2018: LUTEINIZING HORMONE RECEPTOR EXPRESSION IN CANINE T-LYMPHOMACELL LINES


Luteinizing hormone receptor expression in canine t-lymphoma cell lines 
Sabrina Gust,a Alyssa Ettinger,a Takuya Mizuno,b Michelle Kutzlera 

aDepartment of Animal and Rangeland Sciences, Oregon State University, Corvallis, OR; bLaboratory of 
Veterinary Internal Medicine, The United Graduate School of Veterinary Medicine, Yamaguchi 

University, 1677 – 1 Yoshida, Yamaguchi 753 – 8515, Japan 
 

Introduction 
 Canine lymphoma is a common malignant tumor in dogs. Luteinizing hormone receptors (LHR) 
have been found in normal and neoplastic canine lymphatic tissue.1 The LHRs have also been found in 
human leukemia cell lines.2 Therefore, we hypothesized that LHR would also be present in canine 
lymphoma cell lines. The objectives of the study were to determine if LHR is expressed in cultured canine 
T-lymphoma cells and to quantify the level of cellular expression of LHR in 3 different cell lines.  
 
Methods 
 T-lymphoma cell lines (CLC, EMA, CLK) established from primary canine lymphomas were 
generously donated from Yamaguchi University (Yamaguchi, Japan). Following receipt, cells were 
thawed and cultured in R10 complete medium (RPMI1640; VWR Life Science, Visalia, CA) 
supplemented with 10% fetal bovine serum (#10803-034; VWR) and 100 U/ml penicillin with 100 μg/ml 
streptomycin (#SV30082.01; HyClone, South Logan, UT) at 37ºC at a humidified 5% CO2 incubator. 
Cells were cultured in fresh R10 complete media every 2-3 days at a ratio of 1:4 (EMA, CLK) or 1:9 
(CLC). At 75-85% confluence, cells were washed to remove culture media and transferred to Eppendorf 
tubes. Nonspecific binding was blocked with Mouse Seroblock FcR (1:10 dilution; Bio-Rad Antibodies, 
Richmond, CA). Cells were incubated with goat polyclonal LHR (1:50 dilution; SC-26341, Santa Cruz 
Biotechnology, Dallas, TX) for 20 minutes at room temperature. Cells were washed and then incubated 
with goat f(ab’)2 IgG negative control:RPE (1:10 dilution; Bio-Rad Antibodies, Richmond, CA). The 
primary antibody was omitted in the negative controls. The cells were analyzed on a CytoFLEX flow 
cytometer (Beckman Coulter, Indianapolis, IN) at the Oregon State University Core Facility.   
 
Results 
 The CLC, EMA, and CLK cell lines expressed LHR in 45%, 10%, and 35% of cells. In all cell 
lines, the cell population that expressed LHR was smaller in size (forward scatter) and more granular 
(side scatter). There was no positive staining evident in any of the negative control samples.  
 
Discussion 
 This is the first study to show LHR expression in canine lymphoma cell lines. Our laboratory is 
currently investigating the pro-neoplastic effects of LHR activation (e.g. cell proliferation) using these 
cell lines.    
 
Keywords: Cell culture, dog, flow cytometry, luteinizing hormone, lymphoma 
 
Acknowledgment 
 Research funded by Oregon State University Incentives Program 

References 
1. Ettinger A, Zwida K, Kutzler M: Normal and neoplastic canine lymphocytes express luteinizing hormone receptors. 

Clin Therio 2017;9:428. 
2. Abdelbaset-Ismail A, Borkowska S, Janowska-Wieczorek A, et al: Novel evidence that pituitary gonadotropins directly 

stimulate human leukemic cells-studies of myeloid cell lines and primary patient AML and CML cells. Oncotarget 
2016;7:3033-3046.  

 

Clinical Theriogenology • Volume 10,  Number 3 • September 2018321