Protein Kinase CK₂-α' In Adenoid Cystic Carcinoma And Its Relationship With Pulmonary Metastasis

ObjectiveAdenoid cystic carcinoma is one of the most common malignant tumor.It 9.95% of salivary tumor,24%malignant salivary tumor.It occurs in head and neck,and is rare in other part of body.Adenoid cystic carcinoma often happens in small salivary gland and parotid gland,second is submaxillary gland.If it happens in glandula Rivinus,it is maybe adenoid cystic carcinoma.adenoid cystic carcinoma often happens in old human with 40-70 years.There are 3 types:adenoid type,tubular type and parenchymal type.Control of recovery and distant metastasis is an important question in clinical treatment,and now research of pulmonary metastasis of adenoid cystic carcinoma in a focus.Under the microscope,there are two kinds of the tumor cells,myoepithelial cell and vessel epithelial cell.it is maybe conglobate,egg or epithelial mass.it has a lot of capsular spaces that is Inequality of size.cell nucleus are in transparent material which palisade arrangementIt seems that there is clear boundary between adenoid cystic carcinoma and peripheral tissue in salivary gland.But there is no envelope around the tumor,and the tumor often infiltrates tissue.And tumor cell can be found in and out of envelope.Near the tumor,such as muscle,bone and periosteum,the infiltrative tumor cell,which can't be found by eye,can be tested under the microscope.Protein kinase is one of family enzyme,and a catalyst of protein phosphorylation,which is a common mechanism of cellular action.CK₂ is the most effective protein kinase.CK₂ is consist of two catalytic subunit(α,42-44KDa andα',38KDa)and two regulatory subunit(β,28KDa).The structure is stable,and only decomposed in vitro.Except cellar matrix,some virus protein can be phosphorylated by protein kinase CK₂.Protein kinase CK₂ plays a role in transcription,signal growth and development of tumor.Acticity and expression of protein kinase CK₂ is not only in nucleus and plasma of SACC,but also in all parts of SACC.Protein kinase CK₂ can phosphorylate many substrates which involve in cellar fission and differentiation and development of tumor.Through immunofluorescence method,expression of protein kinase CK₂-α'in SACC is evaluated to investigate signify-cancer of interaction of protein kinase CK₂-α'in SACC.To study the relationship of protein kinase CK₂-α'and pulmonary metastasis,we extract nucleus and plasma of SACC-LM and SACC-83,evaluate the expression of protein,and disclose the influence of protein kinase CK₂-α'to pulmonary metastasis in protein level.The fact that protein kinase CK₂-α'is related with pulmonary metastasis of adenoid cystic carcinoma has not been known.Method1.Cell cultureSACC-83 and SACC-LM cell were cultered with Dulbecco's modified Wgle's medium containing 10%(vol/vol)fetal bovine serum.For cell cycle synchronization, SACC-83 and SACC-LM cell were treated with aphidicolin(1ug/ml)for 24h to arrest the cells in the Gl/S phase.Cell were released from the Gl/S cell cycle block by washing the cell three times with phosphate-buffered saline(PBS) and culcure in fresh medium.2.immunofluorescenceSACC-83 and SACC-LM cells(Gl/S)was fixed and perforated,unspecific antigen was prevent by bovine serum.Antibody of protein kinase CK₂-α'was involved for overnight at 4℃.The second antibody with fluorescence were involved away from light.Buffer douched 3 times by step.In control group,the first antibody was replaced of TBS.Mounting by glycerine after stain nuclear.Take pictures.3.Western blottingSACC-83 and SACC-LM cells loaded on to single-dimension SDS-PAGE.For immunobotting,proteins were tansfered to nitrocellulose membranes using boltting techniques Membranes were blocked for 1h in 5%BSA-TTBS.Probed with a specific protein kinase CK₂-α'mouse monoclonal antibody for overnight at 4℃,and the probed with a horseradish peoxidase conjugated secondary antibodies.Antibody reaction was revealed using activator protein(AP)Rusult1,Expression of protein kinase CK₂-α'irfSACCProtein kinase CK₂-α' is located in the nucleus of SACC.2,Expression of protein kinase CK₂-α'in SACC-LM and SACC-83In SACC-LM,expression of protein kinase CK₂-α' in nucleus is higher than in plasma.In SACC-83,expression of protein kinase CK₂-α' in nucleus is higher than in plasma.Comparing with SACC-83,expression of protein kinase CK₂-α' is higher in nucleus and in plasma of SACC-LM.Conclusion1,The high expression of protein kinase CK₂-α' in adenoid cystic carcinoma is related with occurrence of tumor.2,Protein kinase CK₂-α',especially variation of protein kinase CK₂-α' in nucleus,is related with pulmonary metassis of adenoid cystic carcinoma.3,Protein kinase CK₂-α' is positive correlation with pulmonary metassis...