Patent Application: US-15792308-A

Abstract:
the invention relates to β - galactoside binding protein and compositions , including pharmaceutical compositions , comprising βgbp for use in therapy and related applications . in particular , the invention relates to use of βgbp and the manufacture of medicaments for the treatment or prevention of conditions in which disease associated cell division occurs , wherein the cells which result from said disease associated cell division comprise a cell in respect of which the effect of βgbp is not inhibition of growth . the invention also relates to methods of inducing apoptosis , methods of treating or preventing conditions in which disease associated cell division occurs and methods of assessing the suitability of βgbp as a therapeutic agent .

Description:
it has surprisingly been found that βgbp inhibits pi3k activity and reduces expression of the akt gene . unexpectedly , aggressive cancer cells that are treated with βgbp undergo delayed , massive apoptosis after 2 - 3 rounds of cell division , in response to the early event of reduction in akt mrna levels . similarly , βgbp induces apoptosis in activated persistently dividing lymphocytes such as t cells , whilst sparing naïve cells . interestingly , increasing the mitogenic character of : normal - like cells ( to create artificially aggressive or hyperproliferative cells ); cancer cells of a less aggressive type ; or lymphocytes such as activated t cells , renders such cells more susceptible to apoptosis induced by βgbp . equivalent results are seen in colon cancer cells , but at much lower doses than for other cancer types . to determine whether βgbp could overcome the strength of endogenous mitogenic signalling in aggressive cancers , bt474 and skbr3 breast cancer cells which express high levels of erbb2 ( hynes et al ., 1989 ) were examined . it was found that βgbp had virtually no effect on cell replication until , after 2 - 3 generation cycles , abrupt cell death was triggered by an acute sequence of apoptotic events documented by changes in mitochondrial membrane potential , functional alteration of the plasma membrane , caspase 3 activation and dna fragmentation ( fig1 a - d ). there were no changes in erk phosphorylation while cell replication continued unaffected , but it was observed that loss of phosphorylated akt , and loss of akt protein , had preceded the apoptotic process ( fig1 g and j ). in order to determine whether the loss of the akt protein could be attributed to transcriptional or post - transcriptional events akt mrna levels were assessed . fig1 f and i shows that within one day from the addition of βgbp akt mrna had become virtually undetectable . it is reasoned that , while the loss of phosphorylated akt may point to loss of pi3k activity and akt recruitment as one conceivable explanation , the loss of akt mrna does not , unless pi3k activity is required for akt gene expression and unless βgbp can inhibit pi3k activity . as cells &# 39 ; phosphoinositide levels do not directly represent the functional state of the pi3k enzyme , but are the result of pi3k and pten activity , in order to directly assess its functional state , class ia pi3k was isolated by immunoprecipitation using an antibody to the p85α adapter subunit and the ability of the co - precipitated p 110 catalytic subunit ( vanhaesenbroeck et al ., 2001 ) to convert a standard phosphatidylinositol ( 4 , 5 ) p2 ( pip2 ) to phosphatidylinositol ( 3 , 4 , 5 ) p3 ( pip3 ) was assessed in a kinase reaction by measuring the generated pip3 in a competitive elisa . fig1 e and h demonstrates that downregulation of pi3k activity was an early event already present at hour six after the addition of βgbp and maintained throughout the period of observation . framed within a time sequence , these results show that addition of βgbp to the bt474 and skbr3 cells resulted in downregulation of pi3k activity , loss of akt gene expression , loss of akt function and apoptosis . while showing that cancer cells driven by strong mitogenic pressure are unresponsive to the growth inhibitory effect of the βgbp cytokine ( mallucci et al ., 2003 , wells and mallucci 1991 ), the evidence of fig1 suggests that to elevated mitogenic input , corresponding elevated survival signalling may create conditions that would protect mitogenic expansion , but which βgbp can interrupt by downregulation of pi3k activity and consequent block of akt gene function . in order to test the validity of this assumption the inventors experimentally enforced mitogenic pressure in non cancerous cells . the cells used were : spontaneously immortalized mcf10a mammary ductal cells which exhibit normal - like behaviour ( soule et al ., 1990 ), mcf10a cells stably transfected with constitutively active p21 ras mutated at valine 12 ( schulze et al ., 2001 ) ( mcf10a v12ras ), which strongly activates raf - erk signalling ( repasky et al ., 2004 ), and mcf 10a cells where mitogenic input was enhanced by the addition of cholera toxin ( mcf10a ctx ) which increases erk activity via adenyl cyclase upregulation ( alberts et al ., 2002 ). the following concepts were examined : whether mitogenic pressure would affect proliferation rate and cell response to βgbp ; whether there would be correlation between mitogenic input and akt mrna levels ; whether βgbp would affect pi3k activity and whether there would be correlation between pi3k activity and akt gene expression . it was found that cell proliferation was boosted both by v12ras and by cholera toxin and that the response to βgbp , abrupt cell death after 2 - 3 cell replication cycles , mimicked that of the bt474 and skbr3 cells , in contrast with the minor effect exerted by βgbp in the naïve mcf10a cells ( fig2 a - c and d - f ). a correlation was also found between mitogenic pressure and akt gene expression , as shown by changes in akt mrna levels which , barely detectable in the naïve cells , became more markedly expressed where the mitogenic input had been raised , whether by v12 ras or by cholera toxin ( fig2 j - l , left panels ). examination of whether βgbp would affect pi3k activity and akt gene expression showed that in all cases βgbp brought down and maintained pi3k activity below basal levels ( fig2 g - i ) regardless of mitogenic input , with a pattern similar to that observed in the erbb2 positive cells , but with a delay from 6 to 24 hours , in accord with the later disappearance of akt mrna ( fig2 k , right panel ), where the cells were driven by the strong mitogenic signalling imposed by v12 ras . similarly to the cells of fig1 , in all the above cells , downregulation of pi3k activity resulted in virtual absence of akt mrna ( fig2 j - l , right panels ) and consequent loss of akt function ( fig2 m - o ). notably , the time sequence of pi3k inhibition , loss of akt mrna and loss of akt , copied the pattern observed in the bt474 and skbr3 cells . it is of particular significance within a therapeutic context that in the naïve normal - like mcf10a cells , which have low levels of akt mrna , inhibition of pi3k activity and loss of akt gene function did not lead to apoptosis , as it indicates that loss of survival signalling is not harmful in the absence of abnormal mitogenic pressure . without wishing to be bound by theory , this is one conceivable explanation for the selective effect of βgbp which , while killing cancer cells , including their drug resistant derivatives ( mallucci et al ., 2003 , ravatn et al ., 2005 ), causes no harm to normal cells ( wells and mallucci 1991 ). the above data , together with those of fig1 , indicate that strong mitogenic pressure as represented by the degree of erk phosphorylation , whether constitutive or induced ( fig3 ), is coupled to elevated akt gene expression . collectively , the observations described herein endorse a model where enhanced erk activity enhances cell survival by upregulating akt gene expression , for which pi3k activity is a requirement ( fig4 a ), and where , by down - regulating pi3k activity and negating akt gene function , βgbp interrupts cancer cell reliance on survival signalling and leads cells to apoptotic death ( fig4 b ). it is noteworthy within the erk / akt gene context that the observations presented herein bring to attention a new functional aspect in transcriptional control which extends the role of erk from the activation of cell cycle promoting genes ( karin and hunter 1995 ) to the activation of the akt gene , which promotes survival . notably , no evidence was found that raising active erk levels , whether by v12ras or by cholera toxin , had any effect on pi3k activity . the evidence that in the mcf10a cells a shift in phenotypic behaviour as a consequence of enforced mitogenic pressure changed the cells &# 39 ; response to βgbp to mimic that of the skbr3 and bt474 cancer cells , raises the question of whether in cancer cells a shift towards a more aggressive condition would increase their vulnerability to βgbp . mcf - 7 breast cancer cells were therefore examined . these cells have low levels of erbb2 and do not exhibit aggressive behaviour ( karanugaran et al ., 1996 ). mcf - 7 cells were examined in their naïve state and when also treated with cholera toxin ( mcf - 7 ctx ). it was found that cholera toxin raised active erk levels , accelerated cell proliferation and accentuated akt gene expression , thus changing the phenotypic behaviour of the cells ( fig5 a - c ). examination of their response to βgbp showed that , while overriding the growth inhibitory effect that βgbp exerted on the naïve mcf - 7 cells , as reported previously ( ravatn et al ., 2005 ), the mcf - 7 ctx cells succumbed to total death ahead of the naive counterparts ( fig5 d ), thus confirming the proof of the principle that where erk activity and akt gene expression are enhanced , abolition of akt gene function can result in greater cell vulnerability . next it was investigated whether in the mcf - 7 ctx cells pi3k was again a primary responder to the action of βgbp and , if so , whether negation of akt gene expression would be a consequence of the inhibition of pi3k activity . therefore time scale experiments were carried out using βgbp in parallel with wortmannin ( powis et al ., 1994 ) and ly29400 ( vlahos et al ., 1994 ). both compounds are pharmacological inhibitors of the p110 catalytic subunit of pi3k ( walker et al ., 2000 , wymann et al ., 1996 ), and were added at concentrations which would produce an effect similar to that of βgbp . pi3k activity and akt mrna levels were assessed at different time points . evidence was found to indicate that pi3k activity is a necessary requirement for akt gene expression and that basal or close to basal endogenous levels are sufficient . fig5 e - g shows that βgbp lowered pi3k activity to a similar extent as the two inhibitors , but with a more gradual kinetic , in line with the action of a physiological effector molecule , and that akt gene expression was negated when pi3k activity had similarly descended by an approximate 35 % quantum below basal levels , in all three instances . the pi3k / akt signalling cascade is under intensive investigation for its role in oncogenic transformation and for its critical contribution to aggressiveness and resistance to therapies ( lu et al ., 2003 , chen et al ., 2005 , morgensztern and mccleod 2005 ). the present inventors have identified the βgbp cytokine as a selective pi3k targeting agent , provides the first evidence for functional dependence between pi3k and the akt gene and the first evidence for an effective treatment of aggressive cancer cells via therapeutic silencing of the akt gene by a physiological molecule . in conclusion , the present inventors have shown that , contrary to its ability to arrest cell cycle progression both in normal ( wells and mallucci 1991 ) and in some cancer cells ( mallucci et al ., 2003 ), βgbp was unable to affect cell proliferation in cells driven by strong mitogenic input . however , by controlling a functional link between pi3k and the akt gene , βgbp does have strong therapeutic efficacy without compromising cells which exhibit normal - like behaviour . the inventors hypothesise that erk , the akt gene , pi3k and βgbp are mechanistically linked and that pi3k is the pivotal element on which βgbp acts . functional inhibition of the p110 catalytic subunit of class ia pi3k by βgbp is followed by abrogation of akt gene expression and by apoptotic death in cells where erk and akt mrna levels are high , but not otherwise . the similarity of the effect of βgbp with that of wortmannin and ly294002 , in regard of both inhibitory pattern and time required for the inhibitory action to come into effect ( fig5 d - f ), suggests that , similarly to the two inhibitors , βgbp may induce conformational changes which would reduce the functional ability of the regulatory pocket site of the p110 catalytic subunit of pi3k ( walker et al ., 2000 ). pi3 kinase and ras are functionally linked . this link is suppressed by βgbp . the p110 catalytic subunit of pi3 kinase is inhibited by βgbp . thus in normal cells ras - mapk signalling is blocked by βgbp . pi3 kinase activity is downregulated by βgbp , leading to suppression of ras - gtp loading , consequent loss of mapk activation and , in the context of cells which retain a sensitivity to the growth inhibitory properties of βgbp , cell proliferation is blocked . in cells which no longer respond to βgbp with inhibition of growth ( for example , cells as targeted by the present invention , such as persistently dividing cells ), the eventual result of the inhibition of pi3k activity is different . instead , in persistently dividing cells of the present invention , no effect on ras - mapk loading is seen as the mitogentic input is too great . however , in such cells , the silencing of akt gene expression ultimately results in apoptosis . the dependence of gene expression on pi3k activity that is shown herein is a new aspect in gene control which may not be limited to the akt gene . it is of interest within the context of pi3k inhibition and suppression of akt gene function , that the mapping of the gene encoding βgbp ( chiariotti et al ., 1991 ) in the sis / pdgfb homology region ( mu - chromosome 15e / hu - chromosome 22 q12 - q13 ) ( baldini et al ., 1993 ), a syntenic group which undergoes deletion and translocation in a number of human tumors ( aurias et al ., 1984 , turc - carel et al ., 1998 , bridge et al ., 1990 , rey et al ., 1993 ) brings to attention the βgbp gene as a prospective tumor suppressor gene . βgbp can totally prevent the appearance of tumours in scid mice . 5 × 10 6 st4 ( human malignant t cell leukaemia ) cells were injected subcutaneously at day 0 . hu - r - βgbp was administered from day 1 to day 16 by subcutaneous injection . the results are indicated in table 1 below . as described above βgbp is known to inhibit growth and / or promote apoptosis in cells from a range of cancer types . however , in experiments to screen the effects of βgbp in a range of cancer types it was surprisingly noted that βgbp was effective in colon cancer cells at a much lower dose than is observed for other cancer types . in breast cancer , ovarian cancer , oral cancer , prostate cancer and leukaemia cells tested for pro - apoptotic responses to βgbp , the effective dose in cell culture was 15 - 30 nm . in colon cancer cells the effects of βgbp were observed at much lower concentrations . to confirm these observations three colon cancer cell lines were investigated , sw480 , sw620 and lovo . cells were grown in culture and incubated with βgbp at varying concentrations . it was found that all of the colon cancer cell lines were much more susceptible to the pro - apoptotic effects of βgbp than other cancer cell lines . concentrations as low as 1 - 2 nm have been observed as effective in inducing apoptosis ( fig6 ). it is possible that in some circumstances even lower concentrations of βgbp may be effective . similar kinetics for initial growth , followed by a sharp decline in cell numbers were observed ( fig6 ) for each colon cancer cell line , in a manner similar to that previously observed for bt474 cells and skbr3 cells ( fig1 a and c ), mcf10a v12ras cells and mcf10a ctx cells ( fig2 b and c ), and mcf - 7 cells treated with cholera toxin ( fig5 ). this demonstrates that βgbp has an equivalent effect on colon cancer cells as it does on other cancer cell lines , but at concentrations 15 to 30 times lower . this result is unexpected given the broadly similar effective concentration range for the inhibition of growth effect or the induction of apoptosis in other cancer cell lines by βgbp . thus βgbp appears to be particularly suitable for the treatment of colon cancer since therapeutic effects may be possible at much lower βgbp doses . the present invention therefore relates to several therapeutically relevant aspects . firstly , inhibitory effects on the activity of the enzyme pi3 kinase have been demonstrated for βgbp . firstly , the evidence that mitogenic pressure , whether endogenous or enforced , makes cancer cells highly vulnerable to treatment with the βgbp cytokine ( fig1 and 5 ), suggests that this physiological molecule may have particular therapeutic relevance to the treatment of aggressive cancers . secondly , the evidence of fig2 where naïve mcf10a cells , which have relatively low levels of active erk and low levels of akt mrna , are not harmed by βgbp challenge , suggests that βgbp has ability to selectively induce apoptosis according to the particular molecular context of the cell , a property not shared by pharmacological compounds . significantly , the evidence of fig3 also suggests that where an increase in mitogenic signalling is the prime occurrence amongst events that lead to oncogenesis , nascent cancer cells could be eliminated in the healthy organism by the endogenously produced βgbp in a surveillance role ( mallucci et al ., 2003 ). thirdly , while the extent of mutation multiplicity , high in breast cancer ( sjoblom et al ., 2006 ), may require multiple drug combinations with implicit synergistic toxicity , our findings indicate that the disabling by βgbp , a physiological effector molecule , of one single gene , akt , can bypass mutational complexity and chemotherapeutic disadvantages . thus reducing the expression of the akt gene , possibly using alternative agents other than βgbp , is a new therapeutic route for the treatment of cancer , in particular , breast cancer . fourthly , colon cancer cells are particularly susceptible to apoptosis induced by βgbp . thus colon cancer is a particularly suitable target for treatment with pgbp . finally , while providing a model for the clinical use of βgbp in aggressive cancers , the present invention also provides a mechanistic rationale for the use of βgbp in other conditions in which undesirable cell division occurs , in particular , those where said cell division is characterised by aggressive or hyperproliferative behaviour such as self immune responses ( e . g . autoimmune conditions ) and immune responses against transplanted organs , tissues or cells . cell culture : bt474 cells were cultured in dmem / f12 medium with 10 % fetal calf serum ( fcs ) ( invitrogen , uk ) and 20 μg / ml insulin ( sigma , uk ); the skbr3 cells were grown in dmem medium ( invitrogen uk ) plus 10 % fcs . cultures were incubated at 37 ° c . in a humidified atmosphere of 5 % co2 in air . 1 × 10 5 cells were seeded per 25 cm 2 flask , or 3 × 10 5 per 75 cm 2 flask . sufficient numbers of flasks were seeded in parallel to allow for individual flasks to be consumed in the process of monitoring cell numbers ( i . e . growth rates ), apoptosis , pi3k activity , akt mrna levels , akt protein levels and phosphorylated akt ( p - akt ) protein levels as discussed below . unless otherwise indicated results correspond to one flask of cells . growth rates : cells were removed from the flasks by trypsinisation and counted in a haemocytometer to assess cell numbers at time points of 1 , 2 , 3 , 4 , and 5 days for bt474 cells , skbr3 cells , mcf10a v12ras cells and mcf10a ctx cells . mcf10a cells were monitored at 1 , 2 , 3 , and 4 days . cell numbers were determined in triplicate . βgbp : human recombinant βgbp ( hu - r - βgbp ) was expressed in e . coli bl21 ( de3 ) using hgal - 1 cdna in pet21a ( hirabayashi et al ., 1989 ), purified by lactose - agarose ( sigma , uk ) affinity chromatography and purity assessed by maldi - tof . hu - r - βgbp was added to cell cultures at a concentration of 15 nm or 30 nm at hour 3 after seeding cells . control cells ( 0 nm βgbp ) were also prepared . hu - r - βgbp was maintained at the appropriate concentration ( 0 nm , 15 nm or 30 nm respectively ) throughout the time course of the cell culture . cytofluorimetry : apoptosis was monitored using four criteria as outlined below . control cells ( 0 nm hu - r - βgbp ) were compared to cells cultured with 30 nm hu - r - βgbp at a time point of 3 days for bt474 cells and 4 days for skbr3 cells . cells were washed and released from the flasks using trypsin and resuspended in phosphate buffered saline ( pbs ). tetramethylrhodamine ethyl ester ( tmre ) ( molecular probes / invitrogen , uk ) staining was used to assess loss of mitochondrial membrane potential . cells suspended in pbs were incubated at 37 ° c . for 20 minutes in 40 um tmre , centrifuged , washed in pbs and resuspended in 500 ul of 200 ng / ml 4 ′, 6 - diamidino - 2 - phenylindole dihydrochloride ( dapi ) ( sigma , uk ) in pbs and analysed at 575 and 440 nm by flow cytometry ( facs ). redistribution of plasma membrane phosphatidylserine was assessed using annexin v - fitc ( pharmingen , san diego , calif .). cells suspended in 500 ul 10 mm hepes , ph 7 . 4 , 140 mm nacl , 2 . 5 mm cacl2 containing 2 . 5 ug / ml annexin v - fitc were incubated in the dark at room temperature for 20 minutes , 200 ng / ml dapi added and the cells analysed at 540 nm and 440 nm by flow cytometry ( facs ). caspase 3 activity was measured by cleavage of non fluorescent phiphilux to a fluorescent product ( oncoimmunin , mass .). cells in suspension in 500 μl 30 μm phiphilux in pbs were incubated at 37 ° c . for 60 minutes , washed twice with pbs and resuspended in 300 μl pbs containing 200 ng / ml dapi and analysed at 575 nm and 440 nm . strand break dna fragmentation was analysed by terminal deoxynucleotidyl transferase ( tdt )- mediated dutp nick end labelling ( tunel ) using the apo - brdu kit ( phoenix flow systems , phoenix , ariz .) according to the manufacturers instructions and analysed using a facs calibur ( becton dickinson , san jose , calif .). pi3 kinase assay : competitive elisa was used to assess pi3 kinase activity at time points of 0 , 6 , 12 , 18 , and 24 hours from seeding . 30 nm βgbp was added to cells at 3 hours after seeding . assays were performed in triplicate . 5 × 10 6 cells were washed three times with 137 mm nacl , 20 mm tris hcl ph7 . 4 , 1 mm cacl 2 , 1 mm mgcl 2 , 0 . 1 mm na orthovanadate ( sigma , uk ) ( buffer a ) and lysed in 1 ml of the same buffer supplemented with 1 mm pmsf ( sigma , uk ) and 1 % np40 ( calbiochem , san diego , calif .) for 20 minutes on ice . lysates were centrifuged at 13000 rpm for 10 minutes to remove insoluble material and the supernatants stored at − 80 ° c . frozen lysates containing 600 μg protein were thawed on ice and pi3 kinase was immunoprecipitated by incubation with 5 μl anti - pi3 kinase p85 ( upstate biotechnology , lake placid , n . y .) for one hour at 4 ° c . on a rotating wheel , followed by addition of 60 μl of a 50 % slurry of protein a agarose ( sigma , uk ) beads in pbs for one hour at 4 ° c . the immunoprecipitated enzyme was collected by centrifugation at 13000 rpm for 10 seconds . pellets were washed three times in buffer a plus 1 % np40 , three times in 0 . 1m tris - hcl , ph 7 . 4 , 5 mm licl , 0 . 1 mm na orthovanadate and twice with 10 mm tris - hcl , ph7 . 4 , 150 mm nacl , 5 mm edta , 0 . 1 mm na orthovanadate . pellets resuspended in 110 μl kinase reaction buffer ( 5 mm hepes ph 7 . 0 , 2 . 5 mm mgcl2 , 25 μm atp ) were incubated in a water bath for 3 hours at 37 ° c . with 40 pmol pi ( 4 , 5 ) p2 substrate ( echelon biosciences , salt lake city , utah ). the reaction was stopped with edta at a final concentration of 5 mm and the reaction mixture centrifuged at 13000 rpm at 4 ° c . supernatants were transferred to a microtitre plate for a competitive elisa ( echelon biosciences k - 1000 , salt lake city , utah ) to quantify the pi ( 3 , 4 , 5 ) p3 generated in the kinase reaction . duplicate 50 μl volumes of the supernatants were each incubated with 50 μl of anti pi ( 3 , 4 , 5 ) p3 antibody for 1 hour at room temperature . the reaction mixture was then transferred to a microtitre plate coated with pi ( 3 , 4 , 5 ) p3 and incubated for 1 hour in the dark . after 3 washes with tbs plus 0 . 05 % tween 20 ( sigma , uk ), 100 μl of hrp conjugated antibody to the anti pi ( 3 , 4 , 5 ) p3 was added to each well and incubated for 1 hour at room temperature in the dark . following 3 further washes with tbs plus 0 . 05 % tween 20 , 100 μl of tmb substrate ( echelon biosciences , salt lake city , utah ) was added and the reaction was stopped after an appropriate time (˜ 20 minutes ) with 100 μl 0 . 5m h 2 so 4 . absorbance of the samples was measured at 450 nm and the pi ( 3 , 4 , 5 ) p3 was quantified by comparison with a pi ( 3 , 4 , 5 ) p3 standard curve . readings to derive the standard curve were obtained from an elisa using pip3 standard concentrations on in parallel with the elisa of the experimental samples . the standard curve ( not shown ) was plotted on a log scale . the pi ( 3 , 4 , 5 ) p3 concentrations obtained for the experimental samples were thus available for direct comparison to the pip3 standard curve . the experimental samples included samples from control cells and samples from cells treated with βgbp . northern blot analysis : total rna was extracted from cells using trizol reagent ( invitrogen , uk ) according to the manufacturer &# 39 ; s instructions . 10 μg rna was run on 2 . 2m formaldehyde - 1 . 25 % agarose gels . akt mrna was assessed using a cdna probe ( ha . akt ) which recognizes akt gene 1 , 2 , and 3 . a gapdh cdna ) was used for rna loading control . western blot analysis : cells were lysed in 50 mm tris ph7 . 4 . 150 mm nacl , 1 % tritonx - 100 , 1 mmpmsf , 2 . 5 μg / ml leupeptin , 1 % aprotinin , 10 mm sodium fluoride , 1 mm sodium vanadate ( sigma , uk ). 50 μg of protein were loaded onto 12 % polyacrylamide gels . phosphorylated akt was detected using 1 : 1000 anti - phospho - akt ( ser473 ) antibody ( cell signalling technology , boston , mass .) and total akt1 / 2 protein was probed with 1 : 1000 anti - akt1 / 2 ( h136 ) ( santa cruz , santa cruz , calif .). secondary antibodies conjugated to horseradish peroxidase ( hrp ) ( ge healthcare , uk ) were used at 1 : 1000 dilution and visualised by enhanced chemiluminescence ( ge healthcare , uk ). actin ( sigma , uk ) was used as a loading control . changes in mitogenic input in non - cancerous cells and response to treatment with βgbp cell culture : mcf10a , mcf10a v12ras and mcf10a ctx cells were grown in dmem / f12 medium plus 5 % horse serum ( invitrogen uk ), 10 ug / ml insulin , 5 μg / ml hydrocortisone ( calbiochem , san diego , calif .) and 20 μg / ml epidermal growth factor ( calbiochem , san diego , calif . ), plus 100 ng / ml cholera toxin ( sigma , uk ) in the case of the mcf10a ctx cells . cultures were incubated at 37 ° c . in a humidified atmosphere of 5 % co2 in air . growth rates : cells were removed from the flasks by trypsinisation and counted in a haemocytometer to assess cell numbers at time points of 1 , 2 , 3 , 4 , and 5 days for mcf10a v12ras cells and mcf10a ctx cells . mcf10a cells were monitored at 1 , 2 , 3 , and 4 days . cell numbers were determined in triplicate by counting trypsinised cells in a haemocytometer . βgbp : hu - r - βgbp was obtained as described in example 1 and added to cell cultures at a concentration of 15 nm or 30 nm at hour 3 after seeding cells . control cells ( 0 nm βgbp ) were also prepared . hu - r - βgbp was maintained at the appropriate concentration ( 0 nm , 15 nm or 30 nm respectively ) throughout the time course of the cell culture . cytofluorimetry : apoptosis was monitored by reference to annexin v as described in example 1 . cells were analysed at a time point of 3 days . redistribution of plasma membrane phosphatidylserine was assessed using annexin v - fitc ( pharmingen , san diego , calif .). cells suspended in 500 ul 10 mm hepes , ph 7 . 4 , 140 mm nacl , 2 . 5 mm cacl 2 containing 2 . 5 ug / ml annexin v - fitc were incubated in the dark at room temperature for 20 minutes , 200 ng / ml dapi added and the cells analysed at 540 nm and 440 nm . pi3 kinase assays , northern and western blot analysis were carried out on these cells as already described in example 1 . erk levels as a reflection of endogenous and enforced mitogenic input cell culture : bt474 , skbr3 , mcf10a , mcf10av12ras and mcf10actx cells were cultured as described in examples 2 and 3 . no βgbp was included in the culture medium . cells were harvested at 2 days from seeding , except for skbr3 cells which were harvested at 3 days from seeding . western blot analysis : total levels of erk1 and erk2 , and phosphorylated levels of erk1 and erk2 were assessed by western blotting using the same protocol as described in example 1 . phosphorylated erk1 / 2 were probed with 1 : 1000 anti - phospho - p44 erk 1 and p42 erk 2 monoclonal antibody ( cell signalling technology , boston , mass .). non phosphorylated erk1 / 2 proteins were probed with 1 : 1000 anti - erk2 ( santa cruz , santa cruz , calif .) which recognises both p44 erk 1 and p44 erk 2 . actin ( sigma , uk ) was used as a loading control . response of mcf - 7 cells to raised mitogenic input and to treatment with hu - r - βgbp cell culture : in the following procedures mcf - 7 cells were grown in rpmi medium with 10 % fetal calf serum ( invitrogen , uk ). mcf - 7 ctx cells were grown under the same conditions with the addition of cholera toxin at 100 ng / ml . where required , hu - r - βgbp was obtained as described above example 2 . erk levels : the effect of cholera toxin at 100 ng / ml on erk levels was assessed and compared with control cells cultured in the absence of the cholera toxin after 2 days . western blotting was carried out on mcf - 7 cells harvested after 2 days , cultured in the presence or absence of cholera toxin . western blotting to detect erk1 and erk2 and phosphorylated erk1 and phosphorylated erk2 was carried out as described above in examples 1 and 3 . growth rates : cell numbers were measured over a time course of 4 days . cell numbers were assessed at 0 , 1 , 2 , 3 , 4 days for mcf - 7 cells incubated in the presence or absence of cholera toxin at 100 ng / ml . cell numbers were assessed in triplicate by haemocytometer counting as discussed above in example 1 . northern blot analysis : cellular mrna levels were analysed as discussed above in example 1 . akt mrna levels were assessed after mcf - 7 cells had been cultured with cholera toxin at 100 ng / ml at time points of 1 , 2 and 3 days . gadph mrna was used as a loading control as previously . results were compared with control mcf - 7 cells which had been cultured in the absence of cholera toxin . growth rates in response to βgbp : cell numbers were measured over a time course of 6 days . cell numbers were assessed at 0 , 1 , 2 , 3 , 4 , 5 and 6 days for mcf - 7 cells incubated in the presence or absence of cholera toxin at 100 ng / ml . hu - r - βgbp was added to these cell cultures at a concentration of 20 nm at hour 3 after seeding cells . hu - r - βgbp was maintained throughout the time course of the cell culture . cell numbers were assessed in triplicate by haemocytometer counting as discussed above in example 1 . relationship between down regulation of pi3 kinase activity and akt mrna levels : mcf - 7 cells were cultured as discussed above . cells were treated with 20 nm hu - r - βgbp , 10 nm wortmannin or 20 nm ly294002 added at hour 3 from seeding . pip3 levels and akt mrna levels were measured in these cells at time points of 6 , 12 , 18 and 24 hours from seeding . pip3 values were assessed in triplicate according to the protocol for the pi3 kinase assay described in example 1 . cellular mrna levels were analysed following the northern blotting protocol described above in example 1 . female scid mice were housed under standard conditions . mice were divided into four groups of four . at age 6 weeks 5 × 10 6 st4 ( human malignant t cell leukaemia ) cells were injected subcutaneously ( day 0 ). βgbp was administered from day 1 to day 16 by individual daily subcutaneous injection . each group of mice received a different dosage of βgbp . 0 ng / mouse ( control ), 20 ng / mouse , 200 ng / mouse or 400 ng / mouse tumour size was assessed at day 42 or day 90 by calliper measurement . cell culture : sw480 , sw620 and lovo colon cancer cells were grown in leibowitz l - 15 medium with 10 % fetal bovine serum in sealed flasks at 37 ° c . hu - r - βgbp was added 3 hrs after seeding cells at a concentration of 1 nm or 2 nm . control cells were grown without βgbp . growth rates : cell numbers were measured over a time course of 5 days . cell numbers were assessed at 0 , 1 , 2 , 3 , 4 , and 5 days for each cell type incubated in the presence of hu - r - βgbp . cell numbers were assessed in triplicate by counting in a haemocytometer as discussed above in example 1 and compared with cell numbers for control cells grown without βgbp . alberts b , johnson a , raff m , roberts k , walter p ( 2002 ) in molecular biology of the cell , ( garland science , new york ), pp 845 - 856 . aurias a , rimbaut c , buffe d , zucker j m , mazabraud a ( 1984 ) cancer genet cytogenet 12 : 21 - 25 . baldini a , gress , t , patel k , muresu r , chiariotti l , williamson p , boyd l , casciano 1 , wells v , bruni c et al . 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