Patent Application: US-47028304-A

Abstract:
the present invention relates to genes and protein encoded thereby that regulate the secretory pathway and / or the neuroendocrine phenotype in cells and method of isolating same . more particularly , the present invention relates to long - term therapies for diseases or conditions associated with a loss function . more particularly , the present invention relates to the treatment of such diseases using a cell replacement therapy . in particular , the invention relates to genes involved in cellular differentiation and genes that modulate the formation of the regulated secretory pathway . the invention thus also concerns a method to identify such genes , the genes , variants or fragments thereof , vectors comprising same , the products of these genes , variants or fragments thereof and to cells expressing same . in a particular embodiment , the invention relates to the characterization of zis - sr a novel sequence involved in the secretory pathway in cells .

Description:
to identify the genes involved in the differentiation of the neuroendocrine phenotype , a cell line named hya . 15 . 6 . t . 3 ( 6t3 ) was used . the 6t3 cell was derived from the fusion of a mouse myeloma cell line ( 4t001 ) and the anterior pituitary corticotroph cell line ( att - 20 ). previously the 6t3 cell was used to study trafficking of cell membranes and was not considered as a neuroendocrine cell line due to its lack of secretory granules . thus the 6t3 cell line did not have the most important component of the neuroendocrine phenotype . upon appropriate stimulation and correct culture conditions , the 6t3 cells can be made to differentiate into a neuroendocrine phenotype . there is evidence to show that under basal conditions these cells do not produce or store secretory granules and thus do not display the neuroendocrine phenotype . but upon stimulation of these cells with specific neuroendocrine differentiating agents , the cells show all the characteristics of the neuroendocrine phenotype including the presence of secretory granules . in accordance with the method of the present invention , a gene involved in the cellular differentiation leading to the neuroendocrine phenotype was identified . the method involves exposing the cells to camp and then determining all differentially expressed genes as compared to cells that were not exposed to camp . the genes that are isolated are expressed in non - stimulated 6t3 cells to show that they carrying out neuroendocrine differentiation . the isolated genes are also used to block cellular differentiation using anti - sense approaches . the method also permits the identification of genes that modulate the formation of the regulated secretory pathway . the identified genes would then be used to transform embryonic stem cells that would be used for transplantation and replacement cell therapy . as a proof of the concept , a gene that codes for a previously uncharacterized mammalian protein which was named zis - sr was cloned . zis - sr is a homolog of the zis gene , which is involved in rna splicing . we show that zis - r is vital to the formation of secretory granules . stimulation of 6t3 cells with camp results in the formation of functional secretory granules the 6t3 cell line originates from fusion experiments using the mouse pituitary cell line att - 20 and the mouse myeloma 4t001 ( matsuuchi et al ., 1991 ). att - 20 cells are extremely well characterized and known to express a number endocrine - related genes , including proopiomelanocortin ( pomc ), pro - protein convertases , carboxypeptidase e ( cpe ), peptidyl amidating monooxygenase ( pam ), chromogranins ( i . e ., cga , cgb , sgll ), and the neuroendocrine polypeptide 7b2 . each of these molecules is directed to the regulated secretory pathway of endocrine cells . att - 20 cells also package these proteins in electron dense secretory granules . however , the first study describing 6t3 cells ( matsuuchi et al ., 1991 ) revealed that they did not express various soluble markers of the dense core vesicles nor could dense core secretory granules be identified therein . it was therefore of interest to characterize the 6t3 cell line as to expression levels of various endocrine - related proteins ( i . e ., processing enzymes , chromogranins and hormones ) that are found in the regulated secretory pathway of the parental att - 20 cells ( day et al ., 1995 ). it was determined that 6t3 cells were completely devoid of pomc . the 6t3 cells also had much lower levels of cga , cgb and sgll than att - 20 cells . the levels of processing enzymes such as cpe , and furin were unchanged , while pc1 / pc3 levels were reduced . based on these results and others , it was concluded that 6t3 cells retain some characteristics of endocrine cells ( i . e ., since some endocrine markers are still present at various levels ). however , the lack of secretory granules and regulated secretory function suggested the presence of specific defects affecting compartmentalization but not necessarily the expression of all endocrine markers . if these defects could be identified , it could lead us to an understanding of important cellular processes related to the biogenesis and functionality of secretory granules . experiments to investigate the capacity of 6t3 cells to regulate expression of endocrine markers in comparison to the parental att - 20 cells were also conducted . 6t3 cells were stimulated with camp for various periods of time . morphological changes were noted following camp stimulation . similar to att - 20 cells , stimulated 6t3 cells demonstrated the ability to form extensions and processes . it was also noted that pomc mrna expression was restored in stimulated 6t3 cells ( day et al ., 1995 ). this surprising but important clue suggested that defects associated with the 6t3 cell ( i . e ., such as the silencing of pomc expression ) could be restored with camp stimulation . if this was true , then it seemed possible that other defects could also be corrected . it thus seemed possible that camp stimulation could restore a functional regulated secretory pathway in 6t3 cells . to investigate this possibility , camp - stimulated 6t3 cells were examined at the ultrastructural level for any evidence of secretory granules . this analysis revealed the presence of dense - core granules in the cytoplasm and the extremities of treated 6t3 cells ( fig1 ). if these newly formed dense core secretory granules were indeed genuine structures , a detection of the storage of hormones within their dense cores could be carried - out . an immunohistochemical analysis of camp - stimulated 6t3 cells at the ultrastructural level was thus performed . as mentioned above , non - stimulated 6t3 cells do not express pomc , but after camp treatment , pomc expression is induced in 6t3 cells . the distribution of immunoreactive β - lph , a cleavage product of pomc , in camp - stimulated 6t3 cells was therefore examined . this analysis showed β - lph immunoreactivity within newly formed secretory granules , thus demonstrating that they were authentic storage compartments that had been induced by camp stimulation . since it could be shown that camp - stimulated 6t3 cells could re - form dense core secretory granules and correctly cleave and store newly induced pomc protein , it was important to verify if regulated secretion was now functional . in other words , once these dense core secretory granules were formed , did they have the capacity to fuse with the external membrane and release their contents into the media upon stimulation . these experiments were carried out on 6t3 cells that had been induced with camp to produce pomc and dense core secretory granules . after a washout period ( i . e ., 24 hrs to remove all secretory effects of camp stimulation ), the induced 6t3 cells were treated with a short - term depolarization stimulus ( i . e ., 15 min of 56 mm kcl ). the media was examined for the release of p - endorphin immunoreactivity . such a short term stimulus can only release stored products of the cell and will not be sufficient to influence transcriptional changes which could blur the sought after effect . the data showed a rapid release of p - endorphin immunoreactivity in the media as compared to controls . these experiments thus demonstrated that camp differentiated 6t3 cells could release their newly formed dense core secretory granules through a normal regulated secretory mechanism involving exocytosis . these data also suggested that the secretion - defect ( s ) associated with 6t3 cells were related to the formation of secretory granules and not to a defective exocytotic machinery . it therefore seemed possible that the 6t3 secretion defects lied upstream , preventing the formation of secretory granule formation . thus 6t3 cells have specific defects in terms of full neuroendocrine differentiation , which can be repaired after specific stimulation with agents that affect the camp - related signaling pathways . these agents include 8 - bromo - camp , dibutryl camp and ligands which interact with seven transmembrane receptors that activate g proteins and the adenyate cyclase , as for example croticotrophin releasing hormone ( crh ). stimulation by any of these agents results in the differentiation of 6t3 cells as determined by the appearance of distinct secretory granules as observed in ultrastructural studies . furthermore , these newly formed granule storage compartments can be shown to contain authentic peptides and proteins that are normally directed to secretory granules , such as β - lipotrophin ( β - lph ) or β - endorphin . finally , these secretory granule components are released from the 6t3 cells upon cellular depolarization induced with short - term treatment with kcl . this demonstrates that the newly formed secretory granules are fully functional in 6t3 cells . identification of 6t3 defects using differential display polymerase chain reaction ( dd - pcr ). the fact that stimulating 6t3 cells with camp results in the formation of functional secretory granules provides the means to carry - out plus / minus screening . indeed , it now became possible to try to define the molecular factors that are playing a role in the induction of regulated secretion by assessing the difference between non - stimulated and stimulated 6t3 cells . current methods to distinguish mrnas in comparative studies rely largely on differential or subtractive hybridization techniques . several important genes implicated in tumorigenesis have been isolated using these method . although subtraction is quite sensitive and can detect fairly rare mrnas , the method recovers genes incompletely and selects for genes in only one direction at a time during a two - way comparison between a pair of cells . this process can be laborious as well as time - consuming . dd - pcr ( genhunter ) was developed with the goal of identifying differentially expressed genes or detecting individual mrna species that are changed in mammalian cells , then recovering and cloning the cdna . this method utilizes pcr amplification and denaturing polyacrylamide gel electrophoresis , two of the most commonly used molecular biological methods , and provides a sensitive and straightforward approach to detect differentially expressed genes . dd - pcr ( liang et al ., 1998 ) was thus chosen with the aim of identifying genes involved in the formation of secretory granules . 6t3 cells were stimulated with 5 mm camp and non - stimulated 6t3 cells were used as control . total rna was extracted from treated and untreated cells . the rna from each sample was reverse transcribed to prepare cdna from each sample . the cdna was then amplified by pcr with a set of anchored oligo ( dt ) primers and an arbitrary decamer . specifically , an rna sample is reverse transcribed with each of 4 sets of degenerate anchored oligo ( dt ) primers ( t12mn ), where m can be g , a , or c and n is g , a , t and c . each primer set is dictated by the 3 ′ base ( n ), with degeneracy in the penultimate ( m ) position . for example , the primer set where n = g consists of : the resulting cdna population was pcr amplified using the degenerate set , an arbitrary decamer and radioactive nucleotide . the radioactively labeled pcr products that represent a subpopulation of mrnas defined by a given primer set are separated on a denaturing polyacrylamide gel . by changing primer combinations , most of the rna species in a cell may be represented . side by side comparisons of rna samples from treated and untreated samples allows the identification and cloning of differentially expressed genes . this method is extremely rapid and false positives can be rapidly screened out by a simple test on northern blot . in other words once a band is observed as being differentially displayed , that band can be isolated , re - amplified with the same primers and a cdna probe can be made and tested on northern blots that were prepared from the original rna samples . once a band is confirmed and shown to be regulated on the northern blot , it can be sequenced and compared to the existing databases for homologies . if it is a known gene then it can be used to clone in further studies ( e . g . expression in 6t3 cells , precise localization in the pancreas or other tissues by in situ hybridization , etc .). if it is not found in the existing databases , its tissue localization in endocrine tissues ( e . g ., pancreas , pituitary , adrenal ) and in endocrine and non - endocrine cell lines that are available can be examined ( seidah et al ., 1994 ). if the gene is of interest , a full - length clone can be obtained in order to express the protein and further define its function . a differential display - pcr methodology ( liang et al ., 1998 ) to compare stimulated and non - stimulated 6t3 cells was thus used ( fig2 ). various cdna fragments were isolated based on the increased intensity of the observed amplified bands . that these cdnas corresponded to up - regulated genes by further northern blot analysis screening was confirmed ( fig3 ). although a number of candidate genes from this first round of screening experiments were retained , the instant invention focuses on one gene having particular interest . this mouse gene was named zis - sr ( zinc finger splicing with extended ser - arg domain ) to take into account its strong homology with the reported zis gene ( karginova et al ., 1997 ). of note , zis - sr differs from zis by virtue of an extended sr domain ( fig4 and fig5 ), of 68 amino acids starting at amino acid position 243 ( his ) in mzis - sr . using the nucleotide sequence information obtained from the partial dd - pcr fragment , oligo primers were synthesized and were used to obtain the full - length cdna of zis - sr . race - pcr cloning was carried out on marathon - ready cdnas ( clontech laboratories ). the full length zis - sr cdna was 2 , 616 nts which corresponds to the size observed by northern blot analysis . a search of the gene databases revealed that zis - sr is a homolog of zis ( karginova et al ., 1997 ). at the nucleotide level zis - sr and zis are virtually identical , with important differences that result in the translation of very distinct c - terminal domains ( fig5 ). indeed , zis - sr contains an extended sr domain that terminates at the stop codon , while zis has a much shorter sr domain which does not extend into the c - terminal . in essence therefore , it seemed that the sequences of zis shifted the reading frame of the protein such that it was significantly truncated at the c - terminus . indeed , a comparison of the sequences of zis and zis - sr at the nucleotide level shows the presence of three cytosine nucleotides in the zis - sr as compared to two cytosine nucleotides reported in the zis sequence at positions 802 - 803 ( numbering based on zis rat sequence ; genbank accession number af013967 ). in order to verify the sequence data , a specific pcr test was designed so as to distinguish between zis - sr and zis and to confirm that zis - sr is expressed in mouse , rat and human brain tissue ( fig6 ). taken together , the results presented herein enable the conclusion that zis - sr is a unique protein that had not been described previously in mammalian species . a search in non - mammalian species indicates a high homology of zis - sr with the xenopus c4sr both at the nucleotide and protein level . the function of c4sr is undetermined although its relationship to rna splicing has been suggested ( ladomery et al ., 2000 ). in a recent database search , homologs of zis - sr in drosophila and in caenorhabditis elgans ( c . elegans ) were identified . of note , in both species , the fully extended sr domain is conserved . zis - sr has an open reading frame coding for 330 amino acid protein with the following features : 1 ) an n - terminal tandem of zinc finger motifs , 2 ) a nuclear localization signal in the mid - portion of the protein and 3 ) a c - terminal domain rich in serine and arginine ( sr domain ). the sr domain is characteristic of nuclear rna - binding proteins involved in the splicing of pre - mrna ( fu et al ., 1995 ; manley et al ., 1996 ). the sr domain of zis - sr is twice the length of the sr domain of the previously described zis protein . the analysis of the protein structure also shows the presence of two zinc finger motifs at the n - terminal portion of zis - sr , one of which appears to contain a novel consensus motif ( fig7 ). zinc fingers are classically found in transcription factors and typical serve to bind dna . the function of these zinc finger domains is not known but could indicate the capacity of zis - sr to bind dna . this dual feature of zinc fingers and sr - domain makes zis - sr a unique protein , unlike any other pre - mrna splicing factor described to date . unlike other rna splicing factors , zis - sr does not contain an rna recognition motif , that permits the association of the splicing factor with rna . it is also possible that the two zinc finger domains may be involved in rna binding ( arranz et al ., 1997 ; caricasole et al ., 1996 ; clemens et al . 1993 ; finerty et al ., 1997 ; shi et al ., 1995 ). the exact function of zis - sr remains to be formally tested experimentally . without being bound by a particular theory , the most likely argument to date relating zis sr to an rna splicing function is its close homology to the xenopus protein known as c4sr ( ladomery et al ., 2000 ). the c4sr protein was isolated based on its rna - binding properties ( karginova et al ., 1997 ). thus , the zinc fingers could be involved in rna binding , while the extended sr domain of zis - sr could be involved in the promotion of the spliceosome assembly by facilitating specific protein interactions and thus preventing random binding to rna . regulation of these specific molecular events could be dependent on the state of phosphorylation of zis - sr ( yeakley et al ., 1999 ). the analysis of the protein structure shows that the two zinc finger motifs of zis / gap1 - 5a are characteristic of a novel family of zinc finger proteins spread from yeast to mammals ( fig8 ). the consensus sequence of these cys 2 / cys 2 zinc fingers is d - w — x — c — x 4 − c — x n c — n — x 6 c — x 2 — c . interestingly , this zinc finger motifs occurs in the ews ( crozat et al ., 1993 ) and fus oncoproteins , two human rna - binding molecules . unfortunately , the role of these zinc finger structures in ews and fus have not been studied yet in the context of rna binding since ews and fus also bear other more classical rna recognition motifs . this novel zinc finger structure which has been discovered has a defined biological role . the c4sr protein , the xenopus homologue of zis - sr , was isolated from its rna - binding properties . taken together , these observations suggest that both the sr domain and the zinc fingers of zis - sr are related to rna binding purposes . pre - mrna splicing is a fundamental mechanism in all eukaryotic cells . constitutive splicing is required for efficient protein expression by the removal of introns to form coherent coding sequence . furthermore , alternative splicing of pre - mrna is a powerful mechanism for the regulation of gene expression in eucaryotic cells that plays an important role in tissue development and cell differentiation . in drosophila , a differentiation event as fundamental as sex determination is triggered by an alternative splicing mechanism mediated by the family of transformer ( tra ) alternative splicing factors ( tian et al ., 1993 ). genes can also be alternatively spliced to generate endocrine - specific isoforms . as an example , the isoform a of the subtilisin - like pro - protein convertase 5 ( pc5 ) behaves like an endocrine protein , being sorted to the regulated secretory pathway , while the isoform b goes to the constitutive vesicular traffic ( de bie et al ., 1996 ). it appears that a number of genes need an endocrine - specific pattern of splicing , requiring the presence of endocrine splicing factors . since most of the known splicing factors are sr proteins , the discovery of an endocrine - specific protein with an sr domain like zis - sr opens new insights for the study of endocrine differentiation . sr proteins constitute a large family of nuclear phosphoproteins required for constitutive and regulated pre - mrna splicing ( mayeda et al ., 1992 ). the most studied members of this family are the constitutive splicing factors u1 snrnp 70 k and u2af , as well as the positive regulators of alternative splicing srp75 , srp55 , srp40 , asf / sf2 , sc35 , rbp1 and 9g8 ( chabot 1996 ). all of them comprise a domain rich in serines and arginines ( sr ). they also bear a rna - recognition motif ( rrm ) which consists of a four - stranded antiparallel β - pleated sheet and two alpha helices packed on one side of the β sheet ( xu et al ., 1997 ). although some proteins with only the sr domains ( no rrm &# 39 ; s ) have been shown to play a role in splicing mechanisms ( fetzer et al ., 1997 ), only those with rrm &# 39 ; s are called sr proteins . biochemical analysis of nuclear extracts suggests that a large number of different sr proteins are involved in splicing mechanisms ( blencowe et al ., 1995 ; neugebaur et al ., 1995 ). variations in the levels of different sr proteins often lead to a change in alternative mrna splicing patterns ( gallego et al ., 1997 ; zahler et al ., 1993 ). sr proteins have been shown to bind to purine - rich motifs in exons in pre - mrna complexes , where they participate in the spliceosome assembly ( achsel et al ., 1996 ). their effect on pre - mrna splicing is antagonised by type a / b hnrnp , another class of mrna splicing factors ( hanamura et al ., 1998 ). sr proteins display a dynamic intranuclear organization that is regulated by phosphorylation of the serine residues in the rs domain . several sr - protein kinases have been identified from diverse species ( duncan et al ., 1997 ; duncan et al ., 1998 ; gross et al ., 1997 ; gui et al ., 1994b ; nayler et al ., 1997 ). there is a fight linkage between the transcription process and the intranuclear organization of the splicing machinery ( bauren et al , 1996 ). some sr proteins have been shown to directly interact with the highly phosphorylated c - terminal domain of rna polymerase 11 , being associated with the elongating rna polymerase ( du et al ., 1997 ; kim et al ., 1997 ), from where they translocate to the nascent transcripts to ensure efficient splicing , concomitant with transcription ( bourquin et al ., 1997 ; corden et al ., 1997 ). the diversity of sr proteins is further enhanced by alternative splicing mechanisms , which can give several isoforms ( du et al ., 1997 ). some sr proteins have been shown to even regulate the splicing of their own mrna ( jumaa et al ., 1997 ; sarkissian et al ., 1996 ). the role of the sr domain is not clearly understood . while it is clear that the rrm is involved in rna binding , the sr domain seems to play several roles . these include specificity of target rna recognition ( misteli et al ., 1998 ), mediation of the protein - protein interactions into spliceosome assembly ( tronchere et al ., 1997 ; wu et al ., 1993 ), and nuclear sublocalisation of the splicing components ( caceres et al ., 1997 ). in all cases , the degree of phosphorylation of the sr - domain has been shown to be critical for regulating function ( gui et al ., 1994a ; kanopka et al ., 1998 ). the distribution of zis - sr mrna in the mouse brain was examined by in situ hybridization . zis - sr was exclusively expressed in neurons and not in glial cells ( fig9 and 10 ). furthermore , zis - sr was preferentially expressed in all endocrine tissues investigated in the mouse ( fig9 ). within stable cell lines , zis - sr mrna also had a distinct endocrine pattern ( fig9 ). this overall preferential expression pattern within neuro - and endocrine cell types is particularly interesting . based on the strong correlation of zis - sr expression with endocrine cells and tissues , an experiment was designed in order to directly address whether zis - sr was implicated in the biogenesis of secretory granules , using gene silencing methodology in endocrine cell lines . att - 20 cells are typical endocrine cells , that endogenously express zis - sr and contain easily detectable secretory granules . att - 20 cell lines expressing antisense zis sr were thus created . stable att - 20 cell lines expressing antisense cdnas of zis - sr were prepared . different size fragments were tested including 400 , 700 and 1400 nt long cdnas , all inserted into the vector pcdna3 . 1 / hygro . various clones were obtained and have reproducibly created stable att - 20 cells lines that express the antisense zis - sr . zis - sr mrna expression was significantly reduced using different antisense constructs as compared to control att - 20 cell lines ( lacz and vector - transfected controls were also prepared ). stable att - 20 cell lines expressing antisense zis - sr were analyzed by immunocytochemistry to determine if secretory granules had been affected ( fig1 ). briefly , the cells were labeled with antibodies directed against carboxypeptidase e ( cpe ), and analyzed by light microscope ( fricker et al ., 1993 ; song et al ., 1995 ). at the light microscope level , control att - 20 cells were stained ( wild type , lacz - transfected , and vector - transfected ) as well as two different att - 20 cells lines with highly reduced zis - sr expression ( 10 - 20 % of normal levels ) with antibodies directed to carboxypeptidase e ( cpe ). cpe is an enzyme that removes c - terminal charged amino acids ( lysine or arginine ) from peptides or proteins . cpe is also known to be an important component of secretory granules and thus can serve as an excellent marker for the presence of secretory granules . the cpe antibodies were directed to the n - terminal and the c - terminal regions of cpe and gave identical results . the antibodies were used to stain control att - 20 and att - 20 expressing antisense zis - sr ( att - 20 - as ) cell lines . detection was carried out using an anti - rabbit igg second antibody conjugated to fitc . wild type att - 20 cells were heavily stained showing immunoreactivity in extensions and projections as expected . staining was also observed in a perinuclear pattern within the trans golgi network ( tgn ). however , att - 20 - as cells no longer showed any immunoreactivity in their - extensions , suggesting that granule formation had been severely compromised . decreased cpe immunoreactivity was not due to cpe transcriptional / mrna turnover effects or random genomic effects of the antisense zis - sr transfection , since a northern blot analysis of att - 20 zis - sr antisense cell lines demonstrated that cpe mrna expression was identical to that of control att - 20 cells ( fig1 a ). however , the intracellular content of cpe is clearly reduced as shown by western blots of control att - 20 cells compared to att - 20 cells expressing the antisense constructs . the best clone , 700 as - 3 , contains significantly lower levels of cpe than that of att - 20 cells . since rna levels are the same , this strongly suggests that the storage of cpe has been compromised . functional tests of the regulated secretory capacity of the stable att - 20 cell lines expressing antisense zis - sr were conducted ( fig1 ). by western blot analysis , cpe release following short term depolarization conditions ( 56 mm kcl ) were examined . control att - 20 cells responded to depolarization with a large increase in cpe release observed in the media . however cpe levels in the media were not significantly increased in att - 20 cells expressing antisense zis - sr treated with identical depolarization conditions . these data demonstrate that the secretion of cpe is no longer “ regulated ” in the 700 as - 3 clone , like in normal att - 20 cells . an ultrastructural analysis of att - 20 zis - sr antisense cell lines also showed a significant reduction in dense core secretory granules ( fig1 ). taken together , the sum of these data demonstrate that zis - sr expression is vital to the formation of secretory granules and the presence of a functional regulated secretory pathway . the analysis of these clones demonstrates that endogenous zis - sr expression is reduced by 80 - 90 % as compared to wild type att - 20 cells , or att - 20 cells stably transfected with vector alone or with lacz . stable att - 20 cell lines were analyzed . the present invention is illustrated in further detail by the following non - limiting examples . culture media , hygromycin , g - 418 , trypsin and fbs used for cell culture was obtained from gibco - brl life technologies . rnase free dnase was obtained from promega . restriction enzymes , rna polymerases ( spc6 and t7 ), sequencing kits and ecl + kit for western blots were obtained from amersham - pharmacia biotech . t4 ligase was obtained from usb scientific . the vectors pcdna3 . 1 - hygro , pcrii - topo and top10 cells were obtained from invitrogen . the cpe c - terminal antibody was obtained from dr . l . fricker ( albert einstein university , bronx , n . y .). anti - igg antibodies coupled to fitc were purchased from sigma . all electron microcopy materials were obtained from electron microscopy sciences . att - 20 cells are corticotroph cell lines that were cultured in complete d - mem ( 4 . 5 mg / l d - glucose ) containing 10 % inactivated fbs and 28 μg / ml gentamycine . for passages , the cells were washed with 4 ml of pbs ( 0 . 9 % saline containing 2 . 7 mm kcl , 8 . 1 mm na 2 hpo 4 , 1 . 15 mm kh 2 po 4 ) and then treated with a 2 ml of 0 . 05 % trypsin in 1 × versene ( 137 mm nacl , 2 . 7 mm kcl , 8 mm na 2 hpo 4 , 1 mm kh 2 po 4 , 0 . 5 mm edta ). the cells were incubated for 5 min at 37 ° c . the cells are collected and centrifuged and then resuspended in 1 / 4 of the culture media for re - plating . the cells were maintained at 37 ° c . at 100 % humidity and 5 % v / v co 2 . the 6t3 cells originate from the fusion of the mouse myeloma 4t001 and att - 20 cells . they are maintained in d - mem ( 4 . 5 mg / l d - glucose ) supplemented with 10 % v / v inactivated fbs , containing 28 μg / ml gentamycin and 250 μg / ml g418 . in order to differentiate 6t3 cells , the cells are first washed with pbs , followed by stimulation with 8 - bromo - cyclic amp ( 8 - br - camp ) at a final concentration of 5 mm in fresh culture media . three different antisense constructions were tested . all included the initiator codon atg of zis - sr . the insert sizes were of 414 , 737 and 1375 nts . these three fragments were amplified by pcr using distinct sets of oligos and the resulting dna fragment obtained was subcloned into the pcdna3 . 1 - hygro vector . all three amplification reactions used the zis5 primer ( 5 ′ aggtctgctgctggcgtccaagatgtc3 ′). for the 414 fragment the rdf7 primer was used , for the 737 fragment the gp2 - as primer ( 5 ′ ctggactgcgaactggaagagcttctt3 ′) was used and for the 1375 fragment the rdf5 primer was used ( 5 ′ ggcaaccctgtctctgt 3 ′). cells were brought to 60 - 70 % confluence , and washed in opti - mem ™ reduced serum media and transfections were carried out using lipofectamine ™. for a 100 mm dish , 5 μg of plasmid dna and 20 μl of lipofectamine ™ were diluted in separate tubes in opti - mem ™ to a volume of 1900 μl . the dna solution is then added dropwise to the lipofectamine ™ solution . following a 45 min incubation , 800 μl of opti - mem ™ is added and this solution is then added dropwise to the petri dish that contains 4 ml of opti - mem ™. following 6 hrs of incubation at 37 ° c ., the cells are washed with 10 ml of complete media . after 24 hrs post - transfection , the cells were washed with pbs and 48 hrs is post - transfection , the cells were selected with 250 μg / ml of hygromycin . one week following hygromycin selection , cell death is observed by cells that have not incorporated the plasmid dna . the cells are diluted 1 : 3 in 150 mm dishes . selection is carried out for another 3 - 4 weeks . single cell colonies are collected and propagated in 24 well plates . cells are eventually brought to confluence and used for rna extraction , immunocytochemistry , protein extraction or secretion experiments . att - 20 cells were cultured in special chambers for immunocytochemistry ( flacon culture slides ). after 2 days , when the cells had adhered to the slides , they were washed 3 × in pbs and then fixed with 4 % buffered paraformaldehyde for 30 min . the cells were then washed with pbs and submitted to permeabilization treatment for 1 hr with 0 . 1 % triton x - 100 and 2 mg / ml bsa in pbs . the cells were washed 3 × with pbs and 0 . 1 % triton and the primary antibody was then applied . the carboxypeptidase e ( cpe ) antibody was obtained from dr . l . fricker ( albert einstein university ) and the chromogranin a ( cga ) antibody was obtained from dr . sven - ulrik gorr ( university of louisville health sciences center ). the antibodies were incubated overnight at 4 ° c . this is followed by application of the secondary antibody after a 3 × wash with pbs 0 . 1 % triton . the secondary antibody is incubated for 1 - 2 hrs at room temperature . the slides are mounted with antifade solution ( 2 . 5 % dabco , 200 mm tris - hcl ph 8 . 6 , 90 % glycerol ). cells were stimulated with a 4 ml solution of physiological krebs solution containing 56 mm kcl for 30 min . depolarization provokes a release of secretory granules and the short time period precludes the formation of novel proteins through a transcriptional effect . control cells were treated with a similar 4 ml solution , however the kcl concentration was maintained a 4 . 7 mm . the media was collected after 30 min and concentrated on centricons ym - 10 . with a molecular weight exclusion of 10 , 000 kda . cells were extracted in extraction buffer containing protease inhibitors ( 50 mm tris - hcl , 2 . 5 mm edta , 150 mm nacl , 0 . 02 % sodium azide , 2 μg / ml leupeptine , 2 μg / ml aprotinine , 2 mm p - mercaptoethanol , 100 μg / ml pmsf , 100 μm pepstatine ). the cells were extracted using the freeze thaw method . following centrifugation at 15 , 000 × g for 30 min at 4 ° c ., the supernatants were collected and tested for protein content using the bradford assay . the protein samples were loaded on to 10 % sds - page gels and following migration were transferred to hybond ™ p pvdf membranes using electrophoretic transfer . primary antibodies to cpe or cga were applied to the membranes overnight and detection was carried out using the ecl + chemiluminescence kits ( amersham ). the membranes are exposed with x - omat ar ™ x - ray film ( eastman kodak ). cell pellets were resuspended and fixed in a 5 % gluteraldehyde in 0 . 1 m sodium cacodylate buffer ( ph 7 . 4 ) in krebs hepes ( 1 / 1 v / v ) for 20 min at 25 ° c . the cells were then pelleted and resuspended in 2 . 5 % gluteraldehyde in 0 . 1 m sodium cacodylate buffer ( ph 7 . 4 ) in krebs hepes ( 1 / 1 v / v ) for 20 min at 25 ° c . the cells were once again pelleted and a solution of 1 % osmium tetroxide in sodium cacodylatebuffer is added for 5 min . the solution was then replaced with a fresh solution of 1 % osmium tetroxide and fixed for 1 h at 4 ° c . the pellets were carefully resuspended in a 0 . 25 % uranyl acetate solution in 2 % sodium acetate buffer ( ph 6 . 3 ). the pellets were incubated for 45 min at 4 ° c . the pellets were then rinced overnight at 4 ° c . in a 0 . 25 % uranyl acetate dissolved in water ( ph 6 . 3 ). the fragments were re - suspended and dehydrated for 5 min in acetone solutions of 30 , 50 , 70 , 80 , 90 and 100 % at 4 ° c . after a final dehydration step in 100 % acetone , the fragments were placed in a epon - oxide propylene mixture ( 1 / 1 ) at 25 ° c . and then transferred to epon - oxide ( 4 / 1 ) overnight . the fragments were rinced in pure epon and then polymerised for 48 hrs at 60 ° c . sections were stained with 1 % uranyl acetate and 6 % lead citrate ( ph 10 ). total rna was extracted from cell lines using a guanidium isothiocyanate methodology , which includes a lithium chloride precipitation step ( day et al . 1995 ). the rna ( 5 μg ) was separated on agarose gel containing 6 % formaldehyde and blotted on nytran plus ™ membranes ( schleicher & amp ; schuell , keene , nh ). prehybridization was carried out for 2 h at 65 ° c . in hybridization buffer containing 5 % sds , 0 . 4 m sodium phosphate buffer , 1 mm edta , bsa 1 mg / ml , and 50 % formamide . the purified 32 p - utp - labeled crna probe was then added to the prehybridization buffer and incubated with the nytran membrane overnight at 65 ° c . filters were washed in 0 . 1 × ssc , 0 . 1 % sds , 1 mm edta at 72 ° c . for 2 h and exposed to x - ray film ( kodak xar - 5 with intensifying screens ) at − 80 ° c . for 3 hours . the in situ hybridization studies were carried out using antisense and sense labeled crna probes ( 35 s - utp / 36 s - ctp - labeled ). radioactive probes were diluted to 33 × 10 3 dpm / μl and in situ hybridization was performed as previously described ( schafer et al ., 1995 ). cd1 mice were sacrificed by rapid decapitation and tissues were rapidly removed and frozen in isopentane cooled to − 35 ° c . the extracted tissues were stored at − 80 ° c . until cryosectioning . frozen 10 μm sections were cut on a reichert cryostat ™ ( leica microsystems , depew , n . y .) and thaw - mounted on polylysine - coated glass slides and stored at − 80 ° c . until processing . sections were hybridized with zis - sr crna probes . these sections were submitted to standard in situ hybridization procedure ( schafer et al ., 1995 ) followed by x - ray film autoradiography for 1 - 7 days resulting in low resolution images . these slides were also dipped in nbt nuclear emulsion ( kodak ) to obtained cellular resolution . the sections hybridized were counterstained with cresyl violet , cleared in xylene , and mounted with permount ™ histological mounting medium ( fisher scientific ). as a negative control , radioactively labeled sense - strand probes of the same size and specific activity were used instead of the antisense - strand probe . although the present invention has been described hereinabove by way of preferred embodiments thereof , it can be modified without departing from the spirit and nature of the subject invention as defined in the appended claims . achsel , t . & amp ; shimura , y . 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