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Evaluation of stem-like side population cells in a recurrent nasopharyngeal carcinoma cell line.

Hoe SL, Tan LP, Jamal J, Peh SC, Ng CC, Zhang WC, Ahmad M, Khoo AS - Cancer Cell Int. (2014)

Bottom Line: Fumitremorgin C (FTC), as opposed to verapamil, was effective in causing the cells to retain Hoechst 33342 dye.ABCG2, SOX2, TERT, MYC, Hedgehog, Notch, TGFβ and Wnt signalling pathway genes were significantly upregulated in the SP cells.Despite data from in vitro and gene expression experiments suggesting stem-like features, there was no significant difference in tumourigenic potential between SP and NSP cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology Unit, Cancer Research Centre, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia ; Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.

ABSTRACT

Background: Side population (SP) assay identifies cells with dye/drug extrusion ability, a characteristic of stem cells. Here, we determined if SP cells exist in a verified cell line originating from recurrent nasopharyngeal carcinoma (NPC) and a xenograft established from recurrent metastatic NPC. These cells were evaluated for stem-like properties via functional assays as well as for tumourigenicity.

Methods: We used Hoechst 33342 to identify the SP from non-SP (NSP) cells in HK1 NPC cell line and xeno-284 NPC xenograft. The cells were assayed for in vitro characteristics of cancer stem cells (CSC), gene expression and tumourigenicity ability. Student's t test was used to test for significance.

Results: Five to ten percent and less than 0.5% of HK1 and xeno-284 NPC cells, respectively, were SP cells. Fumitremorgin C (FTC), as opposed to verapamil, was effective in causing the cells to retain Hoechst 33342 dye. HK1 SP cells formed more holoclones, had more aldehyde dehydrogenase (ALDH) activity, divided asymmetrically and contained slow-proliferating cells. ABCG2, SOX2, TERT, MYC, Hedgehog, Notch, TGFβ and Wnt signalling pathway genes were significantly upregulated in the SP cells. However, despite these differences in vitro, both HK1 SP and NSP cells had an overall similar tumourigenic potential in vivo.

Conclusions: HK1 SP cells were ABCG2-specific as confirmed by FTC inhibition and gene expression data. Despite data from in vitro and gene expression experiments suggesting stem-like features, there was no significant difference in tumourigenic potential between SP and NSP cells. We conclude that SP assay alone is not sufficient to identify CSCs in HK1 cells. Our work also suggests the presence of a stem-cell like population among NPC cells which do not display increased tumourigenicity.

No MeSH data available.


Related in: MedlinePlus

SP cells grow at a slower rate compared to NSP cells. (A) Upon relabelling with Hoechst 33342 on day 21 post-sorting, recultured SP cells had divided asymmetrically into SP and NSP phenotypes. Recultured NSP cells had poorer asymmetric division ability. (B) Impedance-based cell growth assay indicated that the normalized growth rate for SP cells was lower than NSP cells.
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Fig3: SP cells grow at a slower rate compared to NSP cells. (A) Upon relabelling with Hoechst 33342 on day 21 post-sorting, recultured SP cells had divided asymmetrically into SP and NSP phenotypes. Recultured NSP cells had poorer asymmetric division ability. (B) Impedance-based cell growth assay indicated that the normalized growth rate for SP cells was lower than NSP cells.

Mentions: The ability of SP and NSP cells to grow and divide asymmetrically in vitro was examined by re-evaluating the percentages of SP and NSP fractions in the sorted cells. After three weeks of culture, the SP sorted cells had divided into both SP and NSP cells, with only 24.57 ± 7.97% of the cells still remaining as SP cells. On the other hand, 6.07 ± 1.74% of SP cells had reappeared in the NSP sorted cells (Figure 3A).Figure 3


Evaluation of stem-like side population cells in a recurrent nasopharyngeal carcinoma cell line.

Hoe SL, Tan LP, Jamal J, Peh SC, Ng CC, Zhang WC, Ahmad M, Khoo AS - Cancer Cell Int. (2014)

SP cells grow at a slower rate compared to NSP cells. (A) Upon relabelling with Hoechst 33342 on day 21 post-sorting, recultured SP cells had divided asymmetrically into SP and NSP phenotypes. Recultured NSP cells had poorer asymmetric division ability. (B) Impedance-based cell growth assay indicated that the normalized growth rate for SP cells was lower than NSP cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4195955&req=5

Fig3: SP cells grow at a slower rate compared to NSP cells. (A) Upon relabelling with Hoechst 33342 on day 21 post-sorting, recultured SP cells had divided asymmetrically into SP and NSP phenotypes. Recultured NSP cells had poorer asymmetric division ability. (B) Impedance-based cell growth assay indicated that the normalized growth rate for SP cells was lower than NSP cells.
Mentions: The ability of SP and NSP cells to grow and divide asymmetrically in vitro was examined by re-evaluating the percentages of SP and NSP fractions in the sorted cells. After three weeks of culture, the SP sorted cells had divided into both SP and NSP cells, with only 24.57 ± 7.97% of the cells still remaining as SP cells. On the other hand, 6.07 ± 1.74% of SP cells had reappeared in the NSP sorted cells (Figure 3A).Figure 3

Bottom Line: Fumitremorgin C (FTC), as opposed to verapamil, was effective in causing the cells to retain Hoechst 33342 dye.ABCG2, SOX2, TERT, MYC, Hedgehog, Notch, TGFβ and Wnt signalling pathway genes were significantly upregulated in the SP cells.Despite data from in vitro and gene expression experiments suggesting stem-like features, there was no significant difference in tumourigenic potential between SP and NSP cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology Unit, Cancer Research Centre, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia ; Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.

ABSTRACT

Background: Side population (SP) assay identifies cells with dye/drug extrusion ability, a characteristic of stem cells. Here, we determined if SP cells exist in a verified cell line originating from recurrent nasopharyngeal carcinoma (NPC) and a xenograft established from recurrent metastatic NPC. These cells were evaluated for stem-like properties via functional assays as well as for tumourigenicity.

Methods: We used Hoechst 33342 to identify the SP from non-SP (NSP) cells in HK1 NPC cell line and xeno-284 NPC xenograft. The cells were assayed for in vitro characteristics of cancer stem cells (CSC), gene expression and tumourigenicity ability. Student's t test was used to test for significance.

Results: Five to ten percent and less than 0.5% of HK1 and xeno-284 NPC cells, respectively, were SP cells. Fumitremorgin C (FTC), as opposed to verapamil, was effective in causing the cells to retain Hoechst 33342 dye. HK1 SP cells formed more holoclones, had more aldehyde dehydrogenase (ALDH) activity, divided asymmetrically and contained slow-proliferating cells. ABCG2, SOX2, TERT, MYC, Hedgehog, Notch, TGFβ and Wnt signalling pathway genes were significantly upregulated in the SP cells. However, despite these differences in vitro, both HK1 SP and NSP cells had an overall similar tumourigenic potential in vivo.

Conclusions: HK1 SP cells were ABCG2-specific as confirmed by FTC inhibition and gene expression data. Despite data from in vitro and gene expression experiments suggesting stem-like features, there was no significant difference in tumourigenic potential between SP and NSP cells. We conclude that SP assay alone is not sufficient to identify CSCs in HK1 cells. Our work also suggests the presence of a stem-cell like population among NPC cells which do not display increased tumourigenicity.

No MeSH data available.


Related in: MedlinePlus