- Luteinizing hormone (LH) is secreted in low-amplitude pulses in sexually intact dogs to stimulate synthesis of estrogen and testosterone in females and males, respectively. LH is mainly considered a reproductive hormone, but LH receptors (LHR) have been identified in non-reproductive tissues, including in canine lymphoma and isolated canine T-cell lymphoma (CTCL) cells. The objective of this study was to determine if the LHR in CTCL cells was functional. It was hypothesized that stimulation of the LHR by human chorionic gonadotropin (hCG) or canine LH (cLH) would induce proliferation of CTCL in vitro. Immortalized T-cell lines from three dogs with multicentric T-cell lymphoma donated from Dr. Takuya Mizuno at Yamaguchi University, Japan, were plated in 96-well plates in RPMI 1640 (phenol and protein free) media at 37℃ with 5% CO2. For each cell line, standard curves from 10,000 to 500,000 cells/well were plated in triplicate. Increasing concentrations of hCG (4 U/mL to 40,000 U/mL) or cLH (0.002 ng/mL to 20 ng/mL) were added to wells containing 100,000 cells plated in triplicate. Plates were incubated for 24, 48, and 72 hours for hCG or 48, 72, 96 and 120 hours for cLH before cells were counted using an MTT cell proliferation assay kit following the manufacturer's instructions. Average±SD cell number was compared between hormone concentrations using a one-way ANOVA. Significance was defined as P<0.05.
Activation of LHR in isolated CTCL cells induced significant cell proliferation in all three cell lines with both hCG and cLH but varied between concentrations and incubation times. The highest amount of proliferation from hCG occurred at the 40,000 U/mL concentration following 72 hours of incubation in all three cell lines. The highest amount of proliferation from cLH occurred at the 2 ng/mL concentration following 96 hours of incubation in all three cell lines. However, there was a significant decrease in cell counts following administration of cLH at the highest concentration (20 ng/mL) at all incubation times. This is the first study to provide evidence that LHR in non-reproductive tissues are functional. The current research provides a possible theory as to why gonadectomized dogs are 3-4 times more likely to develop lymphoma. Clinical trials are planned to include LH down-regulation with conventional chemotherapy in efforts to prolong survival times in dogs.
Keywords: cancer, dog, human chorionic gonadotropin, lymphocyte, MTT assay