Expression And Significance Of ERK Protein In Human Breast Carcinoma

IntroductionBreast carcinoma is one of the most common malignancies affecting women. The etiology and pathogenesis of breast carcinoma remain unclear , but several observations suggest a prominent role for the mitogen - activated protein kinase signal pathway in the development of breast carcinoma.Extracellular signal-regulated kinase pathway is one of important and the best characterized and the more strongly related with human cancer among MAPK regulatory network. ERK is a firstly discovered member of MAPK family. ERK1 and ERK2 are two major isozymes, which their molecule weight are 44 -and 42 - kiodalton respectively. It is a kind of highly conserved and ubiquitously expressed serine/threonine kinese which could be phosphorylated and activated by a number of growth factor, cytokines, hormone and mitotic signal, and is involved in cell cycle and cell proliferation , differentiation and resists apoptosis. Recently some observations discovered ERK signal pathway was associated with carcinoma tumorigenesis and progression. Phospho-ERK, or phosphorylated activated ERK may translocate into nucleus to regulate the expression of various oncogene through phosphorylation on transcription level and result in cellular malignant transformation.Some observations suggested that the activated ERK was associated with tumorigenesis and metastases of mouse breast carcinoma. We investigate the expression of ERK and p-ERK protein in human breast cancer and their corresponding tissue , to analyse the relationship between the expression these proteins and the clinical histopathological characteristics or steroid receptors of breast cancer . Our objective is to access the significance of ERK signal pathway in tumorigenesis and progression of breast carcinoma and will provide a beneficial clue for prognosis.Materials and Methods1. Specimens:40 breast cancer cases were used in immunohistochemistry study. These cases were obtained from ShenYang Military Hospital and the First Affiliated Hospital of China Medical University from June 1998 to October 2000. All the specimens were fixed in formalin and embedded with wax. 40 fresh cases with their adjacent normal breast tissues used in Western Blot were obtained from the First Affiliated Hospital of China Medical University from October 2002 to March 2003. These specimens were divided into two parts; one was frozen immediately at - 70 C and the other one was fixed with formalin and made into paraffin block. None of them treated with chemical or radioactivity therapy.2. Reagents:Rabbit polyclonal antibody for ERK1(sc-94) , ERK2(sc - 154) and mouse monoclonal antibody for p-ERK(sc-7383) were purchased from Santa Cruz Biotechnology Company. Alkaline phosphatase conjugated goat anti - rabbit antibody was purchased from Beijing ZhongShan Biotechnology Company. S-P reagent kit (KIT-9710) and DAB agent kit (DAB-0030) were purchased from FuZhou Maxim Company. Alkaline phosphatase conjugated goat anti-mouse antibody and NBT/BCIP kit were purchased from HuaMei Company.3. Methods3. 1 Western Blot. Rapidly homogenize tissues ( about 3mm3) in lysis buffer, then centrifuge the homogenate to pellet insoluble material. Electropho-resis transfer proteins from gel to PVDF blocking incubation with primary antibody and enzyme conjugated substrate incubation. The results were scanned and mensurated by computer image analysis system.3. 2 Immunohistochemistry. To detect the proteins expression of ERK, p-ERK in 40 cases of breast cancer by immunohistochemistry S-P method. 500 tumor cells were observed in view of high magnification per slide at random. ERK1, ERK2 and p-ERK immunostaining was classified in the following three groups according to both intensity and extent: ( 1) Negative, no staining waspresent or immunostaining was present in less than 10% of all 500 cells. (2) Mild positive, immunostaining was weak or similar to that of positive control cells and such cells were more than 10% cells , or strong staining cells were more than 10% cells and less than 50%.