various studies have investigated the role of human papillomavirus ( hpv ) in oral squamous cells carcinoma ( oscc ) but the results are highly controversial . most reports have shown that hpv 16 is the most prevalent type in both oral and genital tumors1 . abnormalities in several components of the cell - cycle regulatory machinery have been observed in oral cancers as well as in other types of cancer . protein p21 is a universal inhibitor of cyclin - dependent kinases ( cdks ) that mediates cell cycle arrest in the g1 and g2/m phase to prevent dna replication in cells in which this molecule is damaged . when cells are stimulated by growth factors , this protein is inactivated by phosphorylation and cells progress from the g1 to the s phase . the hyperphosphorylated form of prb releases e2f transcription factors which , in turn , activate the transcription of various target genes17 . in view of the importance of hpv in carcinogenesis , the objective of this study was to investigate the presence of hpv dna and the most frequent viral types in patients with oscc , and to compare the immunohistochemical expression of the cell - cycle proteins p21 and prb between hpv - positive and hpv - negative cases in order to establish a possible correlation between the expression of these proteins and hpv infection in oscc . thirty - three cases of oscc were retrieved from the 1996 - 2004 pathology service archives of the department of dentistry of the federal university of rio grande do norte and hospital dr . eligible cases were males or females aged 30 years or older ( mean age= 65.45 years ; range= 30 - 93 years ) with newly diagnosed , histologically confirmed oscc and not submitted to any kind of therapy ( chemotherapy , radiotherapy or surgery ) . topographic locations included malignancies of the tongue , gingiva , floor of mouth , palate , buccal mucosa , retromolar area and lip . the presence of hpv dna and immunohistochemical expression of proteins p21 and prb were studied in specimens fixed in formalin and embedded in paraffin . ten 10-m histological sections were used for the study of hpv , two 3-m sections were stained with hematoxylin and eosin for histopathological analysis , and two 3-m sections were submitted to immunohistochemistry . this method was developed by the research team of the laboratory of molecular biology of the dental school of the university of so paulo ( usp)10 and consisted of the initial removal of paraffin by baths in xylene heated to 65c followed by hydration of the tissues in a decreasing ethanol series ( absolute , 95% , 70% and 50% ) . then , 400 l sterile lysis buffer ( 50 mm nacl , 5 mm tris - hcl , ph 8 , 12.5 mm edta , ph 8 , and 0.25% sds ) and proteinase k at a final concentration of 500 g / ml were added to the tissue pellet of each sample and the samples were incubated at 55c for 3 to 5 days until complete dissolution of the material . next , 200 l of a 4 m ammonium acetate solution were added to each sample for protein precipitation , followed by 600 l 100% isopropanol for dna precipitation . finally , the dna pellets obtained were washed with 70% ethanol , dissolved in 50 l te buffer and stored at -20c . the samples were submitted to polymerase chain reaction ( pcr ) to evaluate the efficacy of dna extraction . for this , a pair of primers designated pco3 + ( 5'cttctgacacaactgtgttcactagc3 ' ) and pco4 + ( 5'tcaccgcaac ttcatccacgttcacc3 ' ) was used which flank a 110-bp sequence of the human b - globin gene present in all human cells12 . the reaction was carried out in an eppendorf thermocycler ( eppendorf , netherler , hamburg , germany ) . samples positive for the -globin gene were analyzed by pcr regarding the presence of hpv dna using a pair of generic primers called gp5 + ( 5'tttgttactgtggtagatactac3 ' ) and gp6 + ( 5'gaaaaata aactgtaaatcat attc3')11 , which flank a fragment of about 140 bp of the l1 gene , a highly conserved sequence in the genome of mucosal ( genital and oral ) hpvs . the use of this primer pair permits the detection and amplification of this dna segment from at least 23 individual mucosal ( genital and oral ) hpv types , including high risk hpv types . the reaction mixture contained 1.0 m gp5+/gp6 + ( invitrogen , life technologies , carlsbad , california ) , 1.0 u taq dna polymerase ( biosystems , new jersey , usa ) , 20 mm tris - hcl , ph 8.4 , 50 mm kcl , 1.5 mm mgcl2 , 200 m dntp ( amersham bioscience , bucknghamshire , uk ) , and 0.7 to 7.0 l dna , in a final volume of 50 l . the pcr conditions for detection of hpv were : initial denaturation at 95c for 5 min , followed by 40 cycles of amplification at 95c for 1 min , 45c for 2 min and 72c for 1.5 min , and a final extension step at 72c for 10 min . each dot blot membrane included several negative and positive controls for hpv types , as well as pcr products from patients and controls . the membrane was hybridized overnight as 56c in 2x standard saline citrate ( ssc ) , 0.5% sodium dodecylsulfatte ( sds ) and 200 g / ml dna with 19 hpv types probes ( 6,11 , 16 , 18 , 31 , 33 , 34 , 35 , 39 , 40 , 42 , 43 , 44 , 45 , 51 , 52 , 54 , 56 , 58 ) , most common in mucosal ( genital and oral ) , and label [ p]datp . after hybridization , the unbound probe was of 2x ssc and 0.5% sds at room temperature and two 10-min washes of the same solution at 56 c. the membrane was exposed to x - ray film at -20 c for 24 h. for immunohistochemical analysis , the sections were dewaxed in xylene , rehydrated in decreasing ethanol series , and rinsed in water . in order to inhibit endogenous peroxidade activity , the sections were treated by immersion in 5 changes of 0.3% h2o2 in absolute methanol ( 5 min each change ) and rinsing in water . immunohistochemical analysis was performed using the streptavidin - biotin technique ( streptavidin - biotin complex , sabc ; dako , carpinteria , usa ) . the reaction was incubated with monoclonal antibodies : rb-1 ( dako ) at a dilution 1:50 for 60 min , antigen retrieval by microwaves , 0.1 m ph 10 tris - edta for 10 min ; waf1 ( dako ) at a dilution 1:50 for 20 min , antigen retrieval by steamer , ph 8 edta . careful rinses were performed with several changes of pbs between each stage of the procedure . louis , mo , usa ) , then counterstained lightly with mayer 's hematoxylin , dehydrated in a graded ethanol series , allowed to dry in air and mounted with permount mounting medium ( fisher scientific company , pittsburgh , pa , usa ) . for counting of labeled cells , representative areas of the tumor where staining was constant were selected . p21- and prb - positive cells were counted under a light microscope using a counting grid in an area of 0.25 mm at a final magnification of 1,000 to avoid recounting of the same histological fields . each nucleus was counted as one unit and the number of positive cells for each case was obtained by the sum of labeled nuclei . a sample was considered to be positive for prb and p21 when at least 10% of the cells were stained . the results were analyzed statistically by the student 's t - test , mann - whitney u - test and chi - square test , with the level of significance set at 95% (  = 0.05 ) . the research project was approved by the research ethics committee of the federal university of rio grande do norte ( process # 68/03 ) . of the 33 samples that were positive for the human -globin gene ( figure 1a ) , hpv 16 and/or hpv 18 were detected in 11 ( 33.33% ) ( figure 1b ) . hpv 18 was identified in 9 ( 81.81% ) cases and hpv 16 and 18 were simultaneously present in 2 ( 18.19% ) cases . prb was expressed in 9/11 ( 81.82% ) hpv - positive tumors and in 15/22 ( 68.19% ) hpv - negative cases ( figure 2a ) . expression of p21 was observed in 5/11 ( 45.45% ) hpv - positive tumors and in 7/22 ( 31.82% ) hpv - negative tumors ( figure 2b ) . statistical analysis revealed a significant association between the presence of hpv and immunohistochemical expression of prb ( p=0.044 ) . with respect to the expression of protein p21 , no significant association was observed between the expression of this protein and the presence of the virus ( p=0.416 ) . this study investigated paraffin - embedded oscc specimens for the presence of hpv by pcr . hpv dna was detected in 11 ( 33.33% ) of the 33 oscc cases evaluated . however , it is extremely difficult to compare the results of different reports on hpv in the oral mucosa because of variations in parameters , such as type of sample ( tissue extracted by biopsy , scraping ) , preparation method ( fresh , frozen or fixed ) , ethnic difference between patients , geographic differences , and sensitivity of the methods used . iftner , et al.7 have also reported that differences in dna extraction procedures and in transport and storage of the clinical samples , and especially the use of different dna polymerases for pcr , may affect the performance of the assays . the most widely used primers are the generic primer pairs gp5+/gp6 + and my09/my11 , which are able to amplify various virus types . in the present study , we used the gp5+/gp6 + primer pair because , in addition to detecting a variety of virus types , it amplifies a small fragment ( 140 bp ) . this fact is important when evaluating archived paraffin - embedded tissue , as done in the present study , which may show marked dna fragmentation in some cases . in the present study , only hpv 16 and hpv 18 were detected , which is in agreement with the findings of previous studies3,5,8,10,20 . hpv 18 was present alone in most cases ( 9/11 , 81.81% ) and was detected together with hpv 16 in 2 ( 18.19% ) of the 11 cases . these results agree with those reported by other investigators who detected hpv 18 in most of their samples3,20 . however , hpv 16 was the most frequent type in other studies2,57 . in view of the complexity of the biological behavior of malignant tumors , knowledge of the abnormalities that compromise the study of regulatory proteins , such as p21 and prb , may help identifying malignant tumors in amore accurate manner . in the present study , expression of p21 was detected in 5 ( 45% ) of the 11 hpv - positive tumors and in 7 ( 31.8% ) of the 22 hpv - negative tumors , with no significant difference between groups . these findings suggest that the presence of hpv is not sufficient to evaluate the relationship between viral proteins and cell - cycle regulatory proteins such as p21 . another important cell - cycle regulatory protein is prb , which also shows an important relationship with hpv . viral oncoprotein e7 plays a relevant role in tumor progression by binding to protein prb , thus releasing transcription factor e2f which , in turn , activates the transcription of genes that promote cell - cycle progression and cell proliferation as cited earlier4 . in the present study , a significant difference in the immunohistochemical expression of prb was observed between hpv - positive ( 9/11 , 81.8% ) and hpv - negative tumors ( 15/22 , 68.2% ) . lim , et al.7 examined the alterations of molecules within the prb pathway by looking at the presence of homozygous deletions in p16(ink4a ) and the expression patterns of prb , cyclin d1 and cdk4 , as well as the presence of hpv . prb overexpression was found in 20/20 samples , the expression was mainly observed in all layers of the epithelia , particularly in the basal layer where cells are actively dividing , and aberrant prb expression was found in 12/20 samples . strikingly , hpv was present in all samples , suggesting that it plays a significant role in driving oral carcinogenesis . notably , 17/20 of the samples showed more than one alteration in the prb pathway . however , lim , et al.7 did not find any significant relationship between the presence of hpv , homozygous deletion of p16(ink4a ) and overexpression of prb , cyclin d1 and cdk4 . taken together , these data demonstrate that alterations in the prb pathway are a common event and involve the aberration of more than one molecule within the pathway . furthermore , the presence of hpv in all samples suggests that hpv infection may play an important role in oral carcinogenesis . nevertheless , nemes , et al.9 found no significant difference in the immunohistochemical expression of prb when comparing carcinoma groups with and without hpv . 19 , evaluating cases of colorectal cancer , observed that the increase in prb levels was associated with the hyperphosphorylated form of the protein ( inactive form ) . however , xu , et al.18 stated that antibodies against prb label both the phosphorylated and non - phosphorylated form of the protein . the present results regarding the immunohistochemical expression of prb suggest a higher possibility of hpv infection in oscc cases presenting higher prb expression , and that binding of hpv protein e7 to prb is likely to induce the accumulation of the latter , thus leading to its inactive form . in the present study , hpv dna was detected in a low percentage of oscc cases ( 33.33% ) , suggesting that hpv plays a key role in the development and progression of only a small subgroup of these carcinomas . hpv 18 was the most prevalent type , suggesting that this viral type was specifically related to the development of oscc in the studied sample . the immunohistochemical expression of protein prb was significantly higher in hpv - positive oscc than in hpv - negative tumors , this finding indicating an important relationship between the virus and prb in these tumors . no difference in the immunohistochemical expression of p21 was observed between hpv - positive and hpv - negative oscc cases . it is likely that hpv infection and immunohistochemical expression of p21 represent independent factors in these tumors .
this study investigated the presence of human papillomavirus ( hpv ) dna and viral types in 33 cases of oral squamous cells carcinoma ( oscc ) and compared the immunohistochemical expression of the cell - cycle markers p21 and prb between cases of the disease with and without hpv . 
 dna was extracted from paraffin - embedded tissue and amplified by pcr for the detection of hpv dna . 
 viral typing was performed by dot blot hybridization . 
 immunohistochemistry was performed by the streptavidin - biotin technique . 
 hpv dna was detected in 11 ( 33.33% ) of the 33 cases . 
 the prevalent viral type was hpv 18 ( 81.81% ) . 
 a significant association was observed between the presence of hpv and immunohistochemical expression of prb , but not between p21 expression and the presence of the virus . 
 the low frequency of detection of hpv dna in oscc suggests a possible participation of the virus in the development and progression of only a subgroup of these tumors .