Nocodazole treatment decreases expression of pluripotency markers Nanog and Oct4 in human embryonic stem cells

TY - JOUR

T1 - Nocodazole treatment decreases expression of pluripotency markers Nanog and Oct4 in human embryonic stem cells

AU - Kallas, Ade

AU - Pook, Martin

AU - Maimets, Martti

AU - Zimmermann, Külli

AU - Maimets, Toivo

PY - 2011/4/29

Y1 - 2011/4/29

N2 - Nocodazole is a known destabiliser of microtubule dynamics and arrests cell-cycle at the G2/M phase. In the context of the human embryonic stem cell (hESC) it is important to understand how this arrest influences the pluripotency of cells. Here we report for the first time the changes in the expression of transcription markers Nanog and Oct4 as well as SSEA-3 and SSEA-4 in human embryonic cells after their treatment with nocodazole. Multivariate permeabilised-cell flow cytometry was applied for characterising the expression of Nanog and Oct4 during different cell cycle phases. Among untreated hESC we detected Nanog-expressing cells, which also expressed Oct4, SSEA-3 and SSEA-4. We also found another population expressing SSEA-4, but without Nanog, Oct4 and SSEA-3 expression. Nocodazole treatment resulted in a decrease of cell population positive for all four markers Nanog, Oct4, SSEA-3, SSEA-4. Nocodazole-mediated cell-cycle arrest was accompanied by higher rate of apoptosis and upregulation of p53. Twenty-four hours after the release from nocodazole block, the cell cycle of hESC normalised, but no increase in the expression of transcription markers Nanog and Oct4 was detected. In addition, the presence of ROCK-2 inhibitor Y-27632 in the medium had no effect on increasing the expression of pluripotency markers Nanog and Oct4 or decreasing apoptosis or the level of p53. The expression of SSEA-3 and SSEA-4 increased in Nanog-positive cells after wash-out of nocodazole in the presence and in the absence of Y-27632. Our data show that in hESC nocodazole reversible blocks cell cycle, which is accompanied by irreversible loss of expression of pluripotency markers Nanog and Oct4.

AB - Nocodazole is a known destabiliser of microtubule dynamics and arrests cell-cycle at the G2/M phase. In the context of the human embryonic stem cell (hESC) it is important to understand how this arrest influences the pluripotency of cells. Here we report for the first time the changes in the expression of transcription markers Nanog and Oct4 as well as SSEA-3 and SSEA-4 in human embryonic cells after their treatment with nocodazole. Multivariate permeabilised-cell flow cytometry was applied for characterising the expression of Nanog and Oct4 during different cell cycle phases. Among untreated hESC we detected Nanog-expressing cells, which also expressed Oct4, SSEA-3 and SSEA-4. We also found another population expressing SSEA-4, but without Nanog, Oct4 and SSEA-3 expression. Nocodazole treatment resulted in a decrease of cell population positive for all four markers Nanog, Oct4, SSEA-3, SSEA-4. Nocodazole-mediated cell-cycle arrest was accompanied by higher rate of apoptosis and upregulation of p53. Twenty-four hours after the release from nocodazole block, the cell cycle of hESC normalised, but no increase in the expression of transcription markers Nanog and Oct4 was detected. In addition, the presence of ROCK-2 inhibitor Y-27632 in the medium had no effect on increasing the expression of pluripotency markers Nanog and Oct4 or decreasing apoptosis or the level of p53. The expression of SSEA-3 and SSEA-4 increased in Nanog-positive cells after wash-out of nocodazole in the presence and in the absence of Y-27632. Our data show that in hESC nocodazole reversible blocks cell cycle, which is accompanied by irreversible loss of expression of pluripotency markers Nanog and Oct4.

KW - Animals

KW - Antigens, Tumor-Associated, Carbohydrate/biosynthesis

KW - Antineoplastic Agents/pharmacology

KW - Apoptosis

KW - Cell Cycle/drug effects

KW - Cell Line, Tumor

KW - Embryonic Stem Cells/cytology

KW - Flow Cytometry/methods

KW - Homeodomain Proteins/biosynthesis

KW - Humans

KW - Mice

KW - Mitosis

KW - Nanog Homeobox Protein

KW - Nocodazole/therapeutic use

KW - Octamer Transcription Factor-3/biosynthesis

KW - Stage-Specific Embryonic Antigens/biosynthesis

U2 - 10.1371/journal.pone.0019114

DO - 10.1371/journal.pone.0019114

M3 - Journal article

C2 - 21559451

SN - 1932-6203

VL - 6

SP - e19114

JO - PLoS Computational Biology

JF - PLoS Computational Biology

IS - 4

ER -