| In numerous regulatory procedures of cell signalling transduction, as a character of high-level evolution form and complicated multicell-creature, the phosphorylation of proteins at tyrosine residues, which is covalently modified by protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs), serves as a switch in regulating the activity of biological processes and cellular events, as well as their response to environmental cues. The coordinated regulation of PTPs and PTKs are involved in many crucial procedures of cell signal transduction, such as proliferation, differentiation, adhesion, and immigration etc, and has closely relativity with many pathologies and physiology phenomenon, such as tumor, cardiovascular disease, immunodeficiency, infectious diseases, neurogenic and metabolic diseases. Consequently, many PTPs have become excellent therapeutic targets for mankind's severe diseases in drug development. The widely expressed PTP1B which is a prototype of PTPs superfamily and a typical intracellular PTP, was the first mammalian PTP identified and purified in 1988. PTP1B which has a N-terminal catalytic domain (PTP domain) followed by two proline-rich motifs exists as 435 amino acids form of 49666 Da, and localizes to the endoplasmic reticulum (ER) through a 35 amino-acid residue hydrophobic stretch found in its C-terminus. For the past few years, studies in vivo and vitro show that PTP1B plays a negative key role in regulation of insulin signal transduction, and has become the most hopeful therapeutic target for type 2 diabetes and obesity. PTP1B plays a very important role in signal transduction of a great deal of growth factors. Currently, many major drug discovery efforts focus on PTP1B as a therapeutic target for diabetes and obesity. However, studies on the correlation between PTP1B and cancers are very few.Customarily the activity of hPTP1B holoenzyme is very low and expressing the holoenzyme by means of molecular biology easily form cytorrhyctes, which makes much inconvenience in researching work. As the catalytic domain of PTP1B,â–³PTP1B has the fundamental spatial structure of the holoenzyme and higher activity and stability, thus targetingâ–³PTP1B will benefit the studies on the correlation with the pathologies and physiology phenomenon all the better. Based on this foundation, the datalytic domain of PTP1B was expressed and purfied successfully and anti-â–³PTP1B polyclonal antibody with high titer and specificity was prepared and purified in this study. Breast cancer, one of the most common malignant tumors for female, has become a severe disease endangering women's health and its incidence rate is increasing year by year. In this study, we carried on immunohistochemical examination in 60 human mammary tumors and 15 normal breast tissue samples by applying anti-â–³PTP1B polyclonal antibody, simultaneously the protein expression of estrogen receptor (ER) and progesterone receptor (PR) was evaluated in mammary tumors. Results showed that PTP1B overexpression was observed in 35 tumors (58.33%) as compared to 4 normal breast tissue samples (26.67%) and there is a significant deviation (P<0.05=, ER overexpression was observed in 29 tumors (48.33%) and PR overexpression was observed in 35 tumors ( 58.33%), in ER(+), ER(-), PR(+), and PR(-) groups, PTP1B overexpression was observed in 12(41.38%), 23(74.19%), 14(40.00%), and 21(84.00%) tumors respectively. Statistic analysis showed that PTP1B expression had significant negtive relativity with ER or PR expression (P<0.01= and there was significant positive relativity between ER and PR expression (P<0.01=. Furthermore, PTP1B overexpression had no relativity with age and tumor magnitude (P>0.05), and had remarkable positive relativity with regional lymph nodes metastasis, clinical stages and histological grades (P<0.05=. Therefore, in breast cancer PTP1B may act as one of tumor markers and a detecting index for judging the malignant degree, invasive capability, and prognosis. Breast cancer is a kind of hormone-dependent tumor and its biological behaviors are mainly controlled by estrogen and progestogen. Clinically evaluating the expression level of ER/PR plays a important role in choosing strategy of endocrine therapy, determining plan of combined treatment and judging prognosis. A great deal of research indicate that ER(+) or/and PR (+) always have a significant positive relativity with a better therapeutic efficacy of endocrine therapy and with a better prognosis. In this study, statistic analysis showed that PTP1B expression had significant negtive relativity with ER or PR expression. This result also supported the judgement that PTP1B overexpression associated with poor prognosis. Evaluating the expression of PTP1B protein in cancer tissues may provide patients of breast cancer with a index for the selection of endocrine therapy and chemotherapy, and judging prognosis.ErbB2 is a protein tyrosine kinase (PTK) of the EGF receptor family that is overexpressed in 25% of breast cancer where it associated with poor prognosis. C-Src kinase is a non-receptor protein tyrosine kinase and the elevation of its acitivity has a closely relativity with tumorigenesis. In animal models of transgenic mice with ErbB2-induced mammary tumor, the absence of PTP1B induced delayed tumor development and decreased incidence of lung metastasis accompanied by attenuated signal transduction by which ErbB2 induced mammary tumor, suggesting the crucial role for PTP1B in tumor proliferation and cell apoptosis during development and progression of mammary tumors. PTP1B has been identified as the major PTP catalyzing dephosphorylation of oncoprotein c-Src kinase and activation in breast cancer cell lines, thus it plays a important role in regulating the activity of Src kinase. Regulating these two oncogenic PTKs, ErbB2 and Src is a important mechanism for PTP1B in breast cancer. At this point, overexpression of PTP1B induces increased oncogenic signal transduction and associates with differentiation and infiltration in breast cancer.Furthermore, in vivo PTP1B can dephosphorylate and inactivate IR and IRS located at cytoplasmic membrane, thus it hinders the signal transduction, that is also the major evidence for using PTP1B as a important therapeutic target for type 2 diabetes in recent years. As well as IGF-1R, PTP1B was found to be involved in the dephosphorylation of IGF-1R as a negative regulator. Therefore, in the signal network formed by insuline, IGF-1R, and estrogen induced signallings in breast cancer, PTP1B may act as a regulatory molecule, playing a important role in regulating cell proliferation and differentiation by adjusting the dephosphorylation level of key molecule such as IR, IRS-1, and IGF-1R etc.Overall, we successfully prepared and purified anti-â–³PTP1B polyclonal antibody with high titer and specificity, then we carried on immunohistochemical study in breast cancer by applying the prepared antibody. According to the results, we demonstrated that PTP1B overexpression was a important tumor marker inducing increased oncogenic signalling in breast cancer and associated with poor prognosis. Furthermore, evaluation of PTP1B expression played a important role in diagnosis, choosing treatment plan, and judging prognosis in breast cancer. The prepared anti-â–³PTP1B polyclonal antibody, targeting the catalytic domain of PTP1B directly, was competent for reseaching work as immunoblotting, immunoprecipitation, and immunohistochemistry etc, and its successfully development lay the important foundation for further studying the biological function of PTP1B and the relativity with human diseases.
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