J Vet Sci.  2013 Dec;14(4):481-486. 10.4142/jvs.2013.14.4.481.

Evaluation of a side population of canine lymphoma cells using Hoechst 33342 dye

• Kim MC
• D'Costa S
• Suter S
• Kim Y

• KMID: 1712316
• DOI: http://doi.org/10.4142/jvs.2013.14.4.481

Abstract

Cancer stem cell (CSC) research has increased exponentially to gain further insight into the mechanisms underlying both carcinogenesis and chemotherapy resistance. The present study was performed to explore the potential value of a side population (SP) assay for identifying and characterizing putative CSCs among canine lymphoma cells. Canine lymphoma cells from cell lines and clinical samples were subjected to the SP assay consisting of Hoechst 33342 staining and subsequent flow cytometric analysis. The SP assay revealed various amounts of a SP fraction among the canine lymphoma cells. The percentages of SP were not affected by inhibitors of membrane transporters, verapamil hydrochloride, or fumitremorgin C. Most of the canine lymphoma cells expressed high levels of Bmi-1 and membrane transporter proteins such as ABCG2 and phosphorylated (p)-glycoprotein. This investigation lays the groundwork for further studies of the biological behaviors and molecular characteristics of CSCs in cases of canine lymphoma.

Keyword

dog; flow cytometry; Hoechst 33342; lymphoma; side population

MeSH Terms

Animals
Benzimidazoles/*metabolism
Cell Line, Tumor
Dog Diseases/*diagnosis/drug therapy/pathology
Dogs
Flow Cytometry/*methods/veterinary
Fluorescent Dyes/*metabolism
Gene Expression Regulation, Developmental
Lymphoma/diagnosis/drug therapy/pathology/*veterinary
Neoplastic Stem Cells/drug effects/*metabolism/pathology
Side-Population Cells/drug effects/*metabolism/pathology
Benzimidazoles
Fluorescent Dyes

Figure

• 

  Fig. 1 Flow cytometric analysis of canine lymphoma cells. Hoechst 33342 staining profiles were acquired by detecting Hoechst Blue with a 424/44 broad pass (BP) filter and Hoechst Red with a 675/20 BP filter. The left column (Hoechst) represents Hoechst 33342 staining and flow cytometric analysis. The middle column represents the effects of verapamil hydrochloride on Hoechst 333342 staining. The right column represents the effects of fumitremorgin C (FTC) on Hoechst 33342 staining. The efflux of Hoechst 33342 dye was not significantly inhibited by treatment with verapamil hydrochloride (middle column) or fumitremorgin C (right column). GL-1: a canine B lymphoma cell line, CL-1: a canine T lymphoma cell line, 17-71: a canine B cell lymphoid leukemia cell line, Lymphoma: lymphoma cells obtained from a clinical case of canine B cell lymphoma.

• 

  Fig. 2 Western blot assay for ABCG2 and p-glycoprotein expression in canine lymphoma cell lines. High levels of ABCG2 protein were detected in 17-71 and CL-1 cells but not in GL-1 cells. In contrast, p-glycoprotein was highly expressed in GL-1 cells while 17-71 and CL-1 cells had a lower level of p-glycoprotein. ABCG2: ATP-binding cassette, sub-family G, member 2.

• 

  Fig. 3 Western blot assay for Bmi-1 and p-glycoprotein expression in canine lymphoma cells. High levels of Bmi-1 protein were detected in GL-1 and CL-1 cells as well as one of the five clinical lymphoma cases. Cells from the four other clinical cases of canine lymphomas expressed lower levels of Bmi-1. P-glycoprotein was highly expressed in one clinical lymphoma case, but the four other cases expressed lower levels of this protein. GAPDH was included as a loading control. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 4 Western blot assay for Nanog and ABCG2 expression in canine lymphoma cells. All of the lymphoma cells examined had either low or non-existent levels of Nanog expression. NCCIT cells, an embryonic carcinoma cell line, were included as a positive control for Nanog expression. Higher levels of ABCG2 protein were detected in the clinical cases of canine lymphomas compared to the canine lymphoma cell lines. GAPDH was included as a loading control.

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