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Regulation of nucleotide excision repair by nuclear lamin b1.

Butin-Israeli V, Adam SA, Goldman RD - PLoS ONE (2013)

Bottom Line: Silencing LB1 expression induced G1 cell cycle arrest without significant apoptosis.The arrested cells are unable to mount a timely and effective response to DNA damage induced by UV irradiation.Our findings are relevant to understanding the relationship between the loss of LB1 expression, DNA damage signaling, and replicative senescence.

View Article: PubMed Central - PubMed

Affiliation: The Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.

ABSTRACT
The nuclear lamins play important roles in the structural organization and function of the metazoan cell nucleus. Recent studies on B-type lamins identified a requirement for lamin B1 (LB1) in the regulation of cell proliferation in normal diploid cells. In order to further investigate the function of LB1 in proliferation, we disrupted its normal expression in U-2 OS human osteosarcoma and other tumor cell lines. Silencing LB1 expression induced G1 cell cycle arrest without significant apoptosis. The arrested cells are unable to mount a timely and effective response to DNA damage induced by UV irradiation. Several proteins involved in the detection and repair of UV damage by the nucleotide excision repair (NER) pathway are down-regulated in LB1 silenced cells including DDB1, CSB and PCNA. We propose that LB1 regulates the DNA damage response to UV irradiation by modulating the expression of specific genes and activating persistent DNA damage signaling. Our findings are relevant to understanding the relationship between the loss of LB1 expression, DNA damage signaling, and replicative senescence.

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Activation of key signaling proteins that mediate early G1 arrest.Protein levels in silenced and control cells were detected by immunoblotting at day 3 after LB1 silencing. GAPDH served as a loading control. This experiment was repeated 4 times.
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pone-0069169-g002: Activation of key signaling proteins that mediate early G1 arrest.Protein levels in silenced and control cells were detected by immunoblotting at day 3 after LB1 silencing. GAPDH served as a loading control. This experiment was repeated 4 times.

Mentions: In order to analyze the G1 arrest in more detail, we carried out immunoblotting analyses of factors known to regulate progression through the G1 phase of the cell cycle including p53, ATM, ATR, CHK1 and CHK2 (Fig. 2). We detected a significant increase in p53 levels in LB1 silenced cells (Fig. 2). In addition, we found that the level of ATR increased and that both ATR and its substrate CHK1 showed increased phosphorylation demonstrating their activation [27], [28] (Fig. 2). Phosphorylation of ATM was not significantly altered and the phosphorylation of its downstream effector CHK2 could not be detected. Importantly, we also found that the expression of proliferating cell nuclear antigen (PCNA), a key component of the DNA replication machinery which is normally synthesized at the end of G1 [29], was reduced to ∼10% of controls (Fig. 2). Moreover, PCNA mRNA levels decreased to ∼30% of controls as determined by qRT-PCR. Taken together, these results show that LB1 silenced cells are arrested in the early G1 phase of the cell cycle.


Regulation of nucleotide excision repair by nuclear lamin b1.

Butin-Israeli V, Adam SA, Goldman RD - PLoS ONE (2013)

Activation of key signaling proteins that mediate early G1 arrest.Protein levels in silenced and control cells were detected by immunoblotting at day 3 after LB1 silencing. GAPDH served as a loading control. This experiment was repeated 4 times.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3722182&req=5

pone-0069169-g002: Activation of key signaling proteins that mediate early G1 arrest.Protein levels in silenced and control cells were detected by immunoblotting at day 3 after LB1 silencing. GAPDH served as a loading control. This experiment was repeated 4 times.
Mentions: In order to analyze the G1 arrest in more detail, we carried out immunoblotting analyses of factors known to regulate progression through the G1 phase of the cell cycle including p53, ATM, ATR, CHK1 and CHK2 (Fig. 2). We detected a significant increase in p53 levels in LB1 silenced cells (Fig. 2). In addition, we found that the level of ATR increased and that both ATR and its substrate CHK1 showed increased phosphorylation demonstrating their activation [27], [28] (Fig. 2). Phosphorylation of ATM was not significantly altered and the phosphorylation of its downstream effector CHK2 could not be detected. Importantly, we also found that the expression of proliferating cell nuclear antigen (PCNA), a key component of the DNA replication machinery which is normally synthesized at the end of G1 [29], was reduced to ∼10% of controls (Fig. 2). Moreover, PCNA mRNA levels decreased to ∼30% of controls as determined by qRT-PCR. Taken together, these results show that LB1 silenced cells are arrested in the early G1 phase of the cell cycle.

Bottom Line: Silencing LB1 expression induced G1 cell cycle arrest without significant apoptosis.The arrested cells are unable to mount a timely and effective response to DNA damage induced by UV irradiation.Our findings are relevant to understanding the relationship between the loss of LB1 expression, DNA damage signaling, and replicative senescence.

View Article: PubMed Central - PubMed

Affiliation: The Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.

ABSTRACT
The nuclear lamins play important roles in the structural organization and function of the metazoan cell nucleus. Recent studies on B-type lamins identified a requirement for lamin B1 (LB1) in the regulation of cell proliferation in normal diploid cells. In order to further investigate the function of LB1 in proliferation, we disrupted its normal expression in U-2 OS human osteosarcoma and other tumor cell lines. Silencing LB1 expression induced G1 cell cycle arrest without significant apoptosis. The arrested cells are unable to mount a timely and effective response to DNA damage induced by UV irradiation. Several proteins involved in the detection and repair of UV damage by the nucleotide excision repair (NER) pathway are down-regulated in LB1 silenced cells including DDB1, CSB and PCNA. We propose that LB1 regulates the DNA damage response to UV irradiation by modulating the expression of specific genes and activating persistent DNA damage signaling. Our findings are relevant to understanding the relationship between the loss of LB1 expression, DNA damage signaling, and replicative senescence.

Show MeSH
Related in: MedlinePlus