Expression of Aldehyde dehydrogenase (ALDH) in the breast stem cell line D492 and relations to stem cell properties

Aldehyde dehydrogenases (ALDH) are enzymes expressed in both normal and cancer stem cells (CSC). CSCs are hypothesized to arise from normal stem cells and acquire a malignant phenotype through series of genetic mutations. Current therapies often fail to eradicate CSCs causing metastatic relapse. Further characterization of mammary stem cells (MaSC) may increase the understanding of normal and cancer stem cells and contribute to identification of novel therapeutic targets in breast cancer. The D492 and D492M cell lines were used in the current study. D492 is a breast epithelial cell line with stem cell properties. It generates both luminal and myoepithelial cells and forms branching colonies in 3D culture similar to structures seen in vivo. D492M is a subline of D492 that has adapted mesenchymal character through epithelial-mesenchymal transition (EMT). EMT is a process where epithelial cells gain a mesenchymal phenotype with increased motility and lose their traditional cell-cell contacts and polar epithelial phenotype. EMT is a process occurring naturally during development and tissue repair but has also been linked to invasiveness and metastasis in cancer. In an attempt to characterize the MaSC subpopulation, various markers were used that have been linked to normal and malignant stem cells with focus on ALDH activity. ALDH are enzymes catalyzing the NAD(P)+ dependent oxidation of aldehydes to carboxyl acids. ALDH activity is evaluated with the Aldefluor assay, where ALDH converts a fluorescence substrate to a product that is contained intracellular and detected by FACS. In this study D492 and D492M were analyzed in terms of ALDH activity and stem cell marker expression. When compared, D492 showed higher ALDH activity than D492M. None of the selected surface stem cell marker turned out useful for the characterization and isolation of the stem cell subpopulation with high ALDH activity. A connection was suggested between ALDH activity and side scatter (SSC) that needs further analysis. Increased passage number did not significantly affect ALDH activity in D492. D492 cells were sorted in FACSAria based on their ALDH activity into fractions containing high, medium and low ALDH levels and seeded in 3D culture (Matrigel) with and without breast endothelial cells (BRENC). Colonies formed earlier from ALDH high cells and had a stronger tendency to form branching structures. According to a western blot, ALDH high cells had increased expression of the stem cell marker Thy-1 compared to ALDH low cells and proliferated faster when seeded at a low density. Results from this preliminary study suggest that high expression of ALDH may predict stem cell properties in D492, however further studies including additional stem cell assays are needed to verify these findings.

Author dc:creator

• Katrín Birna Pétursdóttir 1988-

Contributors dc:contributor

• Háskóli Íslands

• Lífeindafræði

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/1946/20463

OAI identifier oai:identifier

oai:skemman.is:1946/20463