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heat shock proteins ( hsps ) or stress proteins , which play an essential role in the maintenance of cellular homeostasis , are the products of several distinct gene families produced under both physiological and stress conditions .
alpha basic - crystallin ( b - c ) belongs to a family of small hsps , and is expressed in response to different stresses including heat shock , oxidative stress , metal ions , and cytokines .
b - c is constitutively expressed in many humans and animals tissues such as the lens of the eyes , heart , and skeletal muscle .
b - c has chaperone - like properties which prevent the aggregation of damaged or proteins misfolded due to cell stress , and has been shown to inhibit apoptosis .
mammary tumors , the most common neoplasms in female dogs , may provide a useful model for human breast cancer research .
both benign and malignant neoplasms account for approximately 50% of all tumors in dogs and show wide pathological and clinical heterogeneity .
after surgical intervention , approximately 48% of the affected dogs die or are euthanized within a 1-year period because of tumor recurrence or metastasis . therefore , additional and reliable prognostic tools are required for effective risk assessment .
the aims of this study were to evaluate the prognostic and/or diagnostic factors associated with canine mammary tumors in dogs by analyzing the immunohistochemical expression of b - c .
formalin - fixed , paraffin - embedded blocks containing 51 naturally occurring canine mammary tumors ( 11 benign and 40 malignant ) were selected retrospectively and acquired from the veterinary laboratory ( italy ) and department of pathology , faculty of veterinary medicine , ondokuz mayis university ( turkey ) .
additionally , eight normal canine mammary glands obtained from the dogs that had died of causes unrelated to tumor development served as healthy control tissues .
three pathologists ( tg , myg and ms ) independently diagnosed the tumors according to the world health organization classifications for canine mammary neoplasm . sections from all the tissue samples were cut ( at a thickness of 4 m ) and stained with haematoxylin and eosin .
additional sections were placed on slides coated with 3-aminopropyltriethoxysilane ( sigma , usa ) and stained using a streptavidin - biotin - peroxidase complex technique ( histostain plus kit ; zymed , usa ) with a monoclonal anti-b - c antibody ( 1/1,000 dilution , spa-222 ; stressgen , canada ) .
incubation with amino ethyl carbazole ( aec substrate kit ; invitrogen , usa ) or 3,3'-diaminobenzidine ( dab chromogen / substrate kit ; scytek , usa ) as a chromogen in h2o2 was performed for 10 min .
the sections were counterstained with mayer 's hematoxylin for 1 min , rinsed with tap water , and mounted with an aqueous mounting medium ( vision mount ; lab vision , usa ) .
the percentages of the total area of the immunohistochemically positive cells were assessed with a microscopy image analysis system ( bs200p ; bab software , turkey ) .
a total of 10 high - power fields were randomly chosen and analyzed at  400 .
the findings were categorized as follows : ( 0 ) no positively stained tumor cells ; ( 1 ) 5~25% ; ( 2 ) 26~50% ; and ( 3 ) greater than 51% positive tumor cells .
the immunohistochemical results of b - c expression and histopathologic diagnoses are summarized in table 1 . in the control mammary tissues , a few luminal epithelial cells were positive but the basal myoepithelial cells were negative ( fig .
muscles ( e.g. , m. supramammarius ) were always positive and were used as positive internal controls when present . in benign or malignant simple type tumors ,
additionally , b - c immunoreactivity of the epithelial cells in malignant tumors was more intense than that observed in the benign type . in both benign and malignant complex type tumors , positive staining
however , b - c - positive mesenchymal elements of the carcinosarcoma , such as cartilage or bone , were not observed ( fig . 1d )
. the numbers of b - c positive cells were statistically significant different between the control canine mammary tissues and benign tumors ( p < 0.05 ) .
statistically , significant differences of b - c immunopositive cells were also found in between benign and malignant tumors ( p < 0.05 ) .
additionally , the most significant difference of immunopositive cell count was observed between normal mammary gland tissues and malignant tumors ( p < 0.001 ) .
the results of the current study indicates that the number of b - c - positive cells were increased in benign and malignant canine mammary tumors when compared to control mammary gland , and the number of b - c - positive cells were higher in malignant tumors when compared with benign tumors .
this factor is expressed in many tissues and organs , and acts as a molecular chaperon .
additionally , b - c is expressed in breast and the other cancers , such as gliomas , and prostate and renal cell carcinomas , in humans .
however , the best of our knowledge , there is no such study performed in animals and the present report documents a connection between b - c and mammary tumors in canines . in the normal human breast tissue , b - c
in contrast to this report , in the current study , very low level expression of b - c was observed in the luminal epithelial cells of normal canine mammary tissues , but b - c immunepositive reactivity in the myoepithelial cells was not observed .
it is possible that this difference might be species - specific or related to the physiological stage of the control mammary gland tissues .
.   reported that b - c may be used as a myoepithelial marker , but it does not have any special advantage over currently used markers such as smooth muscle myosine .
in contrast , b - c immune reactivity in the myoepithelial cells was not observed in our study . therefore , b - c immunostaining in the canine mammary gland tissues could not be suggested as a myoepithelial marker .
chelouche - lev et al .   have reported that out of 672 human breast tumors , 608 ( 90% ) are positive for b - c immunostaining .
in contrast , moyano et al .   noted 39 positively immunostained samples out of 361 ( 11% ) human breast cancer cases .
.   reported that b - c expression is observed in human basal - like ( 81% ) and metaplastic ( 86% ) breast cancer . in the present study ,
the rate of b - c expression in canine mammary gland tumors was 88.2% ( 45 out of 51 ) .
although it is difficult to compare canines to humans , our data are similar to the findings of chelouche - lev et al .   and sitterding et al . ,
differences in the number of b - c - immunolabelled cells were found to be statistically significant between the normal canine mammary tissues and the benign and malignant tumors .
these differences were thought to be evidence that b - c was involved in oncogenesis because of the anti - apoptotic effect of b - c .
this effect probably involves the suppression of caspase-3 activation and/or preventing the mitochondrial translocation of pro - apoptotic bcl-2 family members such as bax . in conclusion ,
we believe that b - c can play a role in the carcinogenesis of canine mammary glands because of the increased immunoreactivity of b - c we observed in neoplastic tissues compared to the normal canine mammary gland tissues .
b - c has an anti - apoptotic effect and this anti - apoptotic effect can be important in chemotherapy resistance of the cancer . additionally , targeted inhibition of b - c may be a new strategy to the cancer therapy .
the aim of this study was to evaluate prognostic and/or diagnostic factors of canine mammary tumors by immunohistochemically analyzing the expression of alpha basic crystallin ( b - c ) . 
 for this , formalin - fixed , paraffin - embedded blocks of 51 naturally - occurring canine mammary tumors ( 11 benign and 40 malignant ) were used . 
 tissue from eight normal canine mammary glands were served as a control . 
 immunohistochemically , in the control mammary tissues , a few luminal epithelial cells were b - c positive but myoepithelial cells were negative . in benign or simple type malignant tumors , 
 b - c expression was observed in luminal epithelial cells while the myoepithelial basal cells were negative . in benign or complex type 
 malign tumors , positive staining was predominantly found in the cytoplasm of epithelial cells . 
 immunoreactivity of b - c was also observed in neoplastic myoepithelial cells . 
 statistically , the number of cells immunolabeled with b - c was found to be significantly different among tissues from normal canine mammary glands , benign lesions , and malignant tumors ( p < 0.05 ) . 
 b - c immunoreactivity was higher in malignant tumors than the control mammary tissues ( p < 0.001 ) . 
 data obtained in the current study revealed a strong association between high expression levels of b - c and primary mammary gland tumors in canines .