Zhang et al. BMC Cancer 2011, 11:32 http://www.biomedcentral.com/1471-2407/11/32 Furthermore, the transient and stable transfection of a RRIG1 expression vector resulted in growth inhibition of esophageal and prostate cancer cells [5,6,8]. Esophageal cancer cells transfected with RRIG1 also showed reduced tumorigenicity in nude mice [6]. These data strongly indicate that RRIG1 plays an important role in suppres- sing the development or progression of human cancers [5,6,8]. At the level of signal transduction, expression of RRIG1 inhibited Src phosphorylation and RhoA activa- tion, which is believed to be causally linked to reduced colony formation, invasion, and proliferation in esopha- geal and prostatic cancer cells. In contrast, transfection of antisense RRIG1 increased RhoA activity and f-actin formation and led to increased colony formation, inva- sion, and proliferation in these cells [5,8]. In this study, we first determined the correlation of RRIG1 expression in breast tissue specimens with the clinicopathologic characteristics of the patients and then examined the effect of RRIG1 expression on breast can- cer growth and invasion. We also explored the changes in expression and phosphorylation or activation of sev- eral relevant proteins following experimental elevation or knockdown of RRIG1 expression in breast cancer cells. Our data indicate that RRIG1 may function as a tumor-suppressor gene in breast cancer. Methods Immunohistochemistry Paraffin blocks from breast tissue specimens were obtained from the Department of Pathology, Anhui Medical University, Hefei, China, guided by a protocol (#Lab08-015) approved by the institutional review board. Patients gave consent for the use of their tissue specimens in this study. These tissue specimens con- sisted of samples from 15 normal mammary glands, 9 atypical hyperplasia lesions, 10 cases of ductal carcinoma in situ, and 77 cases of invasive breast cancer. For immunohistochemical analysis, these paraffin sections together with paraffin sections from the organotypic cul- tures were prepared and stained for RRIG1 as previously reported [6]. Briefly, the tissue sections were deparaffi- nized twice in xylene for 10 min each and rehydrated in a series of ethanol (100%-50%) and then subjected to antigen retrieval with 0.01 M citric buffer in a pressure cooker for 5 min, followed by blocking of tissue endo- genous peroxidase activity with H2O2 treatment. The processed tissue sections were then incubated with 100 μL of 20% normal goat serum in phosphate-buffered sal- ine (PBS) and anti-RRIG1 antibody (custom-made by Lampire Biological Laboratories, Pipersville, PA, with the CAADGLRKPQVHSARAL peptide as the antigen) at 1:500 dilutions with PBS overnight. The next day, the sections were gently washed three times with PBS and once with PBS containing 0.1% Tween 20. They were Page 2 of 12 then subjected to sequential incubations with a second antibody (goat anti-rabbit IgG; Vector Laboratories, Bur- lingame, CA) and the ABC solution (in the dark) for 30 min each, with washing between incubations to remove unbound antibodies. The sections were subject to color development with 9-ethylcarbazol-3-amine for 15 min and counterstained with hematoxylin for 1 min. After being covered with a cover slip, the sections were viewed and scored under a microscope. A semiquantita- tive scoring system was used to score both the staining intensity and the percentage of staining in the tissue sections. The staining intensity was scored as follows: 0, no staining; +, weak staining; ++ positive staining; and + ++, very strong staining. The percentage of staining was scored as follows: 0, no staining; +, less than 10% of tumor cells stained; ++, 10-50%; and +++, more than 50% tumor cells stained positive. Both intensity and per- centage of staining with 0 or + were considered as nega- tive cases, while both intensity and percentage of staining with ++ and +++ were considered as positive cases. Cell lines and culture The human breast cancer cell lines MCF-7, MDA-MB- 231, T47D, SK-Br-3, ZR75-1, and MDA-MB-435 were obtained from ATCC (Manassas, VA) and grown in Dulbecco’s modified Eagle’s minimal essential medium (DMEM), supplemented with 10% fetal bovine serum, at 37ºC in a humidified atmosphere of 95% air and 5% CO2. Esophageal cancer TE-8, the stably RAR-b2- transfected TE-8S22, stably RRIG1-transfected TE-8- RRIG1 cells, and prostate cancer PC3 cells were from our previous study [5]. These cell lines were grown in Dulbecco’s modified Eagle’s minimal essential medium (DMEM), supplemented with 10% fetal bovine serum, at 37ºC in a humidified atmosphere of 95% air and 5% CO2. The growth medium for the stable gene trans- fected cells also contained 200 μg/ml G418 (Invitrogen, Carlsbad, CA). RRIG1 expression vector and transient gene transfection RRIG1 sense and antisense open-reading frames were inserted into pCDNA3.1 plasmid as reported in our pre- vious studies [5,6]. The plasmids were used for transient transfection into MDA-MB-231, MDA-MB-435, and T47D with FuGENE 6 (Roche Diagnostics, Indianapolis, IN) or Lipofectamine™ 2000 (Invitrogen). Thirty-six hours after the transfection, the cells were selected with 1200 μ g/mL G418 (Invitrogen) for 5 days. The total cellular protein was extracted from the cells and sub- jected to Western blotting analysis. In duplicate experi- ments, RNA was extracted from the cells and subjected to semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of gene expression as