Calpain3 is expressed in a proteolitically active form in papillomavirus-associated urothelial tumors of the urinary bladder in cattle.
BACKGROUND
Calpain 3 (Capn3), also named p94, is a skeletal muscle tissue-specific protein known to be responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Recent experimental studies have hypothesized a pro-apoptotic role of Capn3 in some melanoma cell lines. So far the link between calpain3 and tumors comes from in vitro studies. The objective of this study was to describe Capn3 activation in naturally occurring urothelial tumors of the urinary bladder in cattle.


RESULTS
Here we describe, for the first time in veterinary and comparative oncology, the activation of Capn3 in twelve urothelial tumor cells of the urinary bladder of cattle. Capn3 protein was initially identified with nanoscale liquid chromatography coupled with tandem mass spectrometry (nano LC-MS/MS) in a co-immunoprecipitation experiment on E2F3, known to be a transcription factor playing a crucial role in bladder carcinogenesis in humans. Capn3 expression was then confirmed by reverse transcription polymerase chain reaction (RT-PCR). Finally, the Ca(2+)-dependent proteolytic activity of Capn3 was assayed following ion exchange chromatography. Morphologically, Capn3 expression was documented by immunohistochemical methods. In fact numerous tumor cells showed an intracytoplasmic immunoreactivity, which was more rarely evident also at nuclear level. In urothelial tumors, bovine papillomavirus type 2 (BPV-2) DNA was amplified by PCR and the expression of E5 protein, the major oncogenic protein of BVP-2, was detected by western blotting, immunohistochemistry, and immunofluorescence. E2F3 overexpression and pRb protein downregulation were shown by western blotting.


CONCLUSIONS
The role of capn3 protein in urothelial cancer of the urinary bladder remains to be elucidated: further studies would be required to determine the precise function of this protease in tumor development and progression. However, we suggest that activated Capn3 may be involved in molecular pathways leading to the overexpression of E2F3, which in turn could be responsible for urothelial tumor cell proliferation also in cattle, though other mechanisms are likely to exist. If further studies corroborate the important role of Capn3 in urothelial tumors of the urinary bladder, cattle with urinary tumors may prove useful as animal model for bladder carcinogenesis.
Roperto
Sante
S
Department of Pathology and Animal Health, Division of Infectious Diseases, Naples University Federico II, Naples, Italy. sante.roperto@unina.it
De Tullio
Roberta
R
Raso
Cinzia
C
Stifanese
Roberto
R
Russo
Valeria
V
Gaspari
Marco
M
Borzacchiello
Giuseppe
G
Averna
Monica
M
Paciello
Orlando
O
Cuda
Gianni
G
Roperto
Franco
F
D000818:Animals; D052688:Bovine papillomavirus 1; D002118:Calcium; D002154:Calpain / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D050699:E2F3 Transcription Factor / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D009375:Neoplasms, Glandular and Epithelial / Q000201:enzymology* / Q000662:veterinary / Q000821:virology; D030361:Papillomavirus Infections / Q000150:complications; D020133:Reverse Transcriptase Polymerase Chain Reaction; D001749:Urinary Bladder Neoplasms / Q000201:enzymology* / Q000662:veterinary / Q000821:virology; D019459:Urothelium / Q000473:pathology / Q000821:virology
D050699:E2F3 Transcription Factor; D002154:Calpain; D002118:Calcium