this invention relates to the preparation of a peptide compound ( syrapro - 2000 ), its use as an activator of protein phosphatase - 2a1 in vitro and in intact cells and its use for inhibiting cell proliferation and inducing death of cancer cells . the peptide compound ( syrapro - 2000 ), was designed based on the assumption that it is derived from the c - terminus of hiv - 1 vpr which was suspected to be an activator of protein phosphatase - 2a1 . the structure of syrapro - 2000 is hfrigcrhsrigvtrqrrarngasrs . syrapro - 2000 can be synthesized on an automated solid phase peptide synthesizer and the synthesized compound can activate purified protein phosphatase - 2a1 in vitro . the effect of synthesized syrapro - 2000 on purified protein phosphatase - 2a1 can be determined in vitro . protein phosphatase - 2a1 can be assayed using 32 p - labeled phosphorylase a as substrate in the presence of various concentrations of syrapro - 2000 . protein phosphatase - 2a1 is activated in the presence of syrapro - 2000 . the effect of synthesized syrapro - 2000 on protein phosphatase - 2a1 can be determined in intact cells . jurkat cells , a human cd 4 + t transformed cell line . jurkat cells are grown in rpmi 1680 medium supplemented with 5 % ( v / v ) fetal bovine serum and antibiotics and treated or not with 1 μm of syrapro - 2000 for 60 minutes . following treatment with syrapro - 2000 , cells are harvested by centrifugation , washed and homogenized . a cell extract is prepared by centrifugation and loaded onto a deae sepharose column which is washed and eluted with a liner gradient of buffer plus 0 mm nacl to buffer plus 400 mm nacl . the eluted fractions are then assayed using 32 p - labeled phosphorylase a as substrate . protein phosphatase - 2a1 from cells treated with syrapro - 2000 is higher than from cells not treated with syrapro - 2000 . the inhibition of cell proliferation and induction of death of jurkat cells , a human transformed cd 4 + t cell line by syrapro - 2000 can be demonstrated . jurkat cells are grown in rpmi 1680 medium supplemented with 5 % ( v / v ) fetal bovine serum and antibiotics and treated or not with 1 μm of syrapro - 2000 for 1 hour , 2 hours and 4 hours . following treatment with syrapro - 2000 , cell viability are then determined by counting the cells following tryphan blue staining . the number of viable cells are less when cells are treated with syrapro - 2000 . following treatment with syrapro - 2000 , cells are deposited onto glass slides by cytospinning . phase contrast images are then obtained . death of jurkat cells can be observed following treatment with syrapro - 2000 . the inhibition of cell proliferation and induction of death of htb - 3 cells , a transformed human glioblastoma cell line by syrapro - 2000 can be demonstrated . htb - 3 cells are grown in dmem supplemented with 5 % ( v / v ) fetal bovine serum and antibiotics and treated or not with 2 μm syrapro - 2000 for 8 days . following treatment with syrapro - 2000 , cell viability are then determined by counting the cells following tryphan blue staining . the number of viable cells are less when cells are treated with syrapro - 2000 . following treatment with syrapro - 2000 , cells grown on slides by were analyzed by phase contrast microscopy and fluorescence microscopy following staining with fitc labeled annexin v ( an indicator of apoptotic cell death ) or propidium iodide ( an indicator of oncolytic or necrotic cell death ). death of htb - 3 cells can be observed following treatment with syrapro - 2000 . the inhibition of cell proliferation and induction of death of sk — n — sh cells , a transformed human neuroblastoma cell line by syrapro - 2000 can be demonstrated . sk — n — sh cells are grown in dmem supplemented with 5 % ( v / v ) fetal bovine serum and antibiotics and treated or not with 2 μm syrapro - 2000 for 8 days . following treatment with syrapro - 2000 , cell viability are then determined by counting the cells following tryphan blue staining . the number of viable cells are less when cells are treated with syrapro - 2000 . following treatment with syrapro - 2000 , cells grown on slides by were analyzed by phase contrast microscopy and fluorescence microscopy following staining with fitc labeled annexin v ( an indicator of apoptotic cell death ) or propidium iodide ( an indicator of oncolytic or necrotic cell death ). death of sk — n — sh cells can be observed following treatment with syrapro - 2000 . 32 p - labeled phosphorylase a was prepared by phosphorylation of phosphorylase b with phosphorylase kinase as described in ( cohen 1988 ). 32 p - labeled casein was prepared by phosphorylation of casein with protein kinase a as described in ( tung 1986 ). protein phosphatase - 1 was purified from rabbit skeletal muscle as described in ( cohen 1988 ). protein phosphatase - 1 inhibitor - 2 was purified from rabbit skeletal muscle as described in ( yang 1981 ). protein phosphatase - 2a 0 and protein phosphatase - 2a 1 from pig brain and from pig liver were purified as described in ( tung 1985 ). protein phosphatase - 2a 0 and protein phosphatase - 2a 1 from pig adipose tissue were highly purified by successive chromatography of pig subcutaneous adipose tissue extracts on deae sepharose , poly - l - lysine agarose , sephacryl s - 300 hr and thiophosphorylase - a - sepharose - 4b as described in ( tung 1985 ). protein phosphatase - 2b was purified from pig brain as described in ( tung 1986 ). protein phosphatase 2c was highly purified from pig liver by successive chromatography of pig liver extracts on deae sepharose , casein agarose and sephacryl s - 300 hr . peptide activator compound of protein phosphatase - 2a1 ( syrapro - 2000 ) was prepared by chemical synthesis on an automated solid phase peptide synthesizer and purified as described in ( azzi 1992 ). the molecular mass of the peptide activator compound of protein phosphatase - 2a1 was confirmed by maldi tof ms . the sequence syrapro - 2000 is hfrigcrhsrigvtrqrrarngasrs . protein phosphatase - 1 was assayed as described in ( cohen 1988 ). protein phosphatase - 2b was assayed as described in ( tung 1986 ). protein phosphatase - 2c was assayed as described in ( tung 1986 ) except ca 2 + and calmodulin were omitted . the assay of protein phosphatases - 2a1 consisted of 0 . 02 ml of enzyme solution in 50 mm imidazole - cl ph 7 . 2 , 0 . 2 mm egta , 0 . 1 % ( v / v ) 2 - mercaptoethanol and 1 mg / ml bovine serum albumin ( assay buffer ), 0 . 01 ml of inhibitor - 2 at 600 nm in assay buffer , 0 . 01 ml of protamine at 60 μg / ml in assay buffer or 0 . 01 ml of syrapro - 2000 at different concentrations in assay buffer or 0 . 01 ml of assay buffer alone , 0 . 02 ml of 32 p - labeled phosphorylase a at 3 mg / ml in assay buffer containing 15 mm caffeine . the assay components were preincubated for 10 minutes prior to initiating the reaction with 32 p - labeled phosphorylase a . one unit of protein phosphatase activity is that amount of enzyme which catalyzes the release of 1 nmol of phosphate from 32 p - labeled substrate per min at 30 ° c . culture of jurkat cd 4 + t cells and purification of protein phosphatase - 2a 1 . jurkat cells , a human transformed cd 4 + t cell line , were grown in grown in rpmi 1680 supplemented with 5 % ( v / v ) fetal bovine serum at 37 ° c . in 95 % air / 5 % co 2 in a humidified incubator . 2 . 4 liters of cells were collected by centrifugation at 4200 rpm in a low speed centrifuge for 10 minutes . the cells were homogenized in 80 ml of 50 mm imidazole - cl ph 7 . 2 , 2 mm egta , 2 mm edta , 0 . 1 % ( v / v ) 2 - mercaptoethanol , 0 . 2 mm pmsf , 1 mm benzamidine , 4 μg / ml aprotinin , 4 μg / ml leupeptin , 4 μg / ml pepstatin , 0 . 1 mm tlck , 0 . 1 mm tpck , 1 mm sodium orthovanadate and 10 % ( v / v ) glycerol by 40 strokes in a glass hand held homogenizer . the homogenate was centrifuged at 29000 rpm in a high speed centrifuge for 30 minutes . the supernatant ( i . e the extract ) was collected and loaded onto a deae sepharose column ( 1 . 5 × 6 cm ) equilibrated in 25 mm imidazole - cl ph 7 . 2 , 0 . 2 mm egta , 0 . 1 % ( v / v ) 2 - mercaptoethanol , 0 . 1 mm pmsf , 1 mm benzamidine and 10 % ( v / v ) glycerol ( buffer a ). the column was washed with 50 ml of buffer and then eluted with a 200 ml linear gradient of buffer a to buffer a plus 0 . 4 m nacl . the eluted fractions were then assayed for phosphorylase phosphatase activity in the presence of 100 nm inhibitor - 2 and 10 μg / ml of protamine . two major peaks of phosphorylase phosphatase were observed . the first and second peak represent protein phosphatase - 2a 0 and protein phosphatase - 2a 1 respectively . the second peak eluting at around 0 . 2 m nacl , representing the largest proportion of the total protein phosphatase activity and which became activated following treatment of cd 4 + t cells with hiv - 1 vpr 71 - 96 ( fig1 ), was collected and loaded onto a sephacryl s - 300 hr column ( 2 . 5 × 90 cm ) equilibrated in 50 mm imidazole - cl ph 7 . 2 , 0 . 2 mm egta , 0 . 1 % ( v / v ) 2 - mercaptoethanol , 0 . 2 m nacl , 0 . 1 mm pmsf , 1 mm benzamidine and 10 % ( v / v ) glycerol . the major activity eluting as a species of apparent molecular mass 300 kda was collected , diluted four fold in buffer a and loaded onto a poly - l - lysine agarose column ( 1 . 5 × 4 cm ) equilibrated in buffer a . the column was washed with 50 ml of buffer a and then eluted with a 200 ml linear gradient of buffer a to buffer a plus 0 . 5 m nacl . the active fractions eluting at ˜ 0 . 30 m nacl salt concentration was pooled , concentrated by vacuum dialysis and stored at − 20 ° c . in 50 mm imidazole - cl ph 7 . 2 , 0 . 2 mm egta in the presence of 50 % ( v / v ) glycerol . the highly purified enzyme consisted of the characteristic subunits of protein phosphatase - 2a 1 , namely the a , b and c subunits . the purification of the enzyme is summarized in table 1 . the highly purified enzyme had a specific activity of 223 . 5 units per mg of protein . it is difficult to determine the activity of protein phosphatase - 2a 1 in cd 4 + t cell extract . however , assuming that it represented about 66 percent of total type 2a protein phosphatase activity in cd 4 + t cell extract , the specific activity of the enzyme in extract was estimated to be 0 . 3 unit / mg . the enzyme was therefore purified 908 fold . like other previously characterized forms of protein phosphatase - 2a , protein phosphatase - 2a 1 from cd 4 + t cells was not inhibited by inhibitor - 2 but inhibited by okadaic acid and activated by protamine . htb - 3 , a human glioblastoma cell line and sk — n — sh cells , a human neuroblastoma cell line were grown in dmem supplemented with 5 % ( v / v ) fetal bovine serum and antibiotics at 37 ° c . in 95 % air / 5 % co 2 in a humidified incubator . examination of cell viability and cell death in cells treated with syrapro - 2000 . following treatment of cells with syrapro - 2000 , cells were examined by phase contrast microscopy or by fluorescent microscopy following staining with fitc labeled annexin v ( apoptotic cell death ) and propidium iodide ( oncolytic cell death ). cells were analyzed on a zeiss axioplan 2 imaging universal microscope ( thornwood , n . y .). phase contrast images were obtained with white light emission . green ( fitc labeled annexin v ) and red ( propidium iodide ) fluorescence images were obtained . the images were captured and stored digitally with the aid of northern eclipse version 6 software ( zeiss , thornwood , n . y .). the excitation wavelengths were 495 nm ( fitc labeled - annexin v ) and 536 nm ( propidium iodide ) and the emmission wavelengths 520 nm ( fitc labeled - annexin v ) and 617 nm ( propidium iodide ). the images were filtered 510 - 550 nm ( fitc - labeled annexin v ), and 590 - 610 nm ( propidium iodide ) band pass filters . syrapro - 2000 , a peptide compound activator of protein phosphatase - 2a1 was prepared by chemical synthesis on an automated solid phase peptide synthesizer . following synthesis , it was cleaved and purified by reverse phase hplc on a c 18 column . the structure of syrapro - 2000 is : hfrigcrhsrigvtrqrrarngasrs . the molecular mass of syrapro - 2000 was determined by maldi tof ms . the purified material , a white solid was readily dissolved in and reconstituted in water at ˜ 1 - 2 mg / ml . for its use as an in vitro activator of protein phosphatase - 2a1 , it is dissolved in the assay buffer which consisted of 0 . 02 ml of enzyme solution in 50 mm imidazole - cl ph 7 . 2 , 0 . 2 mm egta , 0 . 1 % ( v / v ) 2 - mercaptoethanol and 1 mg / ml bovine serum albumin . for its use on intact cells , it is dissolved in the culture medium . the effect of syrapro - 2000 on the activity of protein phosphatase - 2a 1 , the major form of protein phosphatase - 2a in jurkat cells , was determined in vitro . syrapro - 2000 activated protein phosphatase - 2a 1 by 7 fold with half maximal activation occuring at around 300 nm . the effect of syrapro - 2000 was biphasic . at a concentration of above 1000 nm , there was inhibition of protein phosphatase - 2a 1 by syrapro - 2000 ( fig1 ). the effect of syrapro - 2000 on the activities of other major forms of protein phosphatase namely , protein phosphatase - 1 , protein phosphatase - 2b and protein phosphatase - 2c were determined . fig2 shows that the effect of syrapro - 2000 was quite specific . it had negligible effect on the activities of protein phosphatases - 1 , - 2b and 2c . the effect of syrapro - 2000 on the activity of protein phosphatase - 2a1 in intact cells was investigated . jurkat cells , a cd 4 + t cell line were grown in rpmi 1640 in 10 % ( v / v ) fetal bovine serum and then treated or not with 1000 nm syrapro - 2000 which was added directly to the culture medium in the presence of 0 . 001 % ( v / v ) dmso . extracts were then prepared as described in materials and methods section and chromatographed on deae sepharose column , an efficient procedure for the separation of protein phosphatase - 2a1 . protein phosphatases were then assayed in the presence of protein phosphatase - 1 inhibitor - 2 , a very effective method for the determination of the different forms of protein phosphatase - 2a ( tung 1985 ). as shown in fig3 , protein phosphatase - 2a1 which elutes at around 0 . 18 to 0 . 2 m nacl salt concentration became activated when cells were treated with syrapro - 2000 . inhibition of cell proliferation and induction of cell death in jurkat cells , a human transformed cd 4 + t cell line . treatment of jurkat cells with syrapro - 2000 was accompanied by inhibition of cell proliferation and induction of cell death . after four hours of incubation with syrapro - 2000 , almost 60 percent of the treated cells became non viable as determined by tryphan blue staining ( fig4 a ). examination of cd 4 + t cells treated with syrapro - 2000 by phase contrast microscopy showed that they died as a result of oncolysis ( fig4 b ). inhibition of cell proliferation and induction of cell death in htb - 3 cells , a human glioblastoma cell line . treatment of htb - 3 cells with syrapro - 2000 was accompanied by inhibition of cell proliferation and induction of cell death . after 8 days of incubation with syrapro - 2000 , almost 50 percent of the treated cells became non viable as determined by tryphan blue staining ( fig5 a ). examination of htb - 3 cells treated with syrapro - 2000 by fluorescent microscopy following staining with fitc - annexin v and propidium iodide showed that they died as a result of apoptosis and oncolysis ( fig5 b ). inhibition of cell proliferation and induction of cell death in sk — n — sh cells , a human neuroblastoma cell line . treatment of sk — n — sh cells with syrapro - 2000 was accompanied by inhibition of cell proliferation and induction of cell death . after 4 days of incubation with syrapro - 2000 , almost 50 percent of the treated cells became non viable as determined by tryphan blue staining ( fig6 a ). examination of sk — n — sh cells treated with syrapro - 2000 by fluorescent microscopy following staining with fitc - annexin v and propidium iodide showed that they died as a result of apoptosis and oncolysis ( fig6 b ). azzi , l ., meijer , l ., reed , s . i ., pidikiti , r . and tung , h . y . l . ( 1992 ). “ interaction between the cell - cycle - control poteins p34cdc2 and p9ckshs2 . evidence for two cooperative binding domains in p9ckshs2 . ” eur . j . biochem . 203 : 353 - 360 . chiang , c .- w ., kanies , c ., kim , k . w ., fang , w . b ., parkhurst , c ., xie , m ., henry , t . and yang , e . ( 2003 ). “ protein phosphatase - 2a dephosphorylation of phosphoserine 112 plays the gatekeeper role for bad - mediated apoptosis .” mol . cell biol . 23 : 6350 - 6363 . chiang , c . w ., harris , g ., ellig , c ., masters , s . c ., subramanian , r ., shenolikar , s ., wadszinski , b . e . and yang , e . ( 2002 ). “ protein phosphatase - 2a activates proapoptotic function of bad in interleukin - 3 - dependent lymphoid cells by a mechansim requiring 14 - 3 - 3 dissociation .” blood 2001 ( 97 ): 1289 - 1297 . cohen , p . ( 1989 ). “ the structure and regulation of protein phosphatases .” annu . rev . biochem . 58 : 453 - 508 . cohen , p ., alemany , s ., hemmings , b . a ., resink , t . j ., stralfors , p . and tung , h . y . l . ( 1988 ). “ protein phosphatase - 1 and protein phosphatase - 2a from rabbit skeletal muscle .” methods enzymol . 159 : 390 - 409 . cohen , p . ( 2002 ). “ protein kinase - the major drug targets of teh twenty - first century ?” nat . rev . drug discov . 1 : 309 - 315 . deng , x ., ito , t ., carr , b ., mumby , m . and may , w . s . ( 1998 ). “ reversible phosphorylation of bcl2 following interleukin - 3 and bryostatin 1 is mediated by direct interaction with protein phosphatase - 2a .” j . biol . chem . 273 : 34157 - 34163 . emerman , m . ( 1996 ). “ hiv - 1 , vpr and the cell cycle .” curr . biol . 6 : 1096 - 1103 . garcia , a ., cayla , x ., guergnon , j ., dassauge , f ., hospital , v ., rebollo , m . p ., fleisher , a . and rebollo , a . ( 2003 ). “ serine / threonine protein phosphatases pp1 and pp2a are key players in apoptosis .” biochimie 85 : 721 - 726 . ingebritsen , t . s ., stewart , a . a . and cohen , p . ( 1983 ). “ the protein phosphatases involved in cellular regulation . 6 . measurement of type - 1 and type - 2 protein phosphatases in extracts of mammalian tissues ; an assessment of their physiological roles .” eur . j . biochem . 132 : 297 - 307 . ingebritsen , t . s . and cohen , p . ( 1983 ). “ the protein phosphatases involved in cellular regulation . 1 . classification and substrate specificities .” eur . j . biochem . 132 : 255 - 261 . ingebritsen , t . s . and cohen , p . ( 1983 ). “ protein phosphatases : properties and role in cellular regulation .” science 221 : 331 - 338 . jannssens , v . and goris , j . ( 2001 ). “ protein phosphatase - 2a : a highly regulated family of serine / threonine phosphatases implicated in cell growth and signaling .” biochem . j . 353 : 417 - 439 . janoo , a ., morrow , p . w . and tung , h . y . l . ( 2005 ). “ activation of protein phosphatase - 2a 1 by hiv - 1 vpr cell death causing peptide in intact cd 4 + t cells and in vitro .” j . cell . biochem . 94 : 816 - 825 . li , x ., yost , j ., virshup , d . m . and seeling , j . m . ( 2001 ). “ protein phosphatase - 2a and its b56 regulatory subunit inhibit wnt signalling in xenopus .” embo j . 20 : 4122 - 4131 . mccluskey , a ., sim , a . t . r . and sakoff , j . a . ( 2002 ). serine - threonine protein phosphatase inhibitors : development of potential therapeutic strategies .” j . med . chem . 45 : 1151 - 1175 . meijer , l ., pondaven , p ., tung , h . y . l ., cohen , p . and wallace , r . ( 1986 ). “ protein phosphorylation and oocyte maturation ii . inhibition of starfish oocyte maturation by intracellular microinjection of protein phosphatases 1 and 2a and alkaline phosphatase .” exp . cell res . 163 : 489 - 499 . melnikova , i . and golden , j . ( 2004 ) “ targeting protein kinases .” nat . rev . drug discov . 3 : 993 - 994 . millward , t . a ., zolnierowicz , s . and hemmings , b . a . ( 1999 ). “ regulation of protein kinase cascades by protein phosphatase - 2a .” trends biochem . sci . 24 : 186 - 191 . morrow , p . w ., tung , h . y . l . and hemmings jr ., h . c . ( 2004 ). “ rapamycin causes activation of protein phosphatase - 2a1 and nuclear translocation of pcna in cd 4 + t cells .” biochem biophys . res . commun . 323 : 645 - 651 . pelech , s . and cohen , p . ( 1985 ). “ the protein phosphatases involved in cellular regulation . 1 . modulation of protein phosphatases - 1 and 2a by histone h1 , protamine , polylysine and heparin .” eur . j . biochem . 148 : 245 - 251 . petritsch , c ., beug , h ., balmain , a . and oft , m . ( 2000 ). “ tgf - b inhibits p70 s6 kinase via protein phosphatase - 2a to induce g1 arrest .” genes dev . 14 : 3093 - 3101 . sontag , e ., federov , s ., kamibayashi , c ., robbins , d ., cobb , m ., and mumby , m . ( 1993 ). “ the interaction of sv40 small tumor antigen with protein phosphatase - 2a stimulates map kinase pathway and induces cell proliferation .” cell 75 : 887 - 897 . sontag , e ., sontag , j . m . and garcia , a . ( 1997 ) “ protein phosphatase - 2a is a critical regulator of protein kinase c signaling targeted by sv40 small t to promote cell growth and nf - kb activation .” embo j . 16 : 5662 - 5671 . tung , h . y . l . 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this invention relates to the preparation of a peptide compound , its use to activate protein phosphatase - 2a enzymes for the dephosphorylation of proteins in vitro and in the cell and its use for inhibiting cell proliferation and induction of death of brain cancer cells and other cancer cells and therefore the treatment of proliferative disorder such as brain cancer and other forms of cancer . drugs that target the enzymes of the protein phosphorylation / dephosphorylation apparatus of the cell are mostly inhibitor compounds . the present invention describes for the first time the use of an activating compound of a protein phosphatase to modulate the activity of a cell . in this invention , the peptide compound activator of protein phosphatase - 2a1 is used to activate protein phosphatase - 2a1 , thereby causing inhibition of cell proliferation and induction of cell death of transformed t cells and brain cancer cells .