamous variants , ( p = 0 . 006 ), with 41 % of squamous nsclc expressing htr but only 8 % of adenocarcinoma and large - cell anaplastic nsclc expressing htr . this data suggests that htr may be differentially regulated during the oncogenesis of squamous and non - squamous nsclc . indeed , the low frequency of detectable htr expression in adenocarcinoma of the lung was also observed in adenocarcinomas of ovary and breast , ( table 1 ). in addition , metastatic carcinoma in hilar lymph nodes of 19 of the nsclc cases were available for a comparative study with the paired primary carcinomas . all 6 cases which expressed htr in the primary carcinoma retained expression in the metastasis and all 13 primary carcinomas which lacked detectable htr expression remained negative in the metastasis , ( table 1 ). thus , expression levels appear stable between primary and metastatic carcinomas and expression of htr is not associated with metastasis of pulmonary carcinomas . cancer of the uterine cervix is a heterogeneous group of lesions , which like nsclc can be subdivided into squamous and adenocarcinoma ( benda , 1994 ). the present inventors studied 87 cervical lesions for htr expression , ( table 1 ). htr expression was detected in 44 % of the cervical carcinomas , however in contrast to nsclc , there was no significant difference in frequency of expression between invasive squamous carcinoma , ( 44 %), and invasive adenocarcinoma , ( 32 %). the data for adenocarcinoma of the cervix also contrast those for invasive adenocarcinoma of the breast , ( 13 %), and ovary , ( 17 %), ( table 1 ), and suggest that regulation of htr expression may be different for cervical cancer and therefore relate to the aetiology of the disease ( benda , 1994 ; klingelhutz et al ., 1996 ). interestingly , htr expression was readily detected in preinvasive cervical cancer , ( 40 %, see table 1 ), and there was no significant difference in frequency between invasive and preinvasive lesions . in addition , the case of glandular intraepithelial neoplasia of the cervix has heterogeneous expression of htr ( data not shown ), thus allowing the evolution of htr expressing cells to be followed in their histological context . the primitive germ cells of the male are found in the seminiferous tubules and the present inventors examined 22 sections from normal testis and uninvolved tubules from testicular cancer patients to establish the pattern of htr expression in normal seminiferous tubules , ( data not shown ). of the 22 sections , 21 showed htr expression in the primitive germ cells located in the basal layers of the seminiferous epithelium , ( table 1 ). the intimate relationship between germ cells and the supportive sertoli cells , means that expression of htr in the sertoli cells cannot be excluded . mature germ cells ( spermatids and spermatazoa ) when present , did not express htr . thus , the in situ assay can detect normal levels of htr expression in primitive germ line stem cells and the distribution of htr expression in the testis is consistent with its proposed role in the maintenance of telomere length in the germ line . a series of 22 testicular germ cell tumours were also analysed for htr expression . as shown in table 1 , 73 % of testicular germ cell tumours were positive for htr expression , and there was no significant difference between teratomas and seminomas of the testis . interestingly , within htr - positive teratomas , mature tissues never had detectable htr expression whatever their differentiation . similarly , 15 benign ovarian teratomas composed of fully mature differentiated tissues had no detectable htr expression , ( table 1 ). thus , the differentiated teratomas may recapitulate the down regulation of htr during early embryonic or foetal development , ( wright et al ., 1996 ). in five of the testicular seminomas there was no detectable htr expression , ( table 1 ), but in each seminoma case , germ cells within adjacent normal seminiferous tubules expressed htr , suggesting either that htr expression has been repressed during oncogenesis , or that seminomas without detectable levels of htr arise from germ cells with low or no htr expression . in order to obtain sequences flanking the genes , the p1 genomic clones were digested with ecori and hindiii , subcloned into the plasmid vector , pbluescript , and colonies containing htr or terc sequences identified by hybridisation to pcr generated probes specific for the genes . a 1 . 3 kb genomic clone encompassing htr was isolated as was a 4 kb genomic clone encompassing terc , ( fig3 ). a blast search using the 1 . 3 kb human sequence identified three high - scoring segment pairs : hsu85256 , hsu86046 and mmu33831 . hsu85256 , ( 598 bp of sequence ), and hsu86046 , ( 545 bp of sequence ), are published sequences for the transcribed region of the human telomerase rna gene and confirmed that we had cloned genomic sequences encompassing htr ( bryan et al ., 1997 ; feng et al ., 1995 ). mmu33831 is the sequence of the transcribed region of the mouse telomerase rna gene which has previously been shown to have homology to the human gene ( blasco et al ., 1995 ). a blast search using the 4 kb mouse sequence identified both the published human gene sequences , ( hsu85256 , hsu86046 ), and the published sequence for the transcribed region of the mouse gene , mmu33831 , ( 591 bp of sequence ) ( blasco et al ., 1995 ; bryan et al ., 1997 ; feng et al ., 1995 ). in order to confirm that the genomic sequence obtained from the p1 subclone was genuine , the present inventors cloned 5 ′- flanking sequences using genomic dna from balb / c mice in pcr reactions . sequence analysis of the balb / c clones were identical to the p1 sequence except for minor polymorphism &# 39 ; s , ( fig9 ). a schematic representation of the 4 kb of sequence information encompassing the mouse telomerase rna gene is shown in fig3 . analysis of nucleotide sequence encompassing the human and mouse telomerase rna genes . to investigate the relationship between the human and mouse genomic clones , sequence comparisons were carried out . the transcribed regions of the two genes showed 67 % identity in keeping with the published estimate , ( feng et al ., 1995 ). however , no significant sequence identity could be identified in either the 5 ′- or 3 ′- regions flanking the transcribed sequences . both the human and mouse sequences were analysed for cpg islands by grail . cpg islands were defined as regions larger than 200 bp , with an average gc content greater than 50 % and the ratio of observed versus expected cpgs greater than 0 . 6 ( gardiner - garden & amp ; frommer , 1987 ). interestingly , both the human and mouse genes lie within cpg islands , ( see fig3 ). the human gene is covered by a cpg island 733 bp in length , with a gc content of 66 % and a ratio of observed versus expected cpgs of 0 . 89 . the mouse gene is covered by a cpg island of 659 bp in length , with a gc content of 64 % and a ratio of observed versus expected cpgs of 0 . 81 . the 5 ′- flanking regions of the human and mouse telomerase rna genes were also analysed for potential transcription factor recognition sites . as shown in fig4 , a number of potential binding sites can be identified , including consensus sequences for glucocorticoid / progesterone / androgen receptor binding , ap1 and ets family members . ccaat box &# 39 ; s are found in both genes close to the published transcriptional start sites ( blasco et al ., 1995 ; feng et al ., 1995 ), however , there is no obvious tata box in the mouse gene with the human gene tata box consensus sequence being in the reverse orientation . the mouse promoter region also contains a run of cpa dinucleotide repeats which may be of use in developing microsatellite genetic markers for this gene . transfection assays detect promoter activity in the 5 ′- flanking regions of the human and mouse telomerase rna genes . to identify whether the 5 ′- flanking dna of the telomerase rna genes exhibited promoter activity , sequences were fused to a firefly luciferase reporter gene , ( pgl3 - basic ). the transcriptional start sites for both the human and mouse telomerase rna genes have been established ( blasco et al ., 1995 ; feng et al ., 1995 ). various promoter constructs containing the transcriptional start site were therefore generated , ( see fig4 , 5 ). human promoter constructs containing truncated portions of the 5 ′- flanking region were transiently transfected into hela and gm847 cells , ( fig5 a ). hela is a telomerase positive cervical carcinoma cell line , gm847 is a sv40 - immortalised skin fibroblast cell line which expresses the telomerase rna component but is telomerase - negative , ( bryan et al ., 1997 ). as shown in fig5 a , promoter activity was observed in both cell lines with fragments containing 341 bp or more , ( from position − 272 , see fig4 a , 5 a ). the highest luciferase activity was observed with construct hprom505 which contains a 505 bp fragment , ( position − 436 , see fig4 ). construct hprom111 , which contains only 111 bp of 5 ′- flanking sequence ( position − 42 see fig4 a , 5 a ), produced a dramatically reduced level of luciferase activity ( fig5 a ). thus a minimal promoter sequence can be defined as extending 272 bp upstream of the transcription start site , and that elements responsible for promoter activity must be contained in a 231 bp region between − 272 bp and − 42 bp ( fig4 a and 5 a ). mouse promoter constructs containing various truncated portions of the 5 ′- flanking region were transiently transfected into swiss3t3 and a9 cells , ( fig5 b ). swiss3t3 cells are an embryo derived line and a9 cells are of areolar and adipose origin . both cell lines are telomerase positive . as shown on fig5 b , promoter activity was observed in both cell lines with fragments containing 208 bp or more , ( from position − 94 , see fig4 b ). construct mprom136 , which contains only 136 bp of 5 ′- flanking sequence , ( position − 22 , see fig4 b ), produced dramatically reduced levels of luciferase activity , ( fig4 b ). thus , a minimal promoter sequence can be defined as extending 94 bp upstream of the transcription start site , and that elements responsible for promoter activity must be contained in a 73 bp region between − 94 bp and − 22 bp , ( fig4 b and 5 b ). transfection of the human promoter construct hprom867 into mouse cells gave very strong promoter activity , with up to twice that of the strongest mouse construct , and transfection of the mouse promoter construct , mprom628 into human cells also showed luciferase activity at around 25 % of the strongest human construct , ( data not shown ). identification of transcription factor binding sites in the htr promoter by gel shift assays in order to identify the key sequence elements involved in transcription factor binding , protein extracts from cell lines were assayed for their ability to detect specific dna sequence elements within the htr promoter by gel shift assays . fluorescence assays may also be used and detected by microscopy , microplate reader or cytometry . such elements are then considered candidates for regulating htr in vivo . these studies concentrated on the 176 bp region of the htr promoter shown in fig1 as part of the sequence shown in fig1 and 4 a . this 176 bp region is termed sequence 2923 and contains several potential sp1 transcription regulation sites , retinoblastoma control elements , ( rce ) and the promoter ccaat - box transcriptional regulator site , ( see fig1 ). the oligonucleotide sequence elements used in the analysis of transcriptional regulation are shown in fig1 . a ccaat box element is found upstream of the transcriptional start site for htr , ( see fig1 ). in order to identify whether cellular proteins interact with this site in the htr promoter , protein extract from the hela cell line was used in an electophoretic mobility shift assay , ( emsa ) with oligonucleotide sequence elements corresponding to the normal wild type htr sequence termed h10 in fig1 & amp ; 13 , ( see fig1 , 12 & amp ; 13 ). fig1 shows that the h10 oligonucleotide sequence containing the htr ccaat - box can bind proteins in a specific fashion as competition for binding with the h10 oligonucleotide sequence itself aboloshes the complex as does an oligonucleotide with mutations outside the ccaat region , ( see sequence h10 m2 , fig1 & amp ; 13 ). addition of oligonucleotide sequences with mutations intoduced into the ccaat site cannot compete for protein binding , ( sequence h10m1 in fig1 & amp ; 13 ). this suggests that the ccaat - box sequence element is a functional region for dna binding proteins and may regulate the htr promoter . two sp1 sequence elements are found upstream of the 25 ′ htr transcriptional start site , ( fig1 ). in order to identify whether cellular proteins interact with these sites in the htr promoter , protein extract from the hela cell line was used in an electophoretic mobility shift assay , ( emsa ) with oligonucleotide sequence elements corresponding to the normal wild type htr sequences termed h4 , ( sp1 - 2 , see fig1 & amp ; 12 ), and h9 , ( sp1 - 1 , see fig1 & amp ; 12 ). as can be seen from fig1 , 15 , 16 , htr promoter sequence containing sp1 - 1 and sp1 - 2 sequence elements form dna / protein complexes characteristic of binding of sp1 and sp3 transcription factors . this suggests that the htr promoter may be regulated by the sp family of transcription factors . the gel shift assays discussed above identified a number of sequence elements which form specific dna / protein complexes which may regulate the activity of the htr promoter . in order to confirm these sequence elements function in the regulation of the htr promoter in vivo , mutations were introduced into these sites within the context of the basal promoter region of the htr gene . the ability of these mutations to drive the htr promoter in comparison to the wild type promoter sequence was then analysed . the overall scheme of this approach is shown in fig1 . the wild type promoter sequence used in this study is the htr 2923 sequence shown in fig1 . the promoter activity of this clone relative to the previously described promoter clones , ( see fig5 a ), is shown in fig1 . in order to analyse the function of htr promoter sequence elements in cell lines , constructs with htr promoter sequence element mutations were made and these are shown in fig1 & amp ; 20 . the ability of the mutant promoters to drive gene expression was assayed in comparison to the wild type promoter sequence in cell lines as shown in fig2 . fig2 shows firstly that mutation of the sp1 - 2 sequence element reduces promoter activity , ( construct 29 m223 ), sugesting this element is the site of action for an activator of the htr promoter ; secondly that mutation of the sp1 - 1 sequence element significantly increases basal promoter activity by 4 – 4 . 5 fold , ( construct 92 and 29 m292 ), sugesting this element is the site of action for an inhibitor of htr promoter activity ; and thirdly that mutation or deletion of the ccaat sequence element abolishes htr promoter activity , ( costruct 1011 & amp ; 26n23 ), suggesting this element is the site of action for an activator of htr promoter activity . thus the inventor has identified key regulatory sequence elements involved in htr promoter activation and repression . in order to identify genes regulating telomerase rna gene expression , candidate genes are transfected into cell lines containing the htr promoter / reporter constructs . fluctuations in reporter activity due to candidate genes can are monitored and conclusive evidence presented for their activity . the analysis of htr sequence elements presented above sugests the involvement of the sp family of transcription factors in regulation of the htr promoter . therefore , the inventor co - transfected into cell lines the luciferase reporter gene under control of the htr promoter together with expression vectors for the following three transcriptional regulators : sp1 , sp3 , and prb . the transcriptional regulators sp1 and sp3 both recognise the same dna sequence , the so called sp1 site . transcriptional regulation by the sp family of proteins is complex , but in general sp1 activates genes whereas sp3 can repress promoter activity . the retinoblastoma gene product , prb , is classically thought of as a tumour suppressor gene and has numerous roles as a modulator of gene transcription and cell cycle regulator . the rb gene was the first tumour suppressor gene to be cloned . prb has roles in : mediating transcriptional repression of rna polymerase ii , ( pol ii ), transcribed promoters ; mediating transcriptional repression of rna polymerase iii , ( pol iii ), transcribed promoters ; mediating transcriptional activation of rna polymerase ii , ( pol ii ), dependant promoters . this last role in mediating transcriptional activation is possibly the least well understood and studied . however , many important genes are activated by prb , such as , tgf - b2 , il - 6 , the retinoblastoma gene itself , and cyclin d1 , ( herwig & amp ; strauss , 1997 ; lania et al ., 1997 ; sellers & amp ; kaelin , 1996 ; tenen et al ., 1997 ). the ability of the sp1 transcription factor to modulate the htr promoter was assessed by co - transfecting the htr promoter constructs shown in fig2 , with an sp1 expression vector . from fig2 it can be seen that sp1 is an activator of the htr promoter . the ability of the sp3 transcription factor to modulate the htr promoter was assessed by co - transfecting the htr promoter constructs shown in fig2 , with an sp3 expression vector . from fig2 it can be seen that sp3 is an repressor of the htr promoter . the ability of the prb to modulate the htr promoter was assessed by co - transfecting the htr promoter constructs shown in fig2 , with an prb expression vector . from fig2 it can be seen that prb is an activator of the htr promoter . for a summary of the above data see fig2 . the term “ gdept ” is used to include both viral and non - viral delivery systems . examples of suitable vector systems include vectors based on the molony murine leukaemia virus are known ( ram . z et al , cancer research ( 1993 ) 53 . 83 – 88 ; dalton and treisman , cell ( 1992 ) 68 . 597 – 612 ). these vectors contain the murine leukaemia virus ( mlv ) enhancer system cloned upstream at the β - globin minimal promoter . this vector further contains a polylinker to facilitate cloning , followed by the β - globin 3 ′- untranslated region and polyadenylation sites . suitable viral vectors further include those which are based upon a retrovirus . such vectors are widely availbel in teh art , culver et al ( science ( 1992 ) 256 . 1550 – 1552 ) also describe the use of retroviral vectors in gdept . such vectors or vectors derived from such vectors may also be used . other retroviruses may also be used to make vectors suitable for use in the present invention . such retroviruses include rous sarcoma virus ( rsv ). the promoters from such viruses may be used in vectors in a manner analogous to that described for mlv . in general the vector may be any rna or dna vector used in vdept or gdept therapies . the enzyme may be any enzyme which is not normally expressed in the surface of a cell , nor released into circulation , particularly a mammalian ( especially human ) cell , and which is capable of modifying or killing the target cell e . g . a cancer cell . the enzyme may be linked to a signal sequence which directs the enzyme to the surface of a mammalian cell . this will usually be a mammalian signal sequence or a derivative thereof which retains the ability to direct the enzyme to the cell surface . this will be needed unless the enzyme has an endogenous signal sequence which does this . even if the enzyme does have such a signal sequence , it can be replaced by another signal sequence where this is desirable or appropriate . suitable signal sequences include those found in transmembrane receptor tyrosine kinases such as the c - erbb2 ( her2 / neu ) signal sequence or variants thereof which retain the ability to direct expression of the enzyme at the cell surface . the c - erbb2 signal sequence can be obtained by reference to coussens et al ( 1985 ) science . 230 . 1132 – 1139 . the enzyme may be expressed at the surface of a cell . this means that it will be expressed in such a fashion as to have the enzyme exposed outside the cell so that it may interact with the prodrug , but will still be attached to the plasma membrane by virtue of a suitable plasma membrane anchor . a suitable anchor will be a polypeptide anchor which is expressed by the vector . for example , the enzyme which is linked to a sequence which is a transmembrane region which anchors the enzyme in the membrane of the cell . such a transmembrane region can be derived from transmembrane receptor kinases , such as cerbb2 , egf receptors and csf - 1 receptors . vectors encoding the enzyme , together with , when requird , a signal sequence and / or transmembrane region may be made using recombinant dna techniques known per se in the art . the sequences encoding the enzyme , signal sequence and transmembrane regions may be constructed by splicing synthetic or recombinant nucleic acid sequences together , or by modifying existing sequences by techniques such as site directed mutagenesis . reference may be made to “ molecular cloning ” by sambrook et al ( 1989 ) cold spring harbor ) for discussion for standard recombinant dna techniques . the enzyme will be expressed in the vector using a promoter sequence or functional part thereof according to the present invention capable of activated in the cell to which the vector is targeted . the promoter sequence according to the present invention will be operably linked to the enzyme and its associated sequences . the promoter sequences according to the present invention may be modifed by techniques known in the art . certain regions of the promoter sequence must be retained to ensure cell , e . g . tumour cell , specificity whereas other regions may be modified or delted without significant loss of specificity ( see fig5 ). the degree of regulation of such candidate promoter regions as described herein , can be tected and assessed by techniques known to those skilled in the art . “ operably linked ” refers to a juxtaposition wherein the promoter sequence and the enzyme - coding sequence are in a relationship permitting the coding sequence to be expressed under the control of the promoter sequence . thus , there may be elements such as 5 ′ non - coding sequence between the promoter sequence and coding sequence which is not native to either the promoter or nor the coding sequence . such sequences can be included in the vector if they do not impair the correct control of the coding sequence by the promoter . the vectors for use in gdept or vdept systems comprising the promoter sequence or functional fragment thereof in accordance with the present invention can be used in a method of treatment of the human or animal body . such treatment includes a method of treating the growth of neoplastic cells which comprises administering to a patient in need of treatment the gdept or vdept systems . it is also possible that the vectors may be used to treat cells which are diseased through infection of the human or animal body by bacteria , viruses or parasites . for use of the vectors in therapy , the vectors will usually be packaged into viral particles and the particles delivered to the site of the tumour , as described in for example ram et al ( ibid ). the viral particles maybe modified to include an antibody , fragment thereof ( including a single chain ) or tumour - directed ligand to enhance targetting of the tumour . alternatively the vectors may be packaged into liposomes . the liposomes may be targeted to a particular tumour . this can be achieved by attaching a tumour - directed antibody to the liposome . viral particles may also be incorporated into liposomes . the particles may be delivered to the tumour by any suitable means at the disposal of the physician . preferably , the viral particles will be capable of selectively infecting the tumour cells . by “ selectively infecting ” it is meant that the viral particles will primarily infect tumour cells and that the proportion of non - tumour cells infected is such that the damage to non - tumour cells by administration of a prodrug will be acceptably low , given the nature of the disease being treated . ultimately , this will be determined by the physician . one suitable route of administration is by injection of the particles in a sterile solution . while it is possible for the prodrugs to be administered alone it is preferably to present them as pharmaceutical formulations . the formulations comprise a prodrug , together with one or more acceptable carriers thereof and optionally other therapeutic ingredients . the carrier or carriers must be “ acceptable ” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipients thereof , for example , liposomes . suitable liposomes include , for example , those comprising the positively charged lipid ( n [ 1 -( 2 , 3 - dioleyloxy ) propyl ]- n , n , n - triethylammonium ( dotma ), those comprising dioleoylphosphatidylethanolamine ( dope ), and those comprising 3β [ n -( n ′ n ′- dimethylaminoethane )- carbamoyl ] cholesterol ( dc - chol ). viruses , for example isolated from packaging cell 25 ′ lines may also by administered by regional perfusion or direct intratumoral direction , or direct injection into a body cavity ( intracaviterial administration ), for example by intra - peritoneum injection . formulations suitable for parenteral or intramuscular administration include aqueous and non - aqueous sterile injection solutions which may contain anti - oxidants , buffers , bacteriostats , bactericidal antibiotics and solutes which render the formulation isotonic with the blood of the intended recipient ; and aqueous and non - aqueous sterile suspensions which may include suspending agents and thickening agents , and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs . the formulations may be presented in unit - dose or multi - dose containers , for example sealed ampoules and vials , and may be stored in a freeze - dried ( lyophilized ) condition requiring only the addition of the sterile liquid carrier , for example water , for injections , immediately prior to use . injection solutions and suspensions may be prepared extemporaneously from sterile powders , granules and tablets of the kind previously described . it should be understood that in addition to the ingredients particularly mentioned above the formulations may include other agents conventional in the art having regard to the type of formulation in question . of the possible formulations , sterile pyrogen - free aqueous and non - aqueous solutions are preferred . the doses may be administered sequentially , eg . at daily , weekly or monthly intervals , or in response to a specific need of the patient . preferred routes of administration are oral delivery and injection , typically parenteral or intramuscular injection or intratumoural injection . in using the system of the present invention the prodrug will usually be administered following administration of the vector incorporating the promoter of the present invention and encoding an enzyme . typically , the vector will be administered to the patient and then the uptake of the vector by transfected or infected ( in the case of viral vectors ) cells monitored , for example by recovery and analysis of a biopsy sample of targeted tissue . the exact dosage regime will , of course , need to be determined by individual clinicians for individual patients and this , in turn , will be controlled by the exact nature of the prodrug and the cytotoxic agents to be released from the prodrug but some general guidance can be given . chemotherapy of this type will normally involve parenteral administration of both the prodrug and modified virus and administration by the intravenous route is frequently found to be the most practical . for glioblastoma the route is often intratumoural . a typical dosage range of prodrug generally will be in the range of from about 1 to 150 mg per kg per patient per day , which may be administered in single or multiple doses . preferably the dose range will be in the range from about 10 to 75 , e . g . from about 10 to 40 , mg per kg per patient per day . other doses may be used according to the condition of the patient and other factors at the discretion of the physician . tumours which may be treated using the system of the present invention include any tumours capable or being treated by a gdept or vdept system and thus are not limited to any one particular class of tumours . particularly suitable tumour types include breast , colorectal and ovarian tumours , as well as pancreatic , melanoma , glioblastoma , hepatoma , small cell lung , non - small cell lung , muscle and prostate tumours . the system of the invention may also be used to treat infections diseases , for example , and any other condition which requires eradication of a population of cells . it will be understood that where treatment of tumours is concerned , treatment includes any measure taken by the physician to alleviate the effect of the tumour on a patient . thus , although complete remission of the tumour is a desirable goal , effective treatment will also include any measures capable of achieving partial remission of the tumour as well as a slowing down in the rate of growth of a tumour including metastases . such measures can be effective in prolonging and / or enhancing the quality of life and relieving the symptoms of the disease . tumour specific gene expression for genetic therapy via telomerase rna gene promoters . the present studies point to examples of clear differentials in htr expression between cancerous and adjacent normal tissue which support the application of effective telomerase - based therapy . indeed , the presence of high levels of htr expression in specific cancers suggest the tr promoter may be an interesting focus for genetic therapies designed to target therapeutic genes to tumours , via tumour specific gene expression . therefore , the present inventor provide use of the tr promoters to drive expression of enzyme - prodrug activation systems such as viral thymidine kinase and ganciclovir , although many other systems may also be used ( see above ). targeted gene expression via the telomerase rna gene promoter may also be used in gene replacement strategies for cancer therapy . to this end , the present invention provides a first generation suicide gene therapy vector using the htr promoter sequence , namely pgt52 - codaupptelo , ( fig2 & amp ; 27 ). the htr promoter sequences used to generate pgt52 - codaupptelo are hprom867 as shown in fig4 a . these sequences were inserted into the commercially available gene therapy vector pgt62 - codaupp , ( invivogen , san diago , calif ., usa ), in place of the cmv promoter sequences . the pgt52 - codaupptelo vector consists of two transcriptional units . first transcriptional unit contains the following parts : htr , telomerase rna gene promoter ; plap , placental alkaline phosphatase reporter gene ; tk , hsv thymidine kinase conferring sensitivity to ganciclovir ; sh ble ( streptoalloteichus hindustanus bleomycin gene ), zeocin antibiotic resistance gene . second transcriptional unit contains the following parts : hef1 - htlv promoter , elongation factor 1a and part of htlv promoter ; coda :: upp , bacterial cytosine deaminase and uracil phosphoribosytransferase conferring sensitivity to the drugs 5fu and 5 - fluorocytosine . cells expressing the telomerase rna gene will therefore activate the htr promoter and cause expression of the hsv thymidine kinase gene , ( tk ). expression of the hsv tk gene sensitises cells to the drug ganciclovir . thus , cells activating the htr promoter in the suicide gene vector will be killed on exposure to ganciclovir . the pcr mutagenesis were performed separately by using site - directed mutagenesis kit [ stratagene , quikchange ™]. the primers used for the construction of site - replaced mutants of the sp1 , ccaat , rce and tata binding sites are shown in fig1 . the pcr reactions were performed using the following standard condition ; all pcrs were performed using 2 μl of pc2923 (− 107 /+ 69 ) plasmid dna ( 50 ng / μl ) as template , 1 . 5 mm mgcl 2 , 0 . 2 mm dntp &# 39 ; s , 25 nm of site - replaced primer and 1 . 0 μl ( 2 . 5 u / μl ) pfu dna polymerase in 50 μl reaction volume . a total of 16 cycles were used after originally denaturing at 95 ° c . for 30 seconds . in each cycle , the protocal was as follows : then , mutants of luciferase reporter construct were constructed by subcloning the mutation fragments into xho i / hind iii linearized pgl3 basic vector . to anneal the two complememtary oligonucleotides , the same molar ratio of oligonucleotides were mixed in te buffer , the oligo was treated at 95 ° c . for 5 minutes in either the perkin - elmer cetus dna thermal cycler or the perkin - elmer cetus cycler 9600 , then the cycler was switched off to cool down slowly ( about 4 . 5 hours ). then , the annealing oligo were loaded in the 12 % page gel and band excised . finally , the pellet was resuspended in 25 ul te and quantitated . nuclear protein preparation : approximately 10 10 cells were washed in pbs and then twice with tms ( 5 mm tris - hcl ph 7 . 5 , 2 . 5 mm mgcl 2 , 125 mm sucrose ). cells were lysed in 200 ml of tms plus 0 . 25 % triton x - 100 , and nuclei were harvested by centrifugation ( 1 , 600 × g , 20 minutes ). nuclei were washed three times in 200 ml of tms and resuspended in approximately 5 ml of tms ( 5 to 10 mg of dna per ml ), and 0 . 1 volumes of 4 m nacl was added dropwise with stirring . the solution was centrifuged at 10 , 000 × g for 20 minutes and the supernatant was spun at 100 , 00 × g for 60 minutes . solid ammonium sulfate was added to 0 . 35 g / ml and left on ice for 30 minutes . the precipitate was pelleted at 10 , 000 × g for 30 minutes and redissolved in 5 ml of e50 buffer ( 50 mm ammonium sulfate , 20 mm hepes , ph 7 . 9 , 5 mm mgcl 2 , 0 . 1 mm edta , 0 . 1 %[ v / v ] brij 35 , 20 %[ v / v ] glycerol , 1 mm dtt ) and dialyzed for 16 hours against 1 litter of storage buffer ( 50 mm nacl , 20 mm hepes , ph 7 . 9 , 5 mm mgcl 2 , 20 %[ v / v ] glycerol , 1 mm dtt ). the crude protein extract was cleared by centrifugation at 100 . 00 × g for 60 minutes , and aliquots were stored at − 70 ° c . electrophoresis mobility shift assay ( emsa ) was performed by emsa kit [ promega ]. typical 5 ′- kinase labeling reactions included the dna to be labeled , [ gg - 32 p ] datp [ amersham ], t4 polynucleotide kinase , and buffer [ promega ]. synthesized oligomers were 5 ′- end - labeled with [ gg32p ] datp ( 5 , 000 mci / mmol ) and were used as probes in emsa . 5 . 5 ug of hela nuclear proteins were incubated in 15 ul of reaction containing 4 % glycerol , 1 mm mgcl 2 , 0 . 5 mm dithiothreitol ( dtt ), 0 . 5 mm edta , 50 mm nacl , 10 mm tris - hcl , ph7 . 5 and 2 . 0 ug poly ( di - dc ), with or without excess molar of unlabeled dna competitors , on ice for 10 min , followed by addition of 5 , 000 – 10 , 000 cpm of the probe . for competition experiments , nonradioactive double - stranded oligodeoxynucleotides were added in 100 - fold molar excess prior to addition of the probe . for supershift assays , rabbit anti - human polyclonal antibodies specific against sp1 , sp3 , rb , ap - 2 ( santa cruz biotechnology ) were added to the reaction mixture 25 minutes prior to the addition of the probe . all dna - protein complexes were resolved on 5 % native polyacrylamide gels run in 22 . 3 mm tris , 22 . 3 mm boric acid , and 0 . 5 mm edta . visualization was performed using both a computing phosphorimager with imagequant software analysis ( molecular dynamics ) and autoradiography on kodak xar - 5 films . for co - transfection of wild type ( pl2930 , 107 /+ 69 ) and each individual mutation luciferase reporter construct , 3 ug plasmid dna was mixed with 3 ug of sp1 , or sp3 ( pcmvsp1 and pcmvsp3 ), or sp1 plus sp3 , or pcmvb expression vector , 0 . 25 ug prb ( pcmv - prb ) was used for co - transfection . 7 . 5 μl superfect ™ transfection reagent [ qiagen ] was added to the dna solution and mixed by pipetting up and down 5 times to get complexes . in general , there are no significant sequence homologies between the promoter regions of the human and mouse telomerase rna genes . indeed , there is considerable debate as to whether telomere length is regulated in a similar fashion in humans and mouse ( blasco et al ., 1997 ; kipling , 1997a ; kipling , 1997b ; zakian , 1997 ). however , mouse models represent a valuable resource with which to study the role of telomerase in cellular senescence and tumour progression and mouse models are likely to be required to investigate new therapies based on telomerase inhibition . in addition , the developmental regulation of telomerase will be more easily approached in mice ( bestilny et al ., 1996 ; blasco et al ., 1995 ; blasco et al ., 1997 ; blasco et al ., 1996 ; broccoli et al ., 1996 ; prowse & amp ; greider , 1995 ). thus , any differences between the two species may in fact aid understanding of the function for telomerase in maintaining genome stability and will be important in developing good murine models for human disease or developmental processes . despite the lack of sequence similarity between the human and mouse telomerase rna gene promoter regions , they both have consensus sites for the binding of transcription factors implicated in haematopoiesis and leukaemogenesis such as gata - 1 , pu . 1 , pea2 / pebp2 , c / ebp , and c - ets - 2 ( tenen et al ., 1997 ). this data will therefore be used in the detection of telomerase activity in normal and malignant haematopoietic cells ( bodnar et al ., 1996 ; cheng et al ., 1997 ; holt et al ., 1997 ; norrback & amp ; roos , 1997 ; pan et al ., 1997 ). the human and mouse telomerase rna genes do share an interesting similarity , in that they both lie in cpg islands , and thus their expression may be regulated by methylation . dna methylation is thought to be important for gene regulation during normal development and cellular senescence , and abnormal methylation patterns may be a fundamental change in tumour progression ( baylin et al ., 1991 ; bird , 1996 ; laird & amp ; jaenisch , 1996 ; vertino et al ., 1994 ; wilson & amp ; jones , 1983 ). thus it has been suggested that aberrant cpg island methylation during the normal ageing process , could contribute to immortalisation by interfering with expression of “ mortality ” genes , of which htr and terc can be included ( vertino et al ., 1994 ; wilson & amp ; jones , 1983 ). turning to the functional analysis of the cloned sequences , the minimal promoter for htr resides within a region of 272 bp upstream of the published transcriptional start site , ( feng et al ., 1995 ), ( fig5 a , 4 a ). there are a number of potential transcription factor binding sites in this region including consensus sequences for ap1 , sp1 , pea2 / pebp2 , pea3 and pu . 1 . interestingly , the expression in the fos / jun family of proteins , which determine ap1 activity , are suppresed during the onset of senescence and would be predicted to lead to a reduction in ap1 activity in senescent cells ( campisi , 1997 ; irving et al ., 1992 ; riabowol et al ., 1992 ; seshadri & amp ; campisi , 1990 ). ap1 also responds to protein kinase c , and it has recently been demonstrated that htr expression is induced by protein kinase c during t - cell activation ( bodnar et al ., 1996 ). extending the promoter region to 463 bp upstream of the transcriptional start site , increases the luciferase activity to its maximum level , ( fig5 a , 4 a ). this region contains several consensus binding sites for glucocorticoid / progesterone / androgen receptor binding , which may contribute to the maximal activity demonstrated by hprom505 . a reduction in promoter activity is observed on extending the promoter fragments to include more 5 ′- sequence , ( fig5 a ), suggesting that sequences towards the 5 ′- end of the clone may influence promoter activity in a negative fashion . the minimal promoter for terc resides in a 94 bp region upstream of the published transcriptional start site , ( blasco et al ., 1995 ), ( fig5 b , 4 b ). a striking feature of this region is the presence of three ap - 2 consensus sites , two of which are coupled to c - ets - 2 sites and all these elements are contained in the 73 bp region required for promoter activity , ( fig5 b , 4 b ). oncogenic ras gene signalling has been shown to operate through c - ets - 2 binding sites , thus there is a testable relationship between oncogene activation during tumour progression and telomerase rna gene transcriptional activity ( galang et al ., 1994 ; wasylyk et al ., 1994 ). a reduction in promoter activity is observed on extending the promoter fragments to include more 5 ′- sequence , ( fig5 b , mprom628 ), suggesting that sequences towards the 5 ′- end of the clone may influence promoter activity in a negative fashion . in summary , the present inventor has provided the art with dna sequence elements and gene products which regulate the htr promoter . thus , the inventor has identified the first direct molecular controls on signal transduction pathways affecting telomerase expression . the major findings are firstly , that the retinoblastoma gene product stimulates htr promoter activity ; secondly , that the sp1 transcription factor stimulates htr promoter activity ; thirdly , that the sp3 transcription factor is a potent repressor of htr promoter activity ; fourthly that mutation of the htr ccaat sequence elements abrogates promoter activity ; and fifthly , that the htr sp1 - 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