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. 2009 Jul;130(7):409-19.
doi: 10.1016/j.mad.2009.04.002. Epub 2009 May 3.

Chronic NF-kappaB activation delays RasV12-induced premature senescence of human fibroblasts by suppressing the DNA damage checkpoint response

Christina Batsi  1 , Soultana Markopoulou, George Vartholomatos, Ioannis Georgiou, Panagiotis Kanavaros, Vassilis G Gorgoulis, Kenneth B Marcu, Evangelos Kolettas
Affiliations

Chronic NF-kappaB activation delays RasV12-induced premature senescence of human fibroblasts by suppressing the DNA damage checkpoint response

Christina Batsi et al. Mech Ageing Dev. 2009 Jul.

Abstract

Normal cells divide for a limited number of generations, after which they enter a state of irreversible growth arrest termed replicative senescence. While replicative senescence is due to telomere erosion, normal human fibroblasts can undergo stress-induced senescence in response to oncogene activation, termed oncogene-induced senescence (OIS). Both, replicative and OIS, initiate a DNA damage checkpoint response (DDR) resulting in the activation of the p53-p21(Cip1/Waf1) pathway. However, while the nuclear factor-kappaB (NF-kappaB) signaling pathway has been implicated in DDR, its role in OIS has not been investigated. Here, we show that oncogenic Ha-RasV12 promoted premature senescence of IMR-90 normal human diploid fibroblasts by activating DDR, hence verifying the classical model of OIS. However, enforced expression of a constitutively active IKKbeta T-loop mutant protein (IKKbetaca), significantly delayed OIS of IMR-90 cells by suppressing Ha-RasV12 instigated DDR. Thus, our experiments have uncovered an important selective advantage in chronically activating canonical NF-kappaB signaling to overcome the anti-proliferative OIS response of normal primary human fibroblasts.

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Figures

Fig. 1
Fig. 1
Expression of NF-κB signaling pathway components during replicative senescence of IMR-90. (A) Cytoplasmic and nuclear extracts, ranging from 15 to 40 Pdls, were analyzed for the expression of IKKα, IKKβ, NF-κB p50 and p65 subunits, IκBα and cyclin D1, or lamin B and β-actin. (B) Total cell lysates were analyzed for the expression of IKKα, IKKβ and cyclin D1 or β-actin by immunoblotting.
Fig. 2
Fig. 2
Generation of RasV12- and/or IKKβca-expressing IMR-90. (A) Infection scheme of IMR-90 with the replication defective recombinant retroviruses. (B) Total cell lysates extracted from uninfected IMR-90 and from HygroB- and/or Neo-resistant IMR-90 cells carrying either RasV12 and/or IKKβca or their corresponding control vectors were analyzed for the expression of Ha-RasV12 and IKKβca proteins or β-actin by immunoblotting.
Fig. 3
Fig. 3
IKKβca but not RasV12 activated canonical NF-κB signaling. (A) Total cell lysates extracted from uninfected IMR-90 and from HygroB- and/or Neo-resistant IMR-90 cells carrying either RasV12 and/or IKKβca or their corresponding control vectors were analyzed for the expression of phospho-IκBα (Ser32/36), IκBα or β-actin. (B) Proteins extracted from isolated nuclei of uninfected IMR-90 and from HygroB- and/or Neo-resistant IMR-90 cells carrying either RasV12 and/or IKKβca or their corresponding control vectors were analyzed for the expression of p50, p65 and IκBα, and lamin B by immunoblotting. (C) The relative expression levels of several canonical NF-κB target genes (IκBα, IL-6 and MCP-1/CCL2) were determined by SYBR green real-time RT-PCR in IMR-90 Neo, IMR-90 IKKβca and in IMR-90 cells stimulated with 100 ng/ml TNFα for 2 h. Fold change values are relative to empty vector control (IMR-90 Neo) cells grown under the same conditions. Results shown are representative of several experiments with variation being no more than 5-10%.
Fig. 4
Fig. 4
Analysis of growth properties of RasV12- and/or IKKβca-expressing IMR-90 fibroblasts. (A) HygroB- and/or Neo-resistant IMR-90 fibroblasts expressing either RasV12 and/or IKKβca or their corresponding control vectors were plated into 24-multiwell plates and counted every two days. The experiment was performed twice in duplicates and growth curves were constructed. (B) Flow cytometric analysis of all the different IMR-90 at P25. IMR-90 RasV12 at P18 and senescent IMR-90 at P42 fibroblasts were included in the analysis. The percentages of cells in corresponding phases of the cell cycle are indicated.
Fig. 5
Fig. 5
Morphologies and SA-β-Gal expression in RasV12- and/or IKKβca-expressing IMR-90. Morphologies and SA-β-Gal staining, as a marker for cellular senescence, of senescent IMR-90 (P39) fibroblasts and uninfected or HygroB- and/or Neo-resistant IMR-90 expressing either RasV12 and/or IKKβca or their corresponding control vectors.
Fig. 6
Fig. 6
Analysis of cell cycle regulatory proteins in RasV12- and/or IKKβca-expressing IMR-90 fibroblasts. (A) Total cell lysates extracted from normal IMR-90 and from HygroB- and/or Neo-resistant IMR-90 expressing either RasV12 and/or IKKβca or their corresponding control vectors were analyzed for the expression of selected cell cycle regulatory proteins or β-actin by immunoblotting. (B) Time course of expression of cyclin D1 and p21Cip1/Waf1 in sub-confluent proliferating cultures of IMR-90 Neo and IMR-90 IKKβca fibroblasts following serum stimulation of the cells for 0 - 24 h.
Fig. 7
Fig. 7
Analysis of proteins involved in DNA damage checkpoint response in RasV12- and/or IKKβca-expressing IMR-90 fibroblasts. Total cell lysates extracted from HygroB- and/or Neo-resistant IMR-90 expressing either RasV12 and/or IKKβca or their corresponding control vectors were analyzed for the expression of phospho-Chk2 (Thr68), total Chk2, phospho-p53 (Ser15 and Ser20) and total p53 or β-actin by immunoblotting.
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