human prolactin ( hprl ) , a 23  kda polypeptide hormone with a single chain of 199 residues , is a member of the family of cytokines , which includes erythropoietin , interleukin-6 , and many others , but is most closely related , both evolutionarily and functionally , to human growth hormone and placental lactogen . this polypeptide is known to be involved in a variety of actions     , more than all the other pituitary hormones combined , such as cell proliferation , growth and development , water and electrolyte balance , and several physiological and behavioral aspects of mammal , bird , and reptile reproduction . in humans , apart from the pituitary , many extrapituitary sites for hprl synthesis have been documented , including breast , prostate , and female reproductive tract , where it appears to act locally to regulate cellular growth and differentiation     [ 1 , 4 , 5 ] . the hprl gene has been cloned and the authentic form of the hormone efficiently expressed in bacterial periplasm and in chinese hamster ovary ( cho ) cells in our laboratory     [ 68 ] . since hprl is one of the hormones most frequently determined in routine clinical assays and several therapeutic applications are being considered , an increasing need for pure bioactive hprl can be anticipated     [ 9 , 10 ] . sodium butyrate ( nabu ) is a short chain fatty acid , originally identified as a product of anaerobic bacterial fermentation , which has been shown to alter the structure of chromatin in the nucleus of mammalian cells by reducing the activity of histone deacetylase . one of the possible causes for improved gene expression by nabu cells is histone hyperacetylation , which facilitates the access of general transcription factors in eukaryotic cells     [ 12 , 13 ] . however , nabu can also cause a cellular arrest , leading to increased apoptosis and resulting in an overall reduction in recombinant protein production over longer periods of time     . nabu treatment has been shown , via immunoassay determination , to increase the expression levels of foreign proteins such as human thrombopoietin , interferon--1a , and chimeric igg3 antibodies in cho cell cultures     [ 1517 ] by factors of 2- , 2.5- , and 3.6-fold , respectively , relative to nontreated cells.this led us to investigate , for the first time , via physicochemical techniques , whether addition of nabu would also increase hprl production in these cell cultures . in order to characterize the product obtained , we took advantage of a specific rp - hplc methodology previously developed in our laboratory , which permits qualitative and quantitative analysis of hprl directly in cho - conditioned medium . high - performance size - exclusion chromatography ( hpsec )   methodology was used for both analytical and preparative purposes   and also allowed us to evaluated hprl activity directly in two bioassays , after its purification from cho - conditioned medium     . the clone expressing hprl , derived from cho dhfr cells ( dukx - b11 ) that had been transfected with the vector peddc - hprl , was obtained in our laboratory     . cells were thawed into t - flasks ( 75  cm , from corning costar corporation , cambridge , ma ) containing -minimal essential medium ( -mem , gibco - invitrogen corporation , grand island , ny , usa ) with l - glutamine and without ribonucleosides and deoxyribonucleosides , supplemented with 10% dialysed fetal bovine serum ( dfbs ) , 40  g / ml gentamycin , 4  g / l glucose , and 100  nm methotrexate . cultures were maintained in a humidified incubator at 37c in the presence of 5% co2 . 100  mm nabu was prepared in phosphate - buffered saline ( pbs ) ( 0.007  m na2hpo4 , 0.01  m nah2po4 , ph 7.4 and 0.15  m nacl ) , sterilized by passing through a 0.20  m syringe filter ( corning incorporated , corning , ny , usa ) , and stored in aliquots at 20c . to examine the effect of different concentrations of nabu on hprl production , 1  ml of cho cell suspension ( 5  10  cells / ml ) was seeded into each well of a 24-well plate to obtain a concentration of 2  10  cells / ml . cultures were then rinsed twice in pbs to remove serum and medium . fresh medium was then added containing nabu to a final concentration between 0 and 4  mm . cells were grown for 10 days and cho cell - conditioned medium was collected daily and stored at 80c for subsequent rp - hplc or western blot analysis . for product purification , cho cells were expanded into ten t - flasks , in -mem supplemented with dfbs , until they were confluent . when the cell density was ~2  10  cells / ml , fresh medium was then added to the t - flasks : five contained no nabu , while the chosen amounts of nabu were added to the other five . the hprl - secreting confluent cultures were then maintained for up to ten days by replacing the medium every 24 hours . the conditioned medium was collected and stored , as mentioned above , for sp - sepharose fast flow and hpsec purification . to examine the effect of different concentrations of nabu on cell density and viability , 1  ml of cho cell suspension ( 5  10  cells / ml ) was seeded into each well of a 24-well plate to obtain a concentration of 2  10  cells / ml . nabu was added to a final concentration between 0 and 4  mm and cells were grown for 10 days , as mentioned above . every 24 hours a sample for the different concentrations of nabu was detached from 24-well plate by treatment with 0.025% trypsin - edta in pbs . total cell number and viability were determined by using the trypan blue ( gibco - invitrogen corporation ) exclusion method via haemocytometry and phase contrast microscopy , to distinguish viable and nonviable cells on the basis of dye uptake . 5  10  cells / ml cells were seeded in 24-well plate , treated and detached at regular times , as mentioned above . 1  10  cells / ml per sample were lysed in 400  l of lysis buffer ( 50  mm tris , 10  mm edta ph 8.0 , 1% sds ) and 500  g / ml proteinase k ( fermentas , life sciences , so paulo , brazil ) at 37c for 16 hours . the lysates were extracted with phenol  :  clorophorm  :  isoamylalcohol ( 25  :  24  :  1 ) . the resulting pellets of dna were resuspended in te buffer ( ph 8.0 )      and treated with 10  g / ml rnase a ( fermentas , life sciences ,   brazil ) prior to electrophoresis on a 5% polyacrylamide gel at 70  v . discontinuous sds - page , based on 15% polyacrylamide gels , was carried out under nonreducing conditions as described by laemmli . proteins were revealed by silver or coomassie brilliant blue g-250 ( usb , cleveland , oh ) staining . western blot analysis was performed according to bannerman et al .     , using i - labelled protein a   and anti - hprl antiserum obtained in rabbit and validated in our laboratory against the niddk - anti - hprl-3 from national hormone and pituitary program , torrance , ca , usa     [ 7 , 8 ] . total protein content was determinate by bca protein assay , using bicinchoninic acid ( micro bca protein assay kit , pierce , rockford , il , usa ) . pure bovine serum albumin ( bsa , sigma , so paulo , brazil ) was used as standard . after the treatment with different nabu concentrations , an aliquot of conditioned culture medium ( ~10  ml ) was concentrated ( ~10  fold ) by using a centrifugal membrane filter ultra 4 ( amicon , millipore corporation , billerica , ma , usa ) and applied to a rp - hplc column . a shimadzu model scl-10a hplc apparatus coupled to a spd-10av uv detector ( shimadzu , md , usa ) was used , employing the class vp software , also from shimadzu . the column was a c4 vydac 214tp54 ( 25  cm  4.6  mm i d , pore diameter of 300   and particle diameter of 5  m ) with a silica precolumn packed with lichrosorb si-60 , 7.912.4  m ( merck , darmstadt , germany ) located between the pump and the injector . all vydac columns were purchased from grace vydac ( hesperia , ca , usa ) . the mobile phase consisted of 71% tris - hcl buffer ( 50  mm , ph 7.5 ) and 29%  n - propanol , as described by dalmora et al . , with a flow - rate of 0.5  ml / min , a detector wavelength of 220  nm , a column temperature maintained at 45c , and a sample volume of 25500  l . this methodology was used for analytical purposes to examine the effect of nabu on hprl production . the hpsec is an entropically controlled separation technique in which molecules are separated on the basis of their hydrodynamic molecular volume or size     [ 25 , 26 ] . this methodology was used for both analytical and preparative purposes and allowed us to use purified hprl directly in bioassays , the n - propanol interference being absent due to the rp - hplc technique . hpsec was carried out with the same shimadzu apparatus , processing 25 to 500  l of sample on a tosohaas ( montgomeryville , pa , usa ) g2000 sw column ( 60  cm  7.5  mm i.d . , particle size of 10  m and pore size of 125   ) coupled to a 7.5  cm  7.5  mm i.d . the mobile phase was 0.025  m ammonium bicarbonate , ph 7.0 , with a flow - rate of 1.0  ml / min . the hprl present in conditioned culture medium was purified as described by soares et al .     . a two - step purification process was used : sp - sepharose fast flow followed by a size exclusion chromatography employing hpsec for preparative purposes . the material was then applied onto the sp - sepharose fast flow column ( ge healthcare bio - science corp . , piscataway , nj , usa ) equilibrated in 50  mm sodium acetate , ph 5.0 . uv absorbance was monitored at 280  nm . after washing with the same buffer , the column was treated with 50  mm sodium acetate , ph 5.0 , 90  mm nacl buffer . the protein of interest was then eluted from the column with 25  mm hepes , ph 8.0 . different fractions containing hprl were analysed by hpsec and the most concentrated fractions were purified on the same hpsec column , working this time in a preparative mode . hprl in the final pool obtained via the hpsec purification was quantified for bioassay purposes by hpsec and stored at 80c . the biological activity of purified hprl was determined via the nb2 and ba / f3-llp lymphoma cell - proliferation assays , as previously described , against the first who reference reagent of recombinant hprl , lyophilized in ampoules coded 97/714 . ba / f3-llp cells were routinely maintained in suspension in rpmi-1640 medium supplemented with 10% heat - inactivated fbs , 2  mm glutamine , 50  u / ml of penicillin , 50  g / ml of streptomycin , 700  g / ml of geneticin and 1  ng / ml of hprl . before carrying out the proliferation assay , cells were starved for 6 hours in the rpmi-1640 medium , as mentioned above , without hprl . cells were then distributed in flat bottom 96-well plates at a density of 5  10  cells / well in a final volume of 200  l . after 72 hours at 37c and 5% co2 , the presence of viable cells was assessed using mts assay . briefly , 2  mg / ml mts dye [ 3(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulf - ophenyl)-2h - tetrazolin ( promega corp . , madison , wi , usa ) ] in pbs was mixed in a 20  :  1 ratio ( vol / vol ) with pms ( phenazine methosulphate ) ( sigma , st . twenty microliters of mixture were then added to each well , and 2 hours after incubation at 37c the absorbance at 490  nm was read in a microplate reader ( multiskan ex thermo electron corporation , vantaa , finland ) . nb2 cells also routinely maintained in suspension in rpmi-1640 medium supplemented with 10% heat - inactivated fbs , 2  mm glutamine , 50  u / ml of penicillin , and 50  g / ml of streptomycin mixed in a 50  :  1 ratio ( vol / vol ) with 5  mm 2- mercaptoethanol in pbs . before carrying out the proliferation assay , cells were maintained in the rpmi-1640 , as mentioned above , containing 1% heat - inactivated fbs for 8 hours , constituting the preassay . afterward , the cells were distributed in flat bottom 96-well plates at a density of 2  10  cells / well in a final volume of 200  l , with no heat - inactivated fbs addition . after 72 hours at 37c and 5% co2 , the presence of viable cells was assessed using mts assay , as mentioned before . the influence of different concentrations of nabu on hprl - secreting cho cellular growth and viability during a 10-day culture can be seen in figure 1 . the treatment with either 0.25 or 0.5  mm nabu did not result in significant growth inhibition ( p > 0.05 ) compared to the control without nabu ( figure 1(a ) ) . nonetheless , a dose response - effect on cellular growth was observed in the concentration range of 1 to 4  mm nabu , with a dramatic fall already occurring after day 2 at the concentrations of 2  mm and 4  mm nabu . the treatment with 1  mm nabu produced an apparent decline only 4 days after starting the treatment . with regard to cell viability , none of the concentrations except 4  mm nabu ( p < 0.05 ) showed any significant effect ( figure 1(b ) ) . the rate of apoptosis in nabu - treated and untreated cho cells was examined by polyacrylamide gel electrophoresis and revealed by silver staining . figure 2 shows that nabu - treated and untreated cho cells exhibit oligonucleosomal dna ladder which is the hallmark of apoptosis , resulting from endonuclease activity at internucleosomal sites in an apoptotic cell . the intensity of the oligonucleosomal dna ladder seems nabu dose - dependent . on the other hand , the progressive removal of dfbs from the culture medium could also be associated with apoptotic cell death in nabu - treated and untreated cho cells , leading to a decrease of cellular growth after day 5 . this can be due to the fact that serum contains growth factors that help the culture survival     . a rp - hplc methodology , previously set up in our laboratory specifically for hprl qualitative and quantitative analysis , was then employed to quantify hprl production and detect the presence of possible alteration in hprl structure or hydrophobicity due to the prolonged presence of nabu . the data in table 1 and figure 3 show that 1  mm nabu is capable of greatly increasing hprl synthesis ( p < 0.02 ) , with no significant differences ( p > 0.05 ) in the retention time ( tr ) of the resultant hprl up to 4  mm nabu . human prolactin quantification carried out by rp - hplc in cho cell - conditioned medium ( table 1 ) indicated that the highest production was obtained with 1  mm nabu , which increased the secretion ~2-fold relative to control cultivation without nabu addition . despite an increase in protein expression , the concentrations of 0.5  mm and 2  mm nabu provided lower hprl volumetric yields than 1  mm nabu . the greatest inhibition of cell growth was produced by 4  mm nabu leading to lower hprl synthesis ( figure 1(a ) and table 1 ) . clearly , it will be important to determine the optimal nabu concentration in long - term cell cultures . to confirm the expression and purity level of hprl , equal volumes of cho - conditioned medium , collected on days 1 , 4 , 5 , and 6 , were analyzed by western blot ( figure 4 ) ; the same was done with pools obtained after 10 days of cultivation ( figure 5(a ) ) . these pools were also analyzed via sds - page followed by agno3 staining ( figure 5(b ) ) , which demonstrated the predominance , with 1  mm nabu , of hprl relative to other proteins that might be present in cho cell - conditioned medium . taken together , these rp - hplc , western blot , and sds - page analyses indicate that , under our conditions , 1  mm nabu was the ideal concentration , providing the highest hprl expression level , while still maintaining good cell viability and only a limited decrease in cell growth . sp - sepharose fast flow chromatographic purification of nabu - treated and untreated cells , together with sds - page analysis of applied and eluted fractions , is shown in figure 6 . this confirmed the presence of about twice as much hprl in the medium treated with 1  mm nabu and the efficient adsorption of both the 25  kda glycosylated ( g - hprl ) and the 23  kda nonglycosylated hprl ( ng - hprl ) isoforms by the cationic exchanger . the same partially purified products were also analyzed by hpsec before and after using this same column for preparative purposes ( figures 7(a ) , 7(b ) , and 7(c ) ) . the purity obtained for both products after this second chromatographic step is noteworthy ( > 97% ) ( figure 7(c ) and table 2 ) . from table 2 , on the basis of mass fractions , it is possible to calculate an overall purification factor of ~55 and 122-fold for nabu - treated and untreated cho cells , respectively . the ~2-fold increase in hprl production obtained in the presence of 1 mm nabu was also confirmed after purification . also remarkable are the perfectly coincident tr values of the two cho - derived hprl preparations and of the internal reference of e. coli - derived hprl ( 19.58 minutes < tr < 19.72 minutes ) , confirming their identical hydrodynamic properties in this long ( 60  cm ) hpsec column ( figure 7(c ) ) . it has been reported that these properties can be altered even by subtle modifications in the secondary and tertiary structure of the molecule     [ 32 , 33 ] . finally , the two purified products were tested for their biological activity against the reference reagent for recombinant hprl ( who 97/714 ) in two different in vitro bioassays based on nb2 and ba / f3-llp cells . the data illustrated in figure 8 and reported in table 3 show that apparently there were no differences between the potencies of the purified products and that of the who reference reagent . previous studies have examined the production of recombinant proteins by nabu - treated cho cells . several of these have discussed the benefits or warned against the problems caused by the presence of nabu     [ 1416 ] . nabu has , in fact , been used for increasing the expression of foreign proteins in animal cell cultures . it is recognized that nabu alters primarily chromatin structure through inhibition of histone deacetylases , resulting in hyperacetylation of histones and , consequently , alterations in dna transcription . therefore , these alterations might be correlated with the modulation of gene expression . on the other hand , hyperacetylation of histones in nabu - treated mammalian cancer cell lines has been shown to induce apoptosis mediated by activation of caspase-3 activity , leading to the translation of putative cell death proteins      and resulting in an increased possibility of dna cleavage .   in this study , we examined the expression level and quality of hprl directly in cho cell - conditioned medium , after adding different amounts of nabu . the different nabu concentrations ( 0.25 to 2  mm ) remarkably increased hprl expression . addition of 1  mm nabu to the culture medium resulted in a twofold increase in hprl expression , with only a relatively small decrease in cell growth compared with the untreated control ; this was confirmed by rp - hplc and by western blot and sds - page analysis . although 2  mm and 4  mm nabu apparently induced a higher synthesis of hprl in the first 24 hours of treatment , this synthesis exhibited a decrease in the following days of culture . a higher nabu concentration ( 2 or 4  mm ) may be more effective in inhibiting the activity of histone deacetylases and , consequently , increase the initial expression of proteins . however , a greater modification of the chromatin structure could make the dna more susceptible to apoptotic action of endonucleases , leading to decreased productivity of hprl . the addition of different nabu concentrations apparently did not affect the hydrophobicity or the hydrodynamic proprieties of the hprl molecule . this was inferred from the fact that the tr values determined by rp - hplc and hpsec were not significantly altered by the nabu treatment relative to the untreated control or the internal reference standard of periplasmic e. coli - derived hprl . our analytical determinations , as a difference from other authors that studied nabu influence on heterologous protein production with basis on immunological techniques     [ 1517 ] , have been mostly based on physicochemical methodologies . the cell viability for different concentrations of nabu was similar to that of the untreated control except in the experiments with 4  mm added nabu . therefore , the arrest of cell growth after day 5 and similar decrease in cell viability between 02  mm nabu was probably caused by removal of dfbs from the culture medium and nabu treatment during the production process . this could make the cho cell more susceptible to apoptosis , leading to decreased cellular density and viability . finally , two different in vitro bioassays for purified hprl , based on nb2 and ba / f3-llp cells , were carried out . the first bioassay is the classical heterologous nb2 rat lymphoma cell proliferation assay , while the second is the recently developed homologous ba / f3-llp assay , based on murine pro - b lymphoma cells containing the sequence encoding the human prl receptor and previously utilized in our laboratory     [ 9 , 27 ] . as described by crowell et al . , nabu treatment can potentially modify the oligosaccharide content of glycoproteins in various expression systems . although nabu apparently has no effect on the protein structure of hprl , there might be modifications of the oligosaccharide structure of g - hprl . in this regard , it is known that the oligosaccharide nature and content can influence the in vivo bioactivities of a glycoprotein     [ 3942 ] . it is therefore important to note that the potency of hprl obtained in the presence of nabu was comparable to that of the reference reagent for recombinant hprl ( who 97/714 ) . in conclusion , for the first time recombinant hprl production from genetically modified cho cells has been significant increased ( ~2-fold ) via nabu addition , apparently without compromising either its structure or function ; the quantitative and qualitative effects of this addition have been determined via accurate physicochemical techniques . the same treatment can potentially be applied to other hormones , for example , human thyrotropin ( htsh ) , which is also produced in our laboratory     . the qualitative and quantitative influence of this treatment on the carbohydrate moieties of g - hprl and possibly of other glycohormones requires further investigation .
sodium butyrate ( nabu ) has been used as a productivity enhancer for the synthesis of recombinant proteins in chinese hamster ovary ( cho ) cells . 
 thus , the influence of nabu on the production of recombinant human prolactin ( hprl ) from cho cells was investigated for the first time . 
 cho cell cultures were submitted to a treatment with different concentrations of nabu ( 0.25 to 4  mm ) . quantitative and qualitative analyses by reverse - phase high - performance liquid chromatography ( rp - hplc ) and western blot or sds - page , carried out directly on cho - conditioned medium , showed that the highest hprl expression was obtained with 1  mm nabu . in vitro biological assays based on noble rat lymphoma ( nb2 ) and mouse pro - 
 b lymphoma ( ba / f3-llp ) cells were carried out on purified hprl . 
 its bioactivity in the presence of nabu was not apparently different from that of the first international reference reagent of recombinant hprl ( who 97/714 ) . 
 our results show that nabu increased the synthesis of recombinant hprl in cho cells , apparently without compromising either its structure or function .