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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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Silencing LB1 expression in U-2 OS cells dramatically delays detection and repair of DNA damage induced by UV.Silenced and control cells were irradiated with 20 J/m2 UV, fixed and stained at 8, 24, 48 and 80 hr with antibodies to LB1 (green) and 53BP1 (red); LB1 (green) and pRPA32 (red); and γH2AX (green) and DDB1 (red). No UV samples were from the same transfections. The borders of the nuclei were marked in white in the far right panels. Images of single representative nuclei are shown.
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pone-0069169-g005: Silencing LB1 expression in U-2 OS cells dramatically delays detection and repair of DNA damage induced by UV.Silenced and control cells were irradiated with 20 J/m2 UV, fixed and stained at 8, 24, 48 and 80 hr with antibodies to LB1 (green) and 53BP1 (red); LB1 (green) and pRPA32 (red); and γH2AX (green) and DDB1 (red). No UV samples were from the same transfections. The borders of the nuclei were marked in white in the far right panels. Images of single representative nuclei are shown.

Mentions: In order to determine whether the delay in DNA repair was due the loss or decrease of NER associated factors, we measured the levels of DDB1, CSB, pRPA32, γH2AX and 53BP1 before and at time intervals after UV irradiation. LB1 silencing induced increased expression and post-translational modification of 53BP1 in non-irradiated cells (ct lanes, Fig. 4), suggesting a DNA stress response to a reduction of LB1. Furthermore, UV irradiation of LB1 silenced cells did not induce an increase in 53BP1 expression like that seen in control cells [35], [37]. Both DDB1 and CSB protein expression levels were decreased in LB1 silenced cells compared to control cells without irradiation (Fig. 4). These results suggest that LB1 silencing alone affected the initiation steps of both NER sub-pathways. The accumulation of phosphorylated pRPA32, which binds to the single stranded region opposite the nucleotide lesion during repair [27], [30], [33] was induced by UV. However silenced cells exhibited both a delay in and lower expression level of pRPA32 compared to control cells (Fig. 4). Interestingly, as expected γH2AX was transiently induced between 0 and 8 hours and was not detectable by 24 hours after UV irradiation in control cells. In contrast, γH2AX was induced between 0 and 8 hours in LB1 silenced cells and persisted until at least 48 hours after UV irradiation (Fig. 4 and 5). Taken together these data show that the levels of DNA damage repair factors involved in NER are significantly decreased in LB1 silenced cells. The lack of sufficient repair factors in LB1 silenced cells could explain the delayed response to the DNA damage caused by UV irradiation.


Regulation of nucleotide excision repair by nuclear lamin b1.

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

Silencing LB1 expression in U-2 OS cells dramatically delays detection and repair of DNA damage induced by UV.Silenced and control cells were irradiated with 20 J/m2 UV, fixed and stained at 8, 24, 48 and 80 hr with antibodies to LB1 (green) and 53BP1 (red); LB1 (green) and pRPA32 (red); and γH2AX (green) and DDB1 (red). No UV samples were from the same transfections. The borders of the nuclei were marked in white in the far right panels. Images of single representative nuclei are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0069169-g005: Silencing LB1 expression in U-2 OS cells dramatically delays detection and repair of DNA damage induced by UV.Silenced and control cells were irradiated with 20 J/m2 UV, fixed and stained at 8, 24, 48 and 80 hr with antibodies to LB1 (green) and 53BP1 (red); LB1 (green) and pRPA32 (red); and γH2AX (green) and DDB1 (red). No UV samples were from the same transfections. The borders of the nuclei were marked in white in the far right panels. Images of single representative nuclei are shown.
Mentions: In order to determine whether the delay in DNA repair was due the loss or decrease of NER associated factors, we measured the levels of DDB1, CSB, pRPA32, γH2AX and 53BP1 before and at time intervals after UV irradiation. LB1 silencing induced increased expression and post-translational modification of 53BP1 in non-irradiated cells (ct lanes, Fig. 4), suggesting a DNA stress response to a reduction of LB1. Furthermore, UV irradiation of LB1 silenced cells did not induce an increase in 53BP1 expression like that seen in control cells [35], [37]. Both DDB1 and CSB protein expression levels were decreased in LB1 silenced cells compared to control cells without irradiation (Fig. 4). These results suggest that LB1 silencing alone affected the initiation steps of both NER sub-pathways. The accumulation of phosphorylated pRPA32, which binds to the single stranded region opposite the nucleotide lesion during repair [27], [30], [33] was induced by UV. However silenced cells exhibited both a delay in and lower expression level of pRPA32 compared to control cells (Fig. 4). Interestingly, as expected γH2AX was transiently induced between 0 and 8 hours and was not detectable by 24 hours after UV irradiation in control cells. In contrast, γH2AX was induced between 0 and 8 hours in LB1 silenced cells and persisted until at least 48 hours after UV irradiation (Fig. 4 and 5). Taken together these data show that the levels of DNA damage repair factors involved in NER are significantly decreased in LB1 silenced cells. The lack of sufficient repair factors in LB1 silenced cells could explain the delayed response to the DNA damage caused by UV irradiation.

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