cyclin - dependent kinase inhibitors have the potential to induce cell cycle arrest and apoptosis in cancer cells .
roscovitine , a potent and selective inhibitor of cdk2 and cdc2 , has demonstrated selective inhibition of cdk enzymes over related kinases .
it has been reported that roscovitine does cause not only cell cycle arrest , but also apoptosis in cancer cells [ 2 , 3 ] . in in vitro study , it has been shown that roscovitine has cytotoxic activity against a lot of human tumor cells , as well as in tumor xenograft models [ 4 , 5 ] .
roscovitine is currently undergoing phase ii clinical trials as a treatment for nonsmall cell lung cancer and nasopharyngeal cancer [ 6 , 7 ] .   in this study
, we investigated whether roscovitine could inhibit the tumor growth and exhibit cytotoxicity in cervical carcinoma cell lines : c33a , hce-1 , hela , and siha .
in addition , we are interested in elucidating the biochemistry of apoptosis of roscovitine on these cell lines . our data showed that roscovitine can inhibit tumor cell proliferation in dose- and time - dependent manner in cervical carcinoma cells .
roscovitine can induce cell cycle arrest and apoptosis in 4 cervical cells but showed selective sensitivity .
we estimated that cervical carcinoma cells responded differently to roscovitine because of differences in apoptotic and genetic background .
these results also suggest that roscovitine may be a selective and effective chemotherapeutic agent against cervical carcinoma .
c33a , hce-1 , hela , and siha cell lines were purchased from the institute of basic medical sciences chinese academy of medical sciences and shanghai sanqiang analysis company .
dulbecco 's modified eagle medium ( dmem ) , fetal bovine serum ( fbs ) , and trypsin were purchased from hyclone laboratories inc .
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl - tetrazolium bromide ( mtt ) and acridine orange were purchased from amresco company ( usa ) .
protease inhibitor cocktail ( 1% , cat no : 539134 ) was purchased from merck ( usa ) .
a 10  mmol / l roscovitine stock solution of the compound was prepared in dmso and diluted to a different concentration .
four cervical carcinoma cells ( c33a , hce-1 , hela , and siha ) were maintained in rpmi-1640 media containing 10% fetal bovine serum , 2  mmol / l l - glutamine , 100  u / ml penicillin , and 100  g / ml streptomycin .
c33a , hce-1 , hela , and siha cells ( 5  10/well ) were grown in 24-well plates and treated with roscovitine ( 030  mol / l ) or dmso ( 0.3% , final concentration ) for 48 and 72  h. attached cells were released by trypsin and combined with nonadherent cells .
after 0 , 2 , 4 , 6 , 12 , 24 , and 72  h , 20  mol / l of roscovitine was added into the wells .
cell number and cell viability were determined using haemocytometer and trypan blue dye exclusion test .   
c33a , hce-1 , hela , and siha cells were seeded into 96-well plates and incubated overnight at 37c .
roscovitine was added to cells ( in 5 replicates ) and incubated for 72  h at 37c .
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide ( mtt ) was made up as a stock of 2  mg / ml in cell media and filter sterilized .
media were removed from cells , and mtt solution was then added at 50  l per well and incubated in the dark at 37c for 4  h. mtt solution was removed , and mtt dye was solubilised with 50  l / well of dmso with agitation .
measure the absorbance at 562  nm and then determine the ic50 ( concentration of roscovitine which inhibits cell growth by 50% ) .
cervical carcinoma cells in logarithmic phase of 5  10/ml were incubated into 6-well cell culture plate and were cultured at 37c in an incubator with 5% co2 for 24  h. with the culture medium removed , the volume of 2  ml containing 20  mol / l roscovitine was added into each well .
the cells were cultured at 37c in an incubator with 5% co2 for 0 , 6 , 12 , 24 , 48 , and 72  h , respectively , collected , respectively , and then centrifuged at 2000  g for 5  min .
. the media binding reagent with 10  l of annexinv - fitc was added .
the cells were incubated in darkness for 15  min and centrifuged at 3000  g for 5  min , and then the supernatant was removed .
atp is a sign of cell viability , and it exists in all living cells with metabolism .
the luciferase and d - luciferin contained in the atp lite kit react with atp to produce fluorescence .
the reaction formula is as follows:(1)atp+d - luciferin+o2oxyluciferin+amp+ppi+co2+light 
 fluorescence intensity was measured by perckinelmer 2030 multilabel reader .
100  l cells suspension were seeded at 3  10/ml in 96-well culture plate .
after 24  h , the cells were added with 100  l culture medium containing roscovitine 10 , 20 , 30 , 40 , 50 , and 60  mol / l , respectively .
the blank group was added with media , and the control group was added with 0.5% dmso . each group had 5 repeats .
the cells of c33a and hela were cultured for 48  h. temperatures of atp lite buffer , cell lysis solution , and lyophilized substrate solution were balanced to room temperature ( rt ) .
atp lite buffer with the volume of 5  ml was added into the lyophilized substrate solution .
cell lysis solution with the volume of 50  l was added into each well .
the mixture was vibrated at 1000  g for 5  min by microvibration to dissolve the cells and to stabilize atp . the prepared matrix solution with the volume of 50  l
the mixture was vibrated at 1000  g for 5  min and was protected from light for 10  min to measure its fluorescence intensity .
the concentration of c33a and hela cells in logarithmic phase was diluted to around 5  10/ml .
2  ml of the c33a and hela cell suspension was placed into 6-well culture plate and covered by the cover slips .
the cells were cultured at 37c in an incubator with 5% co2 for 24  h. then the culture medium was removed .
20  mol / l roscovitine was added into each well , respectively , with the group of 0.3% dmso as the control group .
the cover slip with growing cells was selected , washed with pbs for 3 times to remove serum , fixed with 95% ethanol for 15  min , treated by 1% acetic acid for 30  s , then stained by 2  10  mol / l acridine orange for 3060  s , processed by 1  mol / l  cacl2 for 30120  s , and washed by pbs for 3 times .
a slide dropped with 200  l glycerol was covered lightly , as the excess liquid was sucked by filter paper .
the picture was taken by confocal microscope at the excitation wavelength of 488  nm and the emission wavelength of 561  nm .  
. a * p value of < 0.05 was considered statistically significant , and * * p value of < 0.01 was considered very significant .
the growth of cells was inhibited in dose - dependent manner after exposure to roscovitine for 48  h and 72  h ranging from 0 to 30  mol / l ( figure 1 ) .
the antiproliferation effect of roscovitine was evaluated by measuring the growth rates and treated with 20  mol / l roscovitine .
treatment with roscovitine caused a time - dependent inhibition of cell growth in accordance with the cell viability assay during 72  h as compared with control ( figure 2 ) .  
the ic50 values of c33a , hce-1 , hela , and siha cells were 22.09  3.29 , 21.21  1.96 , 13.79  3.30 , and 16.88  7.39  mol / l , respectively , as shown in figure 3 .  
we analyzed the cell cycle profiles of growing cervical cell lines exposed to 20  mol / l roscovitine .
in c33a , hce-1 , and siha cell lines , the sub - g1 dna content was remarkable and apparently increased during 2448  h , indicating that apoptosis occurred ( figure 4 ) .
in hela cell lines , we found that the sub - g1 dna content occurred great increase within 12  h. the results indicated that roscovitine induced apoptosis in four cervical carcinoma cells , and hela is the most sensitive to roscovitine . to understand the reason of the cancer cells exhibiting different sensitivity to roscovitine , the most sensitive hela cells and the most resistant c33a cells were used as the representatives to do the atp lite and morphological study .
with atp assay , it was confirmed that roscovitine significantly inhibited the growth of both c33a and hela cells .
the intracellular atpase activity decreased significantly as the concentration of roscovitine increased , as shown in figure 5 .
the inhibition effect of roscovitine in hela cells is higher than that in c33a cell lines in a dose - dependent manner .  
the c33a and hela cells stained with acridine orange fluorescence were observed by confocal microscopy .
their nuclei presented homogeneous fluorescence of green light while in the apoptotic cells , due to the chromatin pyknosis or the broken fragments of unequal size , the apoptotic body was formed .
they were stained with acridine orange and presented the deep and dense fluorescence or the granular fluorescence of green .
c33a and hela cells treated with roscovitine were taken pictures at 0 , 24  h , the apoptosis feature was demonstrated clearly , as shown in figure 6 .
the results indicated that the cells treated with roscovitine showed cell membrane blister , cell shrinkage , and apoptosis .
those indicated that roscovitine induced apoptosis in both two cell lines , and hela cells were more sensitive to roscovitine .
  roscovitine , an olomoucine - related purine analogue derived from 6-dmap and isopentenyladenine that competes with atp for its binding site on cdks , has been developed as a cdk inhibitor [ 8 , 9 ] .
roscovitine treatment induced not only cell cycle arrest , but also apoptosis in various type cell lines [ 4 , 10 , 11 ] . in this study , we showed for the first time that the novel cdk inhibitor roscovitine inhibits cervical tumor cell proliferation in dose- and time - dependent manner and induced apoptosis rapidly in vitro in cervical carcinoma cells by a mechanism that involved apoptosis .  
we found that roscovitine exhibited selective cytotoxicity towards cervical cells , and the cells showed different morphologic and apoptotic changes at the same concentration .
this observation was confirmed by flow cytometry and indicating the apoptotic mechanism of roscovitine . published paper using roscovitine on head and neck squamous carcinoma cells also showed two possible results [ 1214 ] .
the results suggest that cervical carcinoma cells may respond differently to roscovitine because of the mechanism and relationship of apoptosis and cell cycle arresting . from our study
, roscovitine can inhibit tumor cell proliferation in dose - dependent and time - dependent manner and exhibited difference cytotoxicity in different cervical cell lines .
as such , roscovitine will be a selective and effective chemosensitivity drug for cervical carcinoma therapy .
 the antitumor activity of roscovitine was tested in four cervical carcinoma cells : c33a , hce-1 , hela , and siha . 
 the effects of roscovitine on atp lite assay , cell cycle , and apoptosis were assessed . 
 the sub - g1 dna content occurred great increasing , and this indicates that apoptosis was induced quickly in hela cells , but slowly in the other cells . 
 the morphological observation results showed that roscovitine induced apoptosis and cell death in the cervical carcinoma cells . 
 results revealed that roscovitine exhibited selective cytotoxicity towards 4 cervical carcinoma cells , and the cells showed different morphologic and apoptotic changes at the same concentration . it was estimated that cervical carcinoma cells responded differently to roscovitine because of differences in apoptotic and genetic background in different cervical carcinoma cells . 
 this study suggested that roscovitine had the potential to be a chemotherapeutic agent against cervical carcinoma . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion