
 to assess the expression of p53 in lining epithelium of okcto assess the expression of p53 in lining epithelium of pato compare the expression of p53 in lining epithelium of okc and pato assess the expression of pcna in lining epithelium of okcto assess the expression of pcna in lining epithelium of pato compare the expression of pcna in lining epithelium of okc and pa . 
  to assess the expression of p53 in lining epithelium of okc to assess the expression of p53 in lining epithelium of pa to compare the expression of p53 in lining epithelium of okc and pa to assess the expression of pcna in lining epithelium of okc to assess the expression of pcna in lining epithelium of pa to compare the expression of pcna in lining epithelium of okc and pa
the materials for this retrospective study were retrieved from the records ( 1993 - 2002 ) of the department of oral pathology , ragas dental college and hospital , chennai .
twenty cases of odontogenic cysts were selected in which 10 were okc and 10 were pa .
selection was based on review of previously reported slides in which epithelial lining was intact .
the specimen of these cases , which were fixed in 10% neutral buffered formalin , was taken for the study .
three paraffin sections of 4 um were cut ; one was used for routine hematoxylin and eosin stain , while the other two were used for immunohistochemistry .
the slides with tissue sections were treated with two changes of xylene to remove paraffin wax .
they were then put in descending grades of alcohol that helps in dehydration of the tissue .
slides were then treated with 5% hydrogen peroxide for 20 min to quench endogenous peroxidase activity of cells that would otherwise result in nonspecific staining .
then the slides were transferred to citrate buffer and autoclaved for antigen retrieval at 15 lbs pressure for 15 min .
then the slides were dipped in two changes of phosphate buffered saline ( pbs ) for 5 min each .
the slides were then wiped carefully without touching the tissue section with gauze to remove excess pbs . then primary antibody ( dako - do7 )
was added to p tissue on the slides and then to the n pbs and left in the fridge overnight while staining for p53 and while staining for pcna the slides were flooded with pbs and left in fridge .
following day primary antibody pcna was added on p tissue of slides while staining for pcna and on the n section negative control was added and incubated for 45 min .
then the slides were washed in three changes of cold pbs for 5 min in each change .
then the slides were wiped carefully without touching the tissue section with gauze to remove excess pbs .
then , a drop of biotnylated link from the secondary antibody kit ( dako lsab 2 kit ) was added on both the sections on the slides and slides were incubated for 20 min .
then a drop of streptavidin ( dako lsab 2 kit ) was added on both the tissues on the slide and incubated for 20 min .
then a drop of freshly prepared 3-diaminobenzidine ( 3- dab tetra hydrochloride - a substrate chromogen ) was added on both sections .
slides were then washed in running distilled water to remove excess dab and were counter stained with hematoxylin .
the slides were transferred to 70% alcohol , 100% alcohol , and two changes of xylene in a sequential order .
the slides were checked for positive staining on both tissue marked as p and n. if n has taken positive staining , then the slide was discarded and the sections were restrained .
intensity of staining ( mild , moderate on intense staining ) was noted and graded as + , + + and + + + respectively .
mild staining denoting light brown color , moderate - brown color , and intense - dark brown color .
the materials for this retrospective study were retrieved from the records ( 1993 - 2002 ) of the department of oral pathology , ragas dental college and hospital , chennai .
twenty cases of odontogenic cysts were selected in which 10 were okc and 10 were pa .
selection was based on review of previously reported slides in which epithelial lining was intact .
the specimen of these cases , which were fixed in 10% neutral buffered formalin , was taken for the study .
three paraffin sections of 4 um were cut ; one was used for routine hematoxylin and eosin stain , while the other two were used for immunohistochemistry .
the slides with tissue sections were treated with two changes of xylene to remove paraffin wax .
they were then put in descending grades of alcohol that helps in dehydration of the tissue .
slides were then treated with 5% hydrogen peroxide for 20 min to quench endogenous peroxidase activity of cells that would otherwise result in nonspecific staining .
then the slides were transferred to citrate buffer and autoclaved for antigen retrieval at 15 lbs pressure for 15 min .
then the slides were dipped in two changes of phosphate buffered saline ( pbs ) for 5 min each .
the slides were then wiped carefully without touching the tissue section with gauze to remove excess pbs . then primary antibody ( dako - do7 )
was added to p tissue on the slides and then to the n pbs and left in the fridge overnight while staining for p53 and while staining for pcna the slides were flooded with pbs and left in fridge . following day
primary antibody pcna was added on p tissue of slides while staining for pcna and on the n section negative control was added and incubated for 45 min .
then the slides were washed in three changes of cold pbs for 5 min in each change .
then the slides were wiped carefully without touching the tissue section with gauze to remove excess pbs .
then , a drop of biotnylated link from the secondary antibody kit ( dako lsab 2 kit ) was added on both the sections on the slides and slides were incubated for 20 min .
then a drop of streptavidin ( dako lsab 2 kit ) was added on both the tissues on the slide and incubated for 20 min .
then a drop of freshly prepared 3-diaminobenzidine ( 3- dab tetra hydrochloride - a substrate chromogen ) was added on both sections .
slides were then washed in running distilled water to remove excess dab and were counter stained with hematoxylin .
the slides were transferred to 70% alcohol , 100% alcohol , and two changes of xylene in a sequential order .
the slides were checked for positive staining on both tissue marked as p and n. if n has taken positive staining , then the slide was discarded and the sections were restrained .
intensity of staining ( mild , moderate on intense staining ) was noted and graded as + , + + and + + + respectively .
mild staining denoting light brown color , moderate - brown color , and intense - dark brown color .
the expression of pcna and p53 protein was investigated in 10 cases of okc and 10 cases of pa .
pcna and p53 immunoreactivity was observed in the nuclei of cells to varying extent . staining intensity of pcna and p53 varied from weak to strong , depending on the cells .
nuclei were considered positive when they appeared light brown or dark brown . the staining intensity in okc and pa
of the 10 cases of okc : 
 2 cases showed light brown staining , which were graded + ( 20%)6 cases showed staining + + ( 60%)2 cases showed staining + + + ( 20% ) [ table 1 and graph 1 ] . 
 
2 cases showed light brown staining , which were graded + ( 20% ) 6 cases showed staining + + ( 60% ) 2 cases showed staining + + + ( 20% ) [ table 1 and graph 1 ] .
expression of pcna in okc and periapical cyst pcna in okc and periapical cyst when examining the staining intensity for pa in pcna .
of the 6 positive cases : 
 2 cases showed staining + ( 20%)3 cases showed staining + + ( 30%)1 case showed staining + + + ( 10%)4 cases showed negative staining ( 40% ) [ table 1 and graph 1 ] . 
 
2 cases showed staining + ( 20% ) 3 cases showed staining + + ( 30% ) 1 case showed staining + + + ( 10% ) 4 cases showed negative staining ( 40% ) [ table 1 and graph 1 ] .
six out of 10 cases of okc showed positive staining for p53 and 4/10 cases showed negative staining .
of the 6 positive cases of okc : 
 5 cases showed staining + ( 50%)1 case showed staining + + ( 10%)4 cases showed negative staining ( 40% ) [ table 2 and graph 2 ] . 
 
5 cases showed staining + ( 50% ) 1 case showed staining + + ( 10% ) 4 cases showed negative staining ( 40% ) [ table 2 and graph 2 ] .
expression of p53 in okc and periapical cyst p53 in okc and periapical cyst it was observed that only one out of 10 pa showed staining for p53 ( 10% ) .
9/10 pa showed negative staining ( 90% ) [ table 2 and graph 2 ] .
the expression of p53 and pcna was seen in basal and predominantly in the supra basal layer of all cases of okc and pa .
chi - square test of association revealed the following : 
 the staining intensity of okc using pcna and p53 was compared .
it was found that pcna staining intensity was more significant than that of p53 in okc ( p - 0.013)the staining intensity of pa using pcna and p53 revealed that pcna staining was more significant than p53 . 
 
it was found that pcna staining intensity was more significant than that of p53 in okc ( p - 0.013 ) the staining intensity of pa using pcna and p53 revealed that pcna staining was more significant than p53 .
all 10 cases of okc showed positive staining for pcna with varying intensity . of the 10 cases of okc : 

2 cases showed light brown staining , which were graded + ( 20%)6 cases showed staining + + ( 60%)2 cases showed staining + + + ( 20% ) [ table 1 and graph 1 ] . 
 
2 cases showed light brown staining , which were graded + ( 20% ) 6 cases showed staining + + ( 60% ) 2 cases showed staining + + + ( 20% ) [ table 1 and graph 1 ] .
expression of pcna in okc and periapical cyst pcna in okc and periapical cyst when examining the staining intensity for pa in pcna .
it was observed that 6/10 cases of pa showed positive staining for pcna . of the 6 positive cases : 
 2 cases showed staining + ( 20%)3 cases showed staining + + ( 30%)1 case showed staining + + + ( 10%)4 cases showed negative staining ( 40% ) [ table 1 and graph 1 ] . 
 
2 cases showed staining + ( 20% ) 3 cases showed staining + + ( 30% ) 1 case showed staining + + + ( 10% ) 4 cases showed negative staining ( 40% ) [ table 1 and graph 1 ] .
six out of 10 cases of okc showed positive staining for p53 and 4/10 cases showed negative staining .
of the 6 positive cases of okc : 
 5 cases showed staining + ( 50%)1 case showed staining + + ( 10%)4 cases showed negative staining ( 40% ) [ table 2 and graph 2 ] . 
 
5 cases showed staining + ( 50% ) 1 case showed staining + + ( 10% ) 4 cases showed negative staining ( 40% ) [ table 2 and graph 2 ] .
expression of p53 in okc and periapical cyst p53 in okc and periapical cyst it was observed that only one out of 10 pa showed staining for p53 ( 10% ) .
9/10 pa showed negative staining ( 90% ) [ table 2 and graph 2 ] .
the expression of p53 and pcna was seen in basal and predominantly in the supra basal layer of all cases of okc and pa .
chi - square test of association revealed the following : 
 the staining intensity of okc using pcna and p53 was compared .
it was found that pcna staining intensity was more significant than that of p53 in okc ( p - 0.013)the staining intensity of pa using pcna and p53 revealed that pcna staining was more significant than p53 . 
 
it was found that pcna staining intensity was more significant than that of p53 in okc ( p - 0.013 ) the staining intensity of pa using pcna and p53 revealed that pcna staining was more significant than p53 .
all 10 cases of okc showed positive staining for pcna with varying intensity . of the 10 cases of okc : 

2 cases showed light brown staining , which were graded + ( 20%)6 cases showed staining + + ( 60%)2 cases showed staining + + + ( 20% ) [ table 1 and graph 1 ] . 
 
2 cases showed light brown staining , which were graded + ( 20% ) 6 cases showed staining + + ( 60% ) 2 cases showed staining + + + ( 20% ) [ table 1 and graph 1 ] .
expression of pcna in okc and periapical cyst pcna in okc and periapical cyst when examining the staining intensity for pa in pcna .
it was observed that 6/10 cases of pa showed positive staining for pcna . of the 6 positive cases : 
 2 cases showed staining + ( 20%)3 cases showed staining + + ( 30%)1 case showed staining + + + ( 10%)4 cases showed negative staining ( 40% ) [ table 1 and graph 1 ] . 
 
2 cases showed staining + ( 20% ) 3 cases showed staining + + ( 30% ) 1 case showed staining + + + ( 10% ) 4 cases showed negative staining ( 40% ) [ table 1 and graph 1 ] .
six out of 10 cases of okc showed positive staining for p53 and 4/10 cases showed negative staining .
of the 6 positive cases of okc : 
 5 cases showed staining + ( 50%)1 case showed staining + + ( 10%)4 cases showed negative staining ( 40% ) [ table 2 and graph 2 ] . 
 
5 cases showed staining + ( 50% ) 1 case showed staining + + ( 10% ) 4 cases showed negative staining ( 40% ) [ table 2 and graph 2 ] .
expression of p53 in okc and periapical cyst p53 in okc and periapical cyst it was observed that only one out of 10 pa showed staining for p53 ( 10% ) .
9/10 pa showed negative staining ( 90% ) [ table 2 and graph 2 ] .
the expression of p53 and pcna was seen in basal and predominantly in the supra basal layer of all cases of okc and pa .
chi - square test of association revealed the following : 
 the staining intensity of okc using pcna and p53 was compared .
it was found that pcna staining intensity was more significant than that of p53 in okc ( p - 0.013)the staining intensity of pa using pcna and p53 revealed that pcna staining was more significant than p53 . 
 
it was found that pcna staining intensity was more significant than that of p53 in okc ( p - 0.013 ) the staining intensity of pa using pcna and p53 revealed that pcna staining was more significant than p53 .
classification of odontogenic cysts into developmental and inflammatory origin implies differences in their histogenesis . these presumed histogenic differences together with the clinically more aggressive behavior of okc in comparison with other cyst types has led to a number of studies attempting to characterize possible differences between their epithelial linings , connective tissue capsule and fluid aspirates .
the epithelial lining of okc is considered to have some intrinsic growth potential , which is not present on other odontogenic cyst epithelial lining cell kinetics in the epithelial lining of odontogenic cysts have been reported using many methods , such as mitotic count , tritiated thymidine incorporation , tissue culture , and immunocytochemistry in cryostat section .
immunocytochemical methods of assessing cell proliferation have better advantages over other techniques because of maintenance of cellular and tissue architecture , the relative simplicity of methodology and rapidity of results . in addition , the feasibility of using paraffin embedded sections allows retrospective study of specified lesions within a short time framework . based on this principle ,
the present retrospective study was designed to evaluate the proliferative cell kinetics of the epithelial lining of okc and pa .
proliferating cell nuclear antigen is a 36 kd nuclear protein associated with the cell cycle , increases through g1 , peaks at g1/s interface and decreases through g2 phase .
the p53 gene , encodes a nuclear phosphoprotein , which is thought to control cell growth at g1/s checkpoint .
wild type p53 can suppress or inhibit the transformation of cells in culture by viral or cellular oncogenes and arrest the cell cycle of a transformed cell at the g1 phase . on the contrary , mutant form of p53 shows dominant oncogene properties promoting cell proliferation and malignant transformation .
therefore , these two proliferative markers , namely p53 and pcna were chosen to study the mechanism that regulates cell proliferation and to understand the mechanism underlying the pathogenesis of okc or pas .
immunohistochemical detection of antibodies may show technical variables , like methods of fixation , method of antibody retrieval from the paraffin embedded blocks and type of antibody used . in the present study
the tissues were fixed in 10% buffered formalin , processed and embedded in paraffin and antigen retrieval with autoclave was adapted .
the present study showed , p53 and pcna immunoreactivity in the nuclei of cells in basal and more predominantly in suprabasal layer of okc and pa lining , with varying proportion and varied intensity [ figures 15 ] .
p53 and pcna showed 60% and 100% expression , respectively in okc compared to 10% and 60% in pa .
similarly , the intensity of staining was more ( + + ) in 60% of okc , whereas it was only 30% in pa .
histopathological features of odontogenic keracyst using ( a ) h and e , 10 , ( b ) pcna , 10 histopathological features of odontogenic keracyst using ( a ) pcna , 40 , ( b ) p53 , 40 histopathological features of periapical keracyst using ( a ) h and e , 10 , ( b ) pcna , 10 histopathological features of periapical cryst using ( a ) pcna , 40 , ( b ) p53 , 40 photomicrograph showing various intensity pattern ( a ) mild , ( b ) moderate and ( c ) intense these results are consistent with the findings of li et al . and also consistent with other studies using mitotic counting , tritiated thymidine incorporation and k67 immunostaining methods .
this suggests , greater proliferative activity in okc thus explaining their more aggressive clinical behavior .
although , all the cases of okc epithelial lining ( 100% ) expressed positive staining pattern with pcna , only 60% expression was noted with p53 . in all cases
histological and histochemical studies have suggested the possibilities of basal cells being more differentiated than supra basal cells .
the columnar appearance of basal cell with apparent reverse polarity resembling preameloblasts , indicates that they might have entered to some extent the pathway toward ameloblast differentiation . in this study ,
the observation of more number of positive cells in the suprabasal layer of okc confirms the views of browne 1971 , ogden et al .
immunocytochemical detection of p53 may be influenced by many technical valuables(hall pa 1994 ) , such as alteration in deduction thresholds with different antibodies and different methods of antigen retrieval .
a number of studies have demonstrated a close correlation between increased p53 protein expression and mutation of the p53 gene .
however , recent studies indicate that cellular insult can be a potent inducer of wild type p53 protein , which can be stabilized and become immunocytochemically detectable .
1993 , li 1996 are of opinion that p53 over expression by okc compared to other types of odontogenic cysts is not due to mutation of p53 gene , rather , over production and/or stabilization of normal p53 product appears to be related to proliferation .

 pcna and p53 expression was studied in the epithelial lining of 10 okcs and 10 pasthe level of staining and intensity were assessed in all these casesokcs showed pcna expression in all cases ( 100% ) whereas in pa only 60% of cases exhibited pcna stainingokcs showed p53 expression in 6 cases ( 60% ) whereas in pa only 10% of cases exhibited p53 stainingstaining intensity of pcna and p53 was more significant in okcs than papcna staining was more intense when compared to p53 in both okcs and pain okcs , suprebasal cells showed more intense staining than basal cells with both antibodies . 
 
pcna and p53 expression was studied in the epithelial lining of 10 okcs and 10 pas the level of staining and intensity were assessed in all these cases okcs showed pcna expression in all cases ( 100% ) whereas in pa only 60% of cases exhibited pcna staining okcs showed p53 expression in 6 cases ( 60% ) whereas in pa only 10% of cases exhibited p53 staining staining intensity of pcna and p53 was more significant in okcs than pa pcna staining was more intense when compared to p53 in both okcs and pa in okcs , suprebasal cells showed more intense staining than basal cells with both antibodies .
 introduction : p53 protein is a product of p53 gene , which is now classified as a tumor suppressor gene . the gene is a frequent target for mutation , being seen as a common step in the pathogenesis of many human cancers . 
 proliferating cell nuclear antigen ( pcna ) is an auxiliary protein of dna polymerase delta and plays a critical role in initiation of cell proliferation.aim:the aim of this study is to assess and compare the expression of p53 and pcna in lining epithelium of odontogenic keratocyst ( okc ) and periapical cyst ( pa).materials and methods : a total of 20 cases comprising 10 okc and 10 pa were included in retrospective study . 
 three paraffin section of 4 m were cut , one was used for routine hematoxylin and eosin stain , while the other two were used for immunohistochemistry . 
 statistical analysis was performed using chi - square test.results:the level of staining and intensity were assessed in all these cases . 
 okc showed pcna expression in all cases ( 100% ) , whereas in perapical cyst only 60% of cases exhibited pcna staining . 
 ( 1 ) okc showed p53 expression in 6 cases ( 60% ) whereas in pa only 10% of the cases exhibited p53 staining . 
 chi - square test showed pcna staining intensity was more significant than p53 in okc . 
 ( 2 ) the staining intensity of pa using p53 , pcna revealed that pcna stating intensity was more significant than p53.conclusion:okc shows significant proliferative activity than pa using pcna and p53 . 
 pcna staining was more intense when compared with p53 in both okc and pa . 
None
Aims
Materials and Methods
Tissues
Immunohistochemical procedure
Results
Proliferating cell nuclear antigen in OKC and PA
Staining intensity
Discussion
Summary and Conclusion