purine nucleoside analogs are effective in the treatment of some b - lymphoproliferative disorders ( b - lpds ) .
fludarabine ( fdamp , 9--d - arabinofuranosyl-2-fluoroadenine 5-monophosphate ) induces cell cycle arrest and apoptosis   and is used to treat patients with chronic lymphocytic leukemia ( cll ) and indolent non - hodgkins lymphoma ( nhl ) [ 24 ] .
cladribine ( cda , 2-chloro-2-deoxyadenosine ) is effective against cll , low - grade nhl , and hairy cell leukemia ( hcl ) .
fdamp is administered as the nucleoside 5-monophosphate derivative and is hydrolyzed outside the cell to fludarabine nucleoside ( fda ) then transported and phosphorylated inside the cell ( fda  fdamp  fdadp  fdatp ) .
cda is administered as the nucleoside derivative and converted to the triphosphate ( cdatp ) .
fdatp and cdatp both inhibit ribonucleotide reductase , and at higher concentrations they inhibit dna and rna synthesis [ 3 , 6 ] and induce apoptosis .
the main side - effect of these purine analogs is myelosuppression leading to opportunistic infections [ 8 , 9 ] .  
cell surface molecules are classified as cluster of differentiation ( cd ) antigens ( human cell differentiation molecules - hcdms , http://www.hlda8.org/ ) and have a variety of functions such as cytokine and growth factor receptors , transporters , ion channels , cell - cell interactions , and adhesion molecules .
for example , antibodies against cd20 ( rituximab , mabthera ) , cd52 ( alemtuzumab , campath-1h ) , and cd80 ( galiximab ) are effective for treatment of some b - lpds such as cll and nhl [ 11 , 12 ] .
immunotoxins such as bl22 directed against cd22 may be useful in the treatment of cll , nhl and hcl .
radioimmunoconjugates , such as i - tositumomab ( bexxar ) and y - ibritumomab tiuxetan ( zevalin ) , directed against cd20 are effective for treatment of nhl .  
recent clinical trials support combination of a purine nucleoside analog with a therapeutic antibody for treatment of cll and nhl .
combination therapy with rituximab and fdamp prolongs progression - free survival of patients with previously untreated cll .
fdamp , cyclophosphamide , and rituximab ( fcr ) produce a high rate of complete remissions in untreated cll .
antibody microarray technology is a powerful tool in proteomics , target discovery , and differential analysis .
a cd antibody microarray ( dotscan ) has been developed in our laboratory that enables extensive surface profiles of leukocytes to be obtained in a single , simple assay .
dotscan has been used to determine immunophenotypic changes on human hl60 leukemia cells during differentiation [ 19 , 20 ] . in the present work ,
we have used the microarray to identify cd antigens whose levels change on the human b - lymphoid cell lines mec2 ( derived from cll ) and raji ( from burkitt 's lymphoma ) treated with cda or fda , with subsequent quantitative analysis of those antigens by flow cytometry .
the results show that cda ( 1  m , 24 hours ) changes the surface protein profiles of mec2 and raji cells and fda ( 1  m , 24 hours ) changes the profile of raji but not mec2
. up - regulation of particular cd antigens induced on a b - lpd by a purine analog could provide targets for combined treatment with purine analogs and therapeutic antibodies , directing cytotoxic activity specifically against the neoplastic cells .
rpmi 1640 medium ( hepes modification ) , fda , and trypan blue were purchased from sigma aldrich ( castle hill , australia ) .
cda was a kind of gift from the national cancer institute ( bethesda , usa ) .
gentamicin ( 50  mg gentamicin sulfate / ml ) , l - glutamine ( 200  mm ) , and fetal calf serum were from invitrogen ( mulgrave , australia ) .
the annexin v - pe apoptosis detection kit i was from bd pharmingen ( north ryde , australia ) .
mec2 cells ( derived from a b - cell cll ) were a kind of gift from dr .
raji cells ( from a b - cell burkitt 's lymphoma ) were from the american type culture collection ( manassas , usa ; ) .
mec2 and raji cells were grown in hepes - buffered rpmi 1640 medium with 10% ( v / v ) fetal calf serum , 50  g / ml gentamicin , and 2  mm l - glutamine at 37c in a nonhumidified atmosphere without co2 .  
cultures of mec2 and raji at a density of 3  10  cells / ml were treated with cda or fda ( 1  m , 24 hours ) and harvested for surface profiling of cd antigens .
externalization of phosphatidylserine ( ps ) on cells during apoptosis was quantified by annexin v - pe binding , while the loss of cell membrane integrity indicating dead cells was demonstrated by binding of 7-amino - actinomycin d ( 7-aad ) to dna .
briefly , cells were washed twice with pbs and resuspended at a density of 10  cells / ml in binding buffer ( 10  mm hepes ph 7.4 , 140  mm nacl , and 2.5  mm cacl2 ) .
an aliquot of the suspension ( 50  l , 0.5  10  cells ) was incubated with 2.5  l of annexin v - pe and 2.5  l of 7-aad for 15 minutes at room temperature .
cells were then resuspended in 450  l of binding buffer and analyzed with an facscan flow cytometer ( becton dickinson , san jose , usa ) using cellquest software .
capture of cells on 82-dot cd antibody microarrays , imaging , and data analysis have been described .
dot patterns of cells captured on cd antibody microarrays were imaged and analyzed using a scanner and software from medsaic .
experiments to determine the effects of cda or fda ( 1  m , 24 hours ) on the immunophenotypes of cells were performed in triplicate .
for each cell line , three cell cultures were treated with cda or fda dissolved in dimethylformamide , while the three control cultures were treated with the corresponding amount of dimethylformamide .
cd antigens whose levels changed on raji and mec2 cells using dotscan were also analyzed quantitatively by flow cytometry using fitc- or pe - conjugated monoclonal antibodies and standard procedures .
fluorescently - labelled monoclonal antibodies were obtained from becton - dickinson pharmingen ( north ryde , australia ) , imm , immunotech , beckman coulter ( gladesville , australia ) , and caltag , invitrogen ( mount waverley , australia ) .
samples were analyzed using an facscalibur flow cytometer ( becton dickinson , san jose , usa ) with a 488-nm air - cooled argon - ion laser , running cellquest pro software .
fluorescence detection used logarithmic amplification ( fl1 ( green ) = 530/30  nm ; fl2 ( red ) = 585/40  nm ) .
quantitative data are the averages of 3 determinations with variance expressed as p - values .
cda ( 1  m ) arrested growth of raji and mec2 cells , and fda ( 1  m ) arrested raji cells but not mec2 cells ( data not shown ) .
apoptotic and dead cells were quantified using annexin v - pe and 7-aad with flow cytometry ( figure 1 ) .
cda ( 1  m , 24 hours ) induced for raji : apoptosis ( control 8.4% , + cda 36.3% ) and cell death ( control 9.5% , + cda 12.3% ) , for mec2 cells : apoptosis ( control 7.5% , + cda 23% ) and cell death ( control 3.4% , + cda 6.7% ) .
fda ( 1  m , 24 hours ) induced for raji : apoptosis ( control 8.4% , + fda 21.2% ) and cell death ( control 9.5% , + fda 9.8% ) .
fda ( 1  m , 24 hours ) had no significant effect on mec2 : apoptosis ( control 7.5% , + fda 10% ) and cell death ( control 3.4% , + fda 4.1% ) .  
cd antibody microarrays were used to screen for immunophenotypic changes on raji and mec2 cells induced by cda or fda ( 1  m , 24 hours ) .
the expression profiles in figure 2 show mean cell binding densities from analysis of control and drug - treated cultures ( n = 3 ) .
for raji cells , cda ( 1  m , 24 hours ) increased levels of cd54 , cd80 , cd86 , and cd95 , while cd22 was repressed ( figure 2(a ) ) .
fda ( 1  m , 24 hours ) increased levels of cd20 ( rituximab ) , cd54 , cd80 , cd86 , and cd95 ( figure 2(b ) ) .
although an increase in cd20 was observed with two other clones of cd20 antibody ( b1 , b9e9 ) , these changes were not significant ( p > .05 ) . for mec2 cells ,
cda increased cd20 ( b9e9 ) , cd45 , cd62l , cd80 , cd86 , cd95 , while cd54 was repressed ( figure 2(c ) ) .
the changes for mec2 with cda were consistent , but only cd86 up - regulation was significant ( p < .05 ) .
flow cytometry was used to confirm and quantify levels of cd antigens .   for confirmation , mec2 and
raji cells were treated with cda or fda ( 1  m , 24 hours ) , and cd antigens of interest were analyzed by flow cytometry .
cellquest pro software ( becton dickinson , san jose , usa ) was used to overlay the fluorescence histograms and obtain the median fluorescence intensities for calculation of the fold - changes of cd antigen expression ( drug - treated)/(control ) .  
figure 3 shows overlays of fluorescence histograms of control ( grey ) and drug - treated ( black outline ) samples .
these results confirm that cda ( 1  m , 24 hours ) induced increases on raji cells of cd54 , cd80 , cd86 , and cd95 , and cd22 was decreased ( figure 3(a ) ) . in addition , increased cd10 was detected on raji cells treated with cda .
raji cells treated with fda ( 1  m , 24 hours ) showed increased cd20 , cd54 , cd80 , cd86 , and cd95 ( figure 3(b ) ) .
mec2 cells treated with cda ( 1 m , 24 hours ) showed increased cd20 , cd45 , cd62l , cd80 , cd86 , and cd95 . increased cd11a , cd43 , cd52 , and cd54 were also detected by flow cytometry but not microarrays .
no changes in cd antigen expression were observed for mec2 cells treated with fda ( data not shown ) .  
table 1 summarizes the flow cytometric data for raji and mec2 cells treated with these purine analogs ( figure 3 ) .
recent studies indicate that purine analogs administered in combination with other drugs and/or therapeutic antibodies ( rituximab and alemtuzumab ) may produce higher response rates including complete remissions .
proteomic analysis of raji cells treated with fda using two - dimensional gel electrophoresis and mass spectrometry has identified new nuclear regulatory proteins that may participate in fda - induced apoptosis .
analysis of cytosolic and mitochondrial proteins has identified 25 proteins that differ by more than 2-fold between control cultures and purine analog - treated raji cells ( mactier , henrich , and christopherson , manuscript submitted ) .
these proteins provide further insight into the mechanisms of action of purine analogs and may represent new therapeutic targets .
antibody microarray - based technology is a promising tool in the field of proteomics , in which one of the important applications is to compare proteomic profiles of untreated and drug - treated cancer cells to identify potential drug targets as well as further elucidate mechanisms of drug treatment . in the present study ,
dotscan antibody microarrays were used to screen for changes induced by purine analogs on the surface of leukemia and lymphoma cell lines .
cda and fda induced up - regulation of a number of cd antigens on mec2 and raji cells that could be targets for synergistic treatment with therapeutic antibodies .  
cd10 , a neutral endopeptidase and integral membrane protein whose expression correlates with apoptosis [ 26 , 27 ] , was induced 2.4-fold on raji cells treated with cda ( table 1 ) .
cd54 ( intercellular adhesion molecule-1 , icam-1 ) , a member of the immunoglobulin superfamily of adhesion molecules , was up - regulated 1.4- to 1.8-fold on raji cells treated with cda or fda and on mec2 cells treated with cda ( table 1 ) .
cd11a ( lymphocyte function - associated antigen 1 , lfa-1 integrin ) increased 1.4-fold on mec2 cells treated with cda ( table 1 ) .
when t cells encounter antigen - presenting cells ( apcs ) , cell - cell contact is established between lfa-1 ( cd11a ) and icam-1 ( cd54 ) .
cd20 was induced 1.6-fold on mec2 cells treated with cda and 1.5-fold on raji cells treated with fda ( table 1 ) .
cd20 , a b - cell antigen , is a calcium channel that regulates cell - cycle progression and proliferation .
cd20 is expressed at lower levels on b - cll compared with normal b - cells and other b - lpd .
van meerten et al .   showed that increased cd20 enhances antibody - dependent cellular cytotoxicity ( adcc ) and complement dependent cytotoxicity ( cdc ) .
combination therapy using cda or fdamp with rituximab is clinically synergistic against cll and lymphomas [ 18 , 30 , 31 ] .
the up - regulation of cd20 observed ( table 1 ) may provide an explanation for this synergy .  
cd22 , a regulator of the b - cell receptor ( bcr ) , was repressed 0.51-fold on raji cells treated with cda ( table 1 ) .
cd22 is a b - cell - specific sialoglycoprotein of the immunoglobulin superfamily commonly expressed on nhl   involved with cellular adhesion and homing and regulation of b - cell activation .
monoclonal antibodies against cd22 that block ligand binding inhibit normal and malignant b - cell survival .
repression of cd22 on raji cells by cda may reduce cd22 signaling , decrease proliferation , and promote apoptosis .
cd43 was up - regulated 1.5-fold on mec2 cells treated with cda ( table 1 ) .
overexpression of cd43 induces accumulation of p53 , while monoclonal antibodies binding to cd43 may induce apoptosis [ 3638 ] .  
alemtuzumab ( campath-1h ) , a therapeutic antibody against cd52 , induces cdc and adcc and is effective for treatment of cll .
it was shown that alemtuzumab in combination with a purine nucleoside analog may be effective for treatment of refractory cll .
cd62l ( l - selectin , lecam-1 ) is up - regulated 2-fold on mec2 cells treated with cda ( table 1 ) .
the expression of adhesion molecules may alter the mobility and homing of cll cells and the immune response to cll .
the level of cd62l on b - cll is usually less than half that of normal lymphocytes , possibly contributing to accumulation of b - cll in peripheral blood .  
cd80 and cd86 are up - regulated 1.4- to 2.5-fold on raji and mec2 cells treated with cda or fda ( table 1 ) .
these coregulators of t cell activation induce proliferation , cytokine production , and effector functions .
up - regulation of cd80 and cd86 may enhance a t cell response to cll and lymphoma .
cd80 may also play a role in regulation of normal and malignant b - cells   and may be constitutively expressed on nhl , making it an attractive target for lymphoma therapy .
galiximab is a therapeutic antibody directed against cd80 that has been used to treat nhl [ 11 , 43 ] .
cd95 is up - regulated 1.2- to 1.5-fold on raji and mec2 cells treated with cda or fda ( table 1 ) .
the fas ( cd95)/fas ligand ( cd178 ) system has a potential role in cytotoxic killing of some leukemias .
cda - induced apoptosis may be triggered by the expression of fas and fas - l , and fda induces fas and fas - l mrnas .  
the enhanced expression of cd95 on cll cells treated with cda or fda could promote cell killing .   while cda and fda are structurally similar , there are some differences in their effects . in this study
, cda induced immunophenotypic changes and apoptosis in the cll - derived cell line mec2 that is resistant to fda .
a recent clinical trial showed prolongation of therapeutic response with cda compared to fdamp   suggesting that cda should be further investigated for treatment of cll .
surface molecules up - regulated by a purine analog could be targeted with a therapeutic antibody providing synergy and more effective treatment .
cda and fda upregulate cd antigens on raji and mec2 cells that are targets for therapeutic antibodies such as rituximab ( anti - cd20 ) , alemtuzumab ( anti - cd52 ) , and galiximab ( anti - cd80 ) .
cd10 , cd11a , cd43 , cd54 , cd62l , and cd86 , also up - regulated by fda or cda ( figures 2 and 3 , table 1 ) , are potential targets for development of therapeutic antibodies , for drug - antibody combination therapy . preliminary experiments with primary cells from cll patients showed that cda or fda ( 1  m , 48 hours ) induces increases in cd20 , cd54 , and cd95 ( not shown ) .   in summary ,
microarray analysis of raji cells treated with purine analogs revealed significant changes ( p < .05 ) in expression levels of several cell surface antigens , that were confirmed by flow cytometry ( figures 2 and 3 , table 1 ) . flow cytometry also revealed significant cda - induced increases in cell surface antigens on mec2 cells ( p < .05 , figure 3 , table 1 ) , but not all of these changes were significant by microarray although the same antibody clones were used .
the differences between microarray and flow cytometry results for cda - treated mec2 cells are not fully understood .
flow cytometry uses soluble fluorescent antibodies , which have greater flexibility and freedom of movement than antibodies immobilized on a nitrocellulose surface and therefore have easier access to cell surface antigens , with fluorescence intensity directly reflecting antigen expression levels .
cell viability is not essential for flow cytometry , while cell capture on antibody dots depends not only on the interaction of cell surface antigens with immobilized antibodies but also on energy - dependent processes required for robust attachment of antibody - bound cells to the nitrocellulose surface ; damaged or dead cells bind weakly and are easily washed off during washing and fixation steps .
the tendency of mec2 cells to cluster rapidly in suspension may , at least in part , account for greater variability in replicate dot intensities for mec2 compared to raji ( figure 2(c ) versus 2(a ) ) , because cell clusters are less firmly bound to antibody dots than single cells due to reduced interaction of clustered cells with the immobilized antibodies .  
dotscan microarrays provide a powerful screening tool for the detection of drug - induced modulations of cell surface antigens and could be used to screen patient samples to identify other potential synergistic drug - antibody combinations for cll and other b - lpds .
microarray - based proteomic analysis should also enable rational development of chemoimmunotherapy for less common disease entities and subsets and enable tailored treatment for individual patients .
 the purine analogs , fludarabine nucleoside ( fda ) , and cladribine ( cda ) ( 1  m , 24 hours ) , significantly changed the levels of some surface antigens on the human b - cell lines mec2 and raji . 
 changes in the surface proteins were identified using a cluster of differentiation ( cd ) antibody microarray that captures live cells and confirmed by flow cytometry . for raji cells , cda up - regulated cd10 , cd54 , cd80 , and cd86 , with repression of cd22 , while fda up - regulated cd20 , cd54 , cd80 , cd86 and cd95 . for mec2 cells , cda up - regulated cd11a , cd20 , cd43 , cd45 , cd52 , cd54 , cd62l , cd80 , cd86 , and cd95 , but fda had no effect . 
 up - regulation of particular cd antigens induced on a b - cell lymphoproliferative disorder by a purine analog could provide targets for therapeutic antibodies with synergistic cell killing . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion