Expression Of Tumor Suppressor Gene Product Rb Protein In Breast Cancer And Its Clinical Significance

Breast carcinoma is one of the most common malignanttumor in women, the morbidity rate has continued to rise inmany countries and regions and it has been the critical diseaseharming female health and life. The diagnosis and therapy ofbreast carcinoma has improved, that can inhibite the mortalityof breast cancer to a certain extent. Now special and reliabletumor markers were very little. Immunohistochemistry methodwas conducted to analyze the expression of cyclin D1 proteinand Rb protein in our topic, while we study the relationshipwith MCF-7 apoptosis and the expression of dephosphorylatedRb protein by THP treated. We approached the relationshipwith the expression of cyclin D1 and Rb protein and breastcancer genesis and development;and the relationship withbreast cell apoptosis and dephosphorylated Rb protein, that willprovid a objective standard to determine therapeutic regimen,evaluate pathogenetic condition and prognosis.Material and methods:1. clinical material: Breast tissue specimens were obtainedfrom 72 chinese women accepting operation in Department ofSurgery, China-Japan Hospital affiliated with Jilin Universityfrom June 1999 to January 2001. These patients were randomlyselected, including 52 breast cancer, 15 breast hyperplasias and5 fibroadenomas. All issue specimens were treated in 10%formalin immobilization, paraffinum embedment, cutting sheetand staining, and final diagnosis by pathologic detection results.2. Human breast cancer cells of MCF-7 was cultivanted with0.1FCS-RPMI1640 in the incubator with 5% CO2 and constanttemperature 37℃. Exponential phase of growth cells weremade in the experiment.3. Immunohistochemistry SP method was conducted toanalyze the expression of cyclin D1 protein and Rb protein in52 breast cancer cases and 20 benign breast tissues cases;the46 patients with breast cancer were in follow up, we camparedwith the peritoneal recurrence and metastatic rate in thenegative expression of Rb protein cases and the positiveexpression of Rb protein cases.4. THP as an anticancer drug was treated to chemosensitivecell line MCF-7, MTT assay revealed that THP had effect to theproliferation of chemosensitive cell line MCF-7;FCM assaysdetected the apoptosis of cells;Immunohistochemistry SPmethod detected the expression of dephosphorylated Rb proteinin un-treated and treated groups.Result:1. The relationship with the cyclin D1 overexpression and thehistologic type of breast cancer.The positivity rate of cyclin D1 overexpression in the breastcancer was 34.6% (18/52),while there was a significantdifference compared with that of benign breast tissues10 %( 2 /20),P < 0.05. The positivity rate of cyclin D1 overexpression incatheter infiltrating carcinoma, carcinoma simplex andcarcinoma medullare was 38.46%, 34.6% and 40.00%, P > 0.05.Primary cancer and lymphatic metastasis cancer were all thecyclin D1 overexpression in 3 cases of hemi-breast lymphaticmetastasis and 1 cases of carcinoma simplex.2. The relationship with the cyclin D1 overexpression and theclinical pathological characters of breast cancer.There was no significant difference between cyclin D1overexpression and histologic type, tumor size, age of patient ,histologic grades as well as lymphiod statues.3. The relationship with the negative rate of Rb expressionand the histologic type of breast cancerThe negative rate of Rb expression in breast cancer was76.91%(40/52) while there was a significance compared withthat of benign breast tissues 15.00% (3/20), that indicated thenegative rate of Rb expression in breast cancer was muchhigher than in the benign breast tissues. The negative rate ofRb expression in catheter infiltrating carcinoma , carcinomasimplex and carcinoma medullare was 88.46%,66.67% and60.00%, P > 0.05.4. The relationship with the Rb negative expression and theclinical pathological characters of breast cancerThere was no significant relationship between Rb expressionand histologic type , tumor size , age of patient , histologicgrades as well as lymphiod statues.5. The relationship with cyclin D1 overexpression and RbexpressionThe cyclin D1 overexpression rate was 20.00% in cases of thenegative expression of Rb protein;the cyclin D1 overexpressionrate was 83.33% in cases of positive expression of Rb protein,P<0.05.6. The relation of the negative expression of Rb protein andrecurrence rateThe 46 patients with breast cancer were cured 4-6 courses oftreatments withneoadjuvant chemotherapy of CAF(Cyclophosphamide, Adriamycin and Fluorourac) or CAM andwere in follow up, the peritoneal recurrence and metastatic ratewere 10.53% in 38 cases of the negative of Rb protien , andwhile the rate were 0 in 8 cases of the positive of Rb protien,there was significant differences in two groups (P<0.01).7. THP has effect to the proliferation of chemosensitive cellline MCF-7MTT assay revealed that 1,2.5,5,7.5,10ug/ml THP haddifferent effect to the proliferation of MCF-7 cell line. THP cansignificantly inhibited the proliferation of MCF-7 cell line,thecell survival rate was significantly deseneded with the THPconcentration's increasing,when the THP 's concentration was1ug/ml, the cell survival rate of MCF-7 was 52.8 %, when theTHP 's concentration was 10ug/ml, the cell survival rate ofMCF-7 was 24.4%,not more than 50 %.8. THP has effect to the expression of dephosphorylated Rbprotein of chemosensitive MCF-7 cellThe results of immunocytochemical staining were achieved:Before treatment of THP, phosphorylated Rb protein waspositive and dephosphorylated Rb protein was negative. After24 hours treatment of different concentrations THP(1,2.5,5,7.5,10ug/ml), Rb protein located intranuclear, especially Rbprotein expressed comparative conspicuously in the treatmentof 5ug/ml THP, whlie phosphorylated Rb protein was negative.MOD X areas value of RB protein was 45136723±36471198,and MOD X areas value of phosphorylated RB protein was1459942± 334603.1, we calculated that MOD X areas value ofdephosphorylated Rb protein was 43676781± 36136595.9. Detecting the rate of apoptosis of MCF-7 by THP inducedwith FCM assaysFCM assays indicated: the nature rate of apoptosis of MCF-7was 0.0417, MCF-7 cells apoptosis increased by the treatmentof THP, especially in the treatment of 5ug/ml THP, apoptosiswas very significant and the rate of apoptosis was 0.2321.10. The relationship of the effect of THP inhibiting breast can-cer cell proliferation and the expression ofdephosphorylatedRb proteinThe correlative analysis by SPSS 10 for windows showed thecorrelation confficient of THP inducing the apoptosis rate ofMCF-7 cells and the expression levels of dephosphorylated Rbprotein was 0. 0962. It indicates there were positive correlation(P<0.05)between the expression levels of dephosphorylated Rbprotein and the apoptosis of THP inducement MCF-7/S cells.11.THP has effect to the cell cycle of MCF-7After 24 hours treatment of 0.25,1,2.5,5ug/ml THP, we ana-lyzed the cell cycle made clear : breast cancer MCF-7 were ac-cumulated in G1 phases, and decrease in S phase, that indicatedTHP made MCF-7 block in G1 phase by dephosphorylated Rbprotein.Conclusion:Our study indicated that cyclin D1 protein and Rb proteinhave closely related with the genesis and development of breastcancer;cyclin D1,p16 and Rb acess inactivation may have clo-sely related with the genesis and development of breast cancer;the negative expression of Rb protein have also related with thegenesis of breast cancer. There was no significant relationshipbetween Rb expression and histologic type , tumor size , age ofpatient , histologic grades as well as lymphiod statues. Whenbreast cancer radical correction patients with the nagtive expre-ssion of Rb protein had much higher the peritoneal recurrenceand metastatic rate than that patients with the positive express-ion of Rb protein. This demonstrated that Rb protein as a pro-duct of anti-oncogene can evaluation the breast cancer pathoge-netic condition from the protein level. Rb protein may be a ob-jective standard to determine therapeutic regimen, evaluate pat-hogenetic condition and prognosis;THP can inhibite the proli-feration of MCF-7,with the concentration of THP increasing,the expression of dephosphorylated Rb protein had graduallyenhanced, there were positive correlation between the expre-ssion levels of dephosphorylated Rb protein and the prolifera-tion of THP inhibiting MCF-7 cells, the expression levels ofdephosphorylated Rb protein may predict the apoptosis of thecell of breast cancer.