The Werner syndrome helicase protein is required for cell proliferation, immortalization, and tumorigenesis in Scaffold attachment factor B1 deficient mice

Abstract

Werner syndrome (WS) is a rare disorder characterized by the premature onset of several pathologies associated with aging. The gene responsible for WS codes for a RecQ-type DNA helicase and is believed to be involved in different aspects of DNA repair, replication, and transcription. We recently identified the Scaffold attachment factor B1 (SAFB1) as a potential interactants in human cells. SAFB1 is a multifunctional protein that binds both nucleic acids and is involved in the attachment of chromatin to the nuclear matrix, transcription, and stress response. Mice lacking SAFB1 exhibit developmental abnormalities in their lungs, high incidence of perinatal lethality, and adults develop different types of tumors. Mouse embryonic fibroblasts from Safb1-null animals are immortalized in culture. In this study, mice with a mutation in the helicase domain of the Wrn gene were crossed to Safb1-null mice. Double homozygous mutant mice exhibited increased apoptosis, a lower cell proliferation rate in their lungs and a higher incidence of perinatal death compared to Safb1-null mice. Few double homozygous mutants survived weaning and died before the age of six months. Finally, mouse embryonic fibroblasts lacking a functional Wrn helicase inhibited the immortalization of Safb1-null cells. These results indicate that an intact Wrn protein is required for immortalization and tumorigenesis in Safb1-null mice.

Figure 1.

Co-immunoprecipitation of SAFB1 with the WRN protein in HEK 293 cells. Immunoprecipitation was performed with an antibody against the WRN protein. The immuno-precipitate was analyzed by western blot analyses with an antibody against SAFB1. The lysate represents 10% of total proteins in the immunoprecipitation reaction.

Figure 2.

Hematoxilin and eosin staining of lung tissues from 19 days old embryos from the indicated genotypes. Magnification 400X.

Figure 3.

Apoptotic figures in the lung tissues from 19 days old mutant embryos. (A) Apoptotic cells detection (TUNEL) assay on 19 days post-coitum embryonic lung sections showing a major increase in the number of apoptotic cells in Safb1-null/Wrn^Δhel/Δhel (or Safb1^−/−/Wrn^Δhel/Δhel) embryos compared to the other genotypes. Healthy cells are stained in green (methyl green staining) and apoptotic cells are dark blue. Arrowheads point to representative apoptotic cells. Magnification 400X. (B) Average number of apoptotic figures per area of lung sections containing 1000 cells (n=3 embryos for each genotype; *: unpaired student t−test P-value < 0.05 compared to wild type Safb1^+/+/Wrn^+/+ animals).

Figure 4.

Proliferating cells (stained with an antibody against PCNA) in the lung tissues from 19 days old mutant embryos. (A) Example of PCNA stained cells with DAB (brown color) in 19 days post-coitum embryonic lung sections (stained with hematoxilin) showing a major decrease in cell proliferation in Safb1-null/Wrn^Δhel/Δhelembryos compared to the other genotypes. Magnification 400X. (B) Average number of apoptotic figures per area of lung sections containing 1000 cells (n=3 embryos for each genotype) (*: unpaired student t-test P-value < 0.001 compared to wild type Safb1^+/+/Wrn^+/+animals; **: unpaired student t-test P-value < 0.0001 compared to wild type Safb1^+/+/Wrn^+/+animals).

Figure 5.

Differential saturation density and growth properties of MEFs. (A) Growth curves of MEFs after 7-10 passages in culture (except for Safb1-null MEFs, which were measured at passage 24). Cells (5 × 10⁴) from wild type (Wrn^+/+/Safb1^+/+), Safb1-null (Safb1^−/−/Wrn^+/+), and Safb1-null/Wrn^Δhel/Δhel (Safb1^−/−/Wrn^Δhel/Δhel) embryos were plated in six-well plates as described in materials and methods. Cells were counted by trypan blue exclusion with a hemacytometer. (B) Histogram representing the growth rate of MEFs (from at least three embryos for each genotype) calculated from the growth curves in A. Bars represent the SEM. (Unpaired student t-test: *P < 0.000001 and **P < 0.026577 compared to wild type Wrn^+/+/Safb1^+/+animals). Growth rates were estimated as described in materials and methods.

Figure 6.

Cellular morphology of MEFs. Representative phase-contrast photographs of wild type, Wrn^Δhel/Δhel, Safb1-null, and Safb1-null/Wrn^Δhel/Δhel MEFs after the fifth passage in culture. Magnification 600X.

Figure 7.

Induction of senescence by loss of Wrn helicase activity in Safb1-null MEFs. (A) Example of senescence-associated β-galactosidase staining in Safb1-null and Safb1-null/Wrn^Δhel/Δhel MEFs. Arrowheads point to positive cells. Magnification 100X. (B) Percentage of cells stained with senescence-associated β-galactosidase in wild type, Wrn^Δhel/Δhel, Safb1-null, and Safb1-null/Wrn^Δhel/Δhel MEFs. (Unpaired student t-test; *P < 0.045 and ** P < 0.022 compared to wild type MEFs). Bars represent SEM.

Figure 8.

Protein levels of p53, p21Waf1, p19Arf, PCNA, p38 kinase, PKCδ, and JNK1 in MEFs. Whole cell lysates from MEFs of each genotype were analyzed by immunoblotting with antibodies against the indicated proteins. Proteins were extracted from wild type, Wrn^Δhel/Δhel, Safb1-null, and Safb1-null/Wrn^Δhel/Δhel MEFs. β-tubulin was used as a loading control.

Figure 9.

DNA damage in MEFs. (A) Examples of nuclear foci detected by immunofluorescence with an antibody against γ-H2AX in MEFs of each genotype Magnification 600X. (B) Graph representing the extent of double stranded breaks detected with an antibody against γ-H2AX in MEFs of each genotype. The percentage of cells with more than 0, 10, and 20 γ-H2AX foci were computed from 100 MEFs established from three independent embryos for each genotype (total of 300 cells analyzed/genotype). Bars in the graph represent SEM.

Figure 10.

Correlation between p38 and PKCδ kinase levels and DNA damage and senescence in MEFs. (A) Scanning analyses of Western blots, expressed as ratio of p38 signal to β-tubulin signal. Bars represent SEM. (B) Correlation between the p38 kinase level and DNA damage in the different MEFs. The Pearson's correlation coefficient is indicated. (C) Scanning analyses of Western blots, expressed as ratio of PKCδ signal to β-tubulin signal. Bars represent SEM. (D) Correlation between the PKCδ level and the percentage of senescent MEFs in vitro from the different genotypes. The Pearson's correlation coefficient is indicated.