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α TAT1 downregulation induces mitotic catastrophe in HeLa and A549 cells

View Article: PubMed Central - PubMed

ABSTRACT

α-Tubulin acetyltransferase 1 (αTAT1) controls reversible acetylation on Lys40 of α-tubulin and modulates multiple cellular functions. αTAT1 depletion induced morphological defects of touch receptor neurons in Caenorhabditis elegans and impaired cell adhesion and contact inhibition in mouse embryonic fibroblasts, however, no morphological or proliferation defects in human RPE-hTERT cells were found after αTAT1-specific siRNA treatment. Here, we compared the effect of three αTAT1-specific shRNAs on proliferation and morphology in two human cell lines, HeLa and A549. The more efficient two shRNAs induced mitotic catastrophe in both cell lines and the most efficient one also decreased F-actin and focal adhesions. Further analysis revealed that αTAT1 downregulation increased γ-H2AX, but not other DNA damage markers p-CHK1 and p-CHK2, along with marginal change in microtubule outgrowth speed and inter-kinetochore distance. Overexpression of αTAT1 could not precisely mimic the distribution and concentration of endogenous acetylated α-tubulin (Ac-Tu), although no overt phenotype change was observed, meanwhile, this could not completely prevent αTAT1 downregulation-induced deficiencies. We therefore conclude that efficient αTAT1 downregulation could impair actin architecture and induce mitotic catastrophe in HeLa and A549 cells through mechanisms partly independent of Ac-Tu.

No MeSH data available.


αTAT1 downregulation impaired cell growth, and increased 4N and multiploid population. (a) Phase contrast images of live cells showing increased rounded and abnormally large (arrows) cells after sh #1 treatment. Sacle bar, 100 μm. (b) MTT assay of cell growth. In each treatment, the measurement of day 0 was set as 1 in the arbitrary unit and the fold change of day 1–3 to its day 0 is displayed; data shown in mean±S.D. (N=3). (c and d) Representative flowcytometry results of cell cycle distribution using propidium iodide to reveal the DNA content and histone H3 pSer28 as the M-phase marker. (e–g) Statistical results of flowcytometry data; 30 000 cells were analyzed in each sample and data shown in mean±S.D. (N=4). Proportion of 4N and histone H3 pSer28 positive cells are displayed in g. *P<0.05, **P<0.01.
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fig3: αTAT1 downregulation impaired cell growth, and increased 4N and multiploid population. (a) Phase contrast images of live cells showing increased rounded and abnormally large (arrows) cells after sh #1 treatment. Sacle bar, 100 μm. (b) MTT assay of cell growth. In each treatment, the measurement of day 0 was set as 1 in the arbitrary unit and the fold change of day 1–3 to its day 0 is displayed; data shown in mean±S.D. (N=3). (c and d) Representative flowcytometry results of cell cycle distribution using propidium iodide to reveal the DNA content and histone H3 pSer28 as the M-phase marker. (e–g) Statistical results of flowcytometry data; 30 000 cells were analyzed in each sample and data shown in mean±S.D. (N=4). Proportion of 4N and histone H3 pSer28 positive cells are displayed in g. *P<0.05, **P<0.01.

Mentions: Increased rounded, detached, and abnormally large sized cells were observed after 72 h of sh #1 and sh #2, but not sh #3 treatment in both cell lines (Figure 3a). MTT assay also revealed that sh #1 and sh #2 impaired cell growth (Figure 3b). The effect of sh #3 was comparable to control shRNA, which was consistent with the previous study performed in REP-hTRET cells.7 Analyzing their cell cycle distribution using flowcytometry with propidium iodide staining revealed that only sh #1 and sh #2 increased the 4N as well as multiploid cell population (Figures 3c, e and f). In addition, histone H3 pSer28 staining showed that sh #1 also increased the M-phase population but not sh #2 and HeLa was more susceptible than A549 in this aspect (Figures 3d and g), implying that the increased 4N cells by sh #1 were a combination of normal G2/M and tetraploid G1-phase cells while the increase seen after sh #2 treatment was mostly from normal G2 and tetraploid G1-phase cells.


α TAT1 downregulation induces mitotic catastrophe in HeLa and A549 cells
αTAT1 downregulation impaired cell growth, and increased 4N and multiploid population. (a) Phase contrast images of live cells showing increased rounded and abnormally large (arrows) cells after sh #1 treatment. Sacle bar, 100 μm. (b) MTT assay of cell growth. In each treatment, the measurement of day 0 was set as 1 in the arbitrary unit and the fold change of day 1–3 to its day 0 is displayed; data shown in mean±S.D. (N=3). (c and d) Representative flowcytometry results of cell cycle distribution using propidium iodide to reveal the DNA content and histone H3 pSer28 as the M-phase marker. (e–g) Statistical results of flowcytometry data; 30 000 cells were analyzed in each sample and data shown in mean±S.D. (N=4). Proportion of 4N and histone H3 pSer28 positive cells are displayed in g. *P<0.05, **P<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4979442&req=5

fig3: αTAT1 downregulation impaired cell growth, and increased 4N and multiploid population. (a) Phase contrast images of live cells showing increased rounded and abnormally large (arrows) cells after sh #1 treatment. Sacle bar, 100 μm. (b) MTT assay of cell growth. In each treatment, the measurement of day 0 was set as 1 in the arbitrary unit and the fold change of day 1–3 to its day 0 is displayed; data shown in mean±S.D. (N=3). (c and d) Representative flowcytometry results of cell cycle distribution using propidium iodide to reveal the DNA content and histone H3 pSer28 as the M-phase marker. (e–g) Statistical results of flowcytometry data; 30 000 cells were analyzed in each sample and data shown in mean±S.D. (N=4). Proportion of 4N and histone H3 pSer28 positive cells are displayed in g. *P<0.05, **P<0.01.
Mentions: Increased rounded, detached, and abnormally large sized cells were observed after 72 h of sh #1 and sh #2, but not sh #3 treatment in both cell lines (Figure 3a). MTT assay also revealed that sh #1 and sh #2 impaired cell growth (Figure 3b). The effect of sh #3 was comparable to control shRNA, which was consistent with the previous study performed in REP-hTRET cells.7 Analyzing their cell cycle distribution using flowcytometry with propidium iodide staining revealed that only sh #1 and sh #2 increased the 4N as well as multiploid cell population (Figures 3c, e and f). In addition, histone H3 pSer28 staining showed that sh #1 also increased the M-phase population but not sh #2 and HeLa was more susceptible than A549 in this aspect (Figures 3d and g), implying that the increased 4N cells by sh #1 were a combination of normal G2/M and tetraploid G1-phase cells while the increase seen after sh #2 treatment was mostly from normal G2 and tetraploid G1-phase cells.

View Article: PubMed Central - PubMed

ABSTRACT

&alpha;-Tubulin acetyltransferase 1 (&alpha;TAT1) controls reversible acetylation on Lys40 of &alpha;-tubulin and modulates multiple cellular functions. &alpha;TAT1 depletion induced morphological defects of touch receptor neurons in Caenorhabditis elegans and impaired cell adhesion and contact inhibition in mouse embryonic fibroblasts, however, no morphological or proliferation defects in human RPE-hTERT cells were found after &alpha;TAT1-specific siRNA treatment. Here, we compared the effect of three &alpha;TAT1-specific shRNAs on proliferation and morphology in two human cell lines, HeLa and A549. The more efficient two shRNAs induced mitotic catastrophe in both cell lines and the most efficient one also decreased F-actin and focal adhesions. Further analysis revealed that &alpha;TAT1 downregulation increased &gamma;-H2AX, but not other DNA damage markers p-CHK1 and p-CHK2, along with marginal change in microtubule outgrowth speed and inter-kinetochore distance. Overexpression of &alpha;TAT1 could not precisely mimic the distribution and concentration of endogenous acetylated &alpha;-tubulin (Ac-Tu), although no overt phenotype change was observed, meanwhile, this could not completely prevent &alpha;TAT1 downregulation-induced deficiencies. We therefore conclude that efficient &alpha;TAT1 downregulation could impair actin architecture and induce mitotic catastrophe in HeLa and A549 cells through mechanisms partly independent of Ac-Tu.

No MeSH data available.