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SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed
Andrea Slusser-Nore1,2, Jennifer L Larson-Casey3, Ruowen Zhang1
1Department of Pathology, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota, United States of America.
Background:
This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As(+3)) and cadmium (Cd(+2))-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd(+2)-and As(+3)-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice.
Methods:
Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As(+3)-and Cd(+2)-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres.
Results:
It was shown that the As(+3)-and Cd(+2)-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As(+3)-and Cd(+2)-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells.
Conclusions:
Tumor initiating cells isolated from SPARC-transfected As(+3)-and Cd(+2)-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.
Insights
This study investigated SPARC expression in transformed UROtsa cells. Tumor-initiating cells inherently suppress SPARC mRNA, even after transfection, impacting tumor formation.
Area of Science:
- Oncology
- Cell Biology
- Toxicology
Background:
- SPARC expression was previously found to be downregulated in arsenite (As(+3)) and cadmium (Cd(+2))-transformed UROtsa cells and their derived tumors.
- This downregulation was observed compared to parental UROtsa cells.
Purpose of the Study:
- To determine the effect of SPARC re-expression on tumor formation in immune-compromised mice.
- To investigate the ability of SPARC-transfected cells to form tumors.
Main Methods:
- Stable transfection of As(+3)- and Cd(+2)-transformed UROtsa cell lines with a SPARC expression vector.
- Analysis of SPARC expression using real-time PCR, western blotting, immunohistochemistry, and immunofluorescence.
- Tumorigenicity assessment via subcutaneous injection into immune-compromised mice.
Main Results:
- SPARC-transfected cells successfully expressed SPARC mRNA and secreted protein.
- Tumors derived from SPARC-transfected cells showed no SPARC expression.
- Urospheres from SPARC-transfected cells exhibited only background SPARC levels, yet remained tumorigenic.
Conclusions:
- Tumor-initiating cells derived from SPARC-transfected transformed cell lines possess an intrinsic mechanism to suppress SPARC mRNA expression.
- This inherent suppression suggests a role for SPARC in regulating tumor-initiating cell populations.
