cervical cancer ( cc ) is the third most common cancer worldwide with 530 000 new cases and 275 000 deaths each year .
it is the sixth most common cancer in women in developed countries and the second most common cancer in underdeveloped countries 1 .
current treatment for cc is suboptimal and the identification of new carcinogenic pathways could provide new therapeutic targets . during the different stages of cc development ,
altered mechanisms of epigenetic regulation in cc include dna methylation , post - translational modifications ( ptms ) of histones , and noncoding rnas . frequently in cc ,
hypermethylation within the promoter region of genes are involved in cell cycle , apoptosis , dna repair , proliferation , cell adhesion , and metabolism leading to transcriptional repression 2 .
dna methylation in the promoter region of genes enables the recruitment of the histone deacetylases ( hdacs ) resulting in gene silencing in cc 3 - 5 .
additionally , it has been reported that histone modifying enzymes like hdac1 and hdac2 are over - expressed in cervical dysplasia and invasive carcinoma 6 , suggesting that the dysregulation of enzymes that modify histones in cc is a mechanism by were gene silencing occurs .
recently an increasing number of publications have reported histone h3 clipping in human cells such as embryonic stem cells ( hescs ) 7 , senescent fibroblasts and melanocytes 8 , peripheral blood mononuclear cells ( pbmcs ) 9 , raji cells 10 , and hepatocytes 11 .
analysis of the proteolytic clipping of histone h3 in the differentiation of hescs evidenced that the n - terminal tail of h3 can be cleaved in different sites and it was mediated by a serine protease 7 .
in addition , proteolytic clipping of histone h3 was performed by a cysteine protease , cathepsin l , in models of oncogene - induced and replicative senescence .
notably , it was suggested that cleaved products of h3 could contribute to regulate genes implicated in cell cycle - promotion , dna replication , cellular proliferation , apoptosis and migration in senescence 8 . since histone h3 clipping only has been identify in a hepatocellular carcinoma cell line 11 and ptms play an important role in cancer development , the aim of this study was to investigate if proteolytic clipping of histone h3 occurs in cc .
in the present work 5 samples of patients diagnosed with cc and 1 patient non - cervical cancer ( ncc ) were used .
ncc patient was negative to hpv presented normal pathology and was submitted to hysterectomy due to diagnosis of uterine myoma .
the protocols were previously approved by the ethics committee of the hospital juarez de mexico ( hjm2231/13-b ) .
after signing the informed consent , a biopsy was taken of each patient and divided into three parts ; the first one for pathological confirmation of at least 80% of tumor cells , the second one for obtaining dna and the remaining part was used to obtain total proteins extracts .
all patients diagnosed with cc were classified according to the international federation of gynecology and obstetrics ( figo ) .
however , the information concerning to figo classification was obtained only of 3 patients and it is included in the table 1 .
hela cells were obtained from the american type culture collection ( atcc ) and cultivated in advanced dmem ( gibco ) supplemented with 8 % fbs ( gibco ) , 100 u / ml penicillin , 100 g / ml streptomycin ( gibco ) and 4 mm l - glutamine .
cells were grown at 37 c in a humidified atmosphere containing 10% co2 . for inhibition experiments ,
cells were treated independently with 0.5 mm of 4-(2-aminoethyl ) benzen esulfonyl fluoride hydrochloride ( aebsf ) , 27 m of trans - epoxysuccinyl - l - leucylamido-(4-guanidino ) butane ( e64 ) or 15 m pepstatin a ( dissolved in 10% ( v / v ) acetic acid in dmso ) ( 9:1 dmso : acetic acid ) for 12 hours .
doses of e64 and pepstatin a inhibitors for hela cells are described in the literature 12 .
immunoblotting assays were performed using total proteins extracts from hela cells , ncc and cc .
all samples were obtained with rsb buffer ( 10 mm tris - cl , ph 7.4 , 10 mm nacl , 3 mm mgcl2 , 1% np40 ) supplemented with 
briefly , samples were dissolved in 2x laemmli - buffer with -mercaptoethanol , heated to 95c for 8 min , and loaded onto a 15% sds - polyacrylamide gel .
proteins were transferred to a pvdf membrane by damp blotting at 40 ma for 3 h. the pvdf membrane was blocked with 5% milk in tbs - t ( 10x tbs - t : 198 mm tris , 1400 mm nacl , 0.01% tween 20 , ph 7.6 ) for 1 h. then the pvdf membrane was incubated overnight at 4c with the primary h3 antibody ( thermo fisher scientific ) or hrp conjugated - gapdh antibody ( cell signaling technology ) .
we use a h3 primary antibody that recognizes the residues of the c - terminal of the human histone h3 .
incubation with hrp - conjugated secondary antibody ( jackson immunoresearch ) was performed by 1 h at room temperature for only h3 .
proteins were visualized using the supersignal west femto maximum sensitivity substrate ( thermo fisher scientific ) and gel doc xr system ( bio - rad ) .
dna extraction from the patient 's tissue was performed following manufactures instructions ( invitrogen thermo fisher scientific ) .
after this , genotyping and copy number determination of hpv was carried out through anyplex ii hpv28 detection kit ( seegene ) following the manufactures instructions .
briefly , in 20 l pcr reaction 1x of hpv28 primer mix a ( 14 high - risk hpv types : 16 , 18 , 31 , 33 , 35 , 39 , 45 , 51 , 52 , 56 , 58 , 59 , 66 , 68 ) or b ( 5 high risk hpv types : 26 , 53 , 69 , 73 , 82 and 9 low risk types : 6 , 11 , 40 , 42 , 43 , 44 , 54 , 61 , 70 ) , 1x anyplex master mix , and approximately 50 ng of dna were added .
the thermocycling conditions was as follow : an initial step at 50 c for 4 minutes and a denaturation step at 95 c for 15 minutes .
the samples underwent 30 cycles at 95 c for 30 seconds , 60 c for 60 seconds and 72 c for 30 seconds .
after this , the reaction mix was followed by a 55 c step for 30 seconds and a melting curve analysis at 55 to 85 c ( 5s/0.5c ) .
the pcr reactions continue cycling at the same temperature for 10 cycles followed by melting curve analysis .
this last step was repeated for another 10 cycles , so the melting curve analysis were obtained at 30 , 40 , and 50 cycles . the pcr reaction were done in a cfx96 real time pcr system ( bio - rad ) as recommended by the manufacturer . as positive controls , three plasmid constructs with all hpv genotypes provided by the kit were used .
this is a semi quantitative test where the results are indicated at 30 cycles ( equivalent of 10 copies ) , 40 cycles ( 10 copies ) or 50 cycles ( 10copies ) .
in the present work 5 samples of patients diagnosed with cc and 1 patient non - cervical cancer ( ncc ) were used .
ncc patient was negative to hpv presented normal pathology and was submitted to hysterectomy due to diagnosis of uterine myoma .
the protocols were previously approved by the ethics committee of the hospital juarez de mexico ( hjm2231/13-b ) .
after signing the informed consent , a biopsy was taken of each patient and divided into three parts ; the first one for pathological confirmation of at least 80% of tumor cells , the second one for obtaining dna and the remaining part was used to obtain total proteins extracts .
all patients diagnosed with cc were classified according to the international federation of gynecology and obstetrics ( figo ) .
however , the information concerning to figo classification was obtained only of 3 patients and it is included in the table 1 .
hela cells were obtained from the american type culture collection ( atcc ) and cultivated in advanced dmem ( gibco ) supplemented with 8 % fbs ( gibco ) , 100 u / ml penicillin , 100 g / ml streptomycin ( gibco ) and 4 mm l - glutamine .
cells were grown at 37 c in a humidified atmosphere containing 10% co2 . for inhibition experiments ,
cells were treated independently with 0.5 mm of 4-(2-aminoethyl ) benzen esulfonyl fluoride hydrochloride ( aebsf ) , 27 m of trans - epoxysuccinyl - l - leucylamido-(4-guanidino ) butane ( e64 ) or 15 m pepstatin a ( dissolved in 10% ( v / v ) acetic acid in dmso ) ( 9:1 dmso : acetic acid ) for 12 hours .
doses of e64 and pepstatin a inhibitors for hela cells are described in the literature 12 .
immunoblotting assays were performed using total proteins extracts from hela cells , ncc and cc .
all samples were obtained with rsb buffer ( 10 mm tris - cl , ph 7.4 , 10 mm nacl , 3 mm mgcl2 , 1% np40 ) supplemented with 
briefly , samples were dissolved in 2x laemmli - buffer with -mercaptoethanol , heated to 95c for 8 min , and loaded onto a 15% sds - polyacrylamide gel .
proteins were transferred to a pvdf membrane by damp blotting at 40 ma for 3 h. the pvdf membrane was blocked with 5% milk in tbs - t ( 10x tbs - t : 198 mm tris , 1400 mm nacl , 0.01% tween 20 , ph 7.6 ) for 1 h. then the pvdf membrane was incubated overnight at 4c with the primary h3 antibody ( thermo fisher scientific ) or hrp conjugated - gapdh antibody ( cell signaling technology ) .
we use a h3 primary antibody that recognizes the residues of the c - terminal of the human histone h3 .
incubation with hrp - conjugated secondary antibody ( jackson immunoresearch ) was performed by 1 h at room temperature for only h3 .
proteins were visualized using the supersignal west femto maximum sensitivity substrate ( thermo fisher scientific ) and gel doc xr system ( bio - rad ) .
dna extraction from the patient 's tissue was performed following manufactures instructions ( invitrogen thermo fisher scientific ) . after this ,
genotyping and copy number determination of hpv was carried out through anyplex ii hpv28 detection kit ( seegene ) following the manufactures instructions .
briefly , in 20 l pcr reaction 1x of hpv28 primer mix a ( 14 high - risk hpv types : 16 , 18 , 31 , 33 , 35 , 39 , 45 , 51 , 52 , 56 , 58 , 59 , 66 , 68 ) or b ( 5 high risk hpv types : 26 , 53 , 69 , 73 , 82 and 9 low risk types : 6 , 11 , 40 , 42 , 43 , 44 , 54 , 61 , 70 ) , 1x anyplex master mix , and approximately 50 ng of dna were added .
the thermocycling conditions was as follow : an initial step at 50 c for 4 minutes and a denaturation step at 95 c for 15 minutes .
the samples underwent 30 cycles at 95 c for 30 seconds , 60 c for 60 seconds and 72 c for 30 seconds .
after this , the reaction mix was followed by a 55 c step for 30 seconds and a melting curve analysis at 55 to 85 c ( 5s/0.5c ) .
the pcr reactions continue cycling at the same temperature for 10 cycles followed by melting curve analysis .
this last step was repeated for another 10 cycles , so the melting curve analysis were obtained at 30 , 40 , and 50 cycles . the pcr reaction were done in a cfx96 real time pcr system ( bio - rad ) as recommended by the manufacturer . as positive controls , three plasmid constructs with all hpv genotypes provided by the kit were used .
this is a semi quantitative test where the results are indicated at 30 cycles ( equivalent of 10 copies ) , 40 cycles ( 10 copies ) or 50 cycles ( 10copies ) .
h3 truncated products generated by cleavage in distinct sites of h3 n - terminal tail has been reported in different cellular types ( fig .
1a ) 7 , 8 ; nevertheless , proteolytic clipping of histone h3 has not been identified in cc .
using hela cells , a cellular line of cc , we identified reduced levels of a cleaved h3 product in comparison to ncc , ( fig .
1b ) , suggesting that histone h3 is lower cleave in cc cells compared with control tissue .
this observation is interesting because a recent study did not observe the cleavage of histone h3 in hela cells 11 .
we used rsb buffer , which contains 10 mm tris - cl ph 7.4 , 10 mm nacl , 3 mm mgcl2 and 1 % np40 supplemented with 
tvardovskiy a , et al 11 used modified ripa buffer , that contain : 50 mm tris - cl ph 8.0 , 150 mm nacl , 1 mm edta , 0.5 mm egta , 0.5 % sodium deoxycholate and 0.5% sds supplemented with 
complete  protease inhibitor cocktail and  phosstop  phosphatase inhibitors . to last , a monoclonal antibody against c - termini of h3 histone ( e.960.2 , thermo fisher scientific ) , was used in the present study compared to a polyclonal antibody ( anti - histone h3 antibody , ab1791 , abcam ) used in tvardovskiy a , et al 11 .
monoclonal antibodies have been documented to possess higher specificity and it may explain why tvardovskiy a , et al 11 did not inmunodetect the cleaved h3 product . additionally , the truncated product of h3 ( 13 kda ) that we observed in hela cells had similar molecular weight as the h3 cleavage product reported in hescs 7 and the proximal cleavage observed in senescent fibroblasts and melanocytes 8 .
on the other hand , the band indicating complete histone h3 was not detected in ncc ( fig .
we also explore the proteolytic cleavage of the histone h3 in samples of patients with cc .
the characteristics of the samples used in this study are indicated in table 1 . in agreement with what we observed in hela cells ,
the western blot analysis showed a decreased in the cleavage product of h3 in cc samples of patients as compared with ncc ( fig .
the intensity of the bands corresponding to complete and cleavage product of h3 , was reduced in samples of adenocarcinoma ( fig.1c ) .
the h3 cleavage could be reduced independently of the cc type ( adenocarcinoma or squamous cell carcinoma ) , genotype or copy number of hpv .
we do recognize that the number of human samples used here are low and the interpretation of this data should take cautiously . to identify the enzymes participating in the histone h3 clipping ,
hela cells were treated with inhibitors of different protease classes as described in material and methods .
western blot indicated that the cleavage of h3 was inhibited when hela cells were pre - treated with aebsf , a serine protease inhibitor , since the lower weight band was not observed . of note
, the intensity of the band indicating complete h3 was also reduced in treated cells ( fig .
consistent with this , it has been suggested that the peptide released during the histone h3 clipping participates regulating the expression of the h3 20 .
similar to aebsf , the pre - treatment with pepstatin a ( pep a ) reduced the levels of h3 cleavage product ( fig
in contrast , pre - treatment with e64 did not inhibit the cleavage of h3 ( fig .
together , these results suggest that the proteolytic clipping of histone h3 occurs in cc and this is exerted by serine and aspartyl proteases . in accordance
, it has been reported that h3 cleavage was performed by a serine protease in hesc 7 . to our knowledge ,
the aspartyl protease activity participating in cleavage of h3 has not been reported heretofore and it may be a peculiarity in cc .
it was previously showed in vitro that human recombinant legumain or purified bovine kidney legumain can cleave recombinant histone h3 , additionally , even though this enzyme is expressed in nucleus of colon cancer cell lines , the proteolytic clipping of endogenous h3 histone has not been reported in these cell lines 13 .
others enzymes participating in this process are cathepsin l and d 8 , 14 , 15 , granzyme a 10 , glutamate dehydrogenase ( gdh ) 16 , prb1 17 , the viral protease 3c 18 , and matrix metalloproteinase 9 ( mmp-9 ) 19 .
we did not investigate if these other enzymes classes are implicated in the cleavage of h3 in cc cells .
histones modifications regulate gene expression and drastic changes in gene expression take place in cc 2 .
thus , it is not surprising that alterations in histone modifications occurs in different types of cancer 20 .
furthermore , different reports account the potential used of histone modifications as biomarkers or as a target for epigenetic therapy 2 , 20 , 21 .
it has been suggested that h3 cleavage in humans regulates different biological process 7 , 8 . in this work
thus , it is valid to speculate that histone h3 clipping may be implicated in the genetic regulation during development of cc .
 chromatin in cervical cancer ( cc ) undergoes chemical and structural changes that alter the expression pattern of genes . recently , 
 a potential mechanism , which regulates gene expression at transcriptional levels is the proteolytic clipping of histone h3 . 
 however , until now this process in cc has not been reported . 
 using hela cells as a model of cc and human samples from patients with cc , we identify that the h3 cleavage was lower in cc compared with control tissue . 
 additionally , the histone h3 clipping was performed by serine and aspartyl proteases in hela cells . 
 these results suggest that histone h3 clipping operates as part of post - translational modification system in cc . 
Introduction
Material and Methods
Patients
Cell culture
Immunoblot analysis
Genotyping and load of HPV
Results and discussion