Genomic instability in Gadd45a-deficient mice - Nature

article

© 1999 Nature America Inc. • http://genetics.nature.com

Genomic instability in Gadd45a-deficient mice M. Christine Hollander1, M. Saeed Sheikh1, Dmitry V. Bulavin1, Karen Lundgren2, Laura Augeri-Henmueller1, Ronald Shehee2, Thomas A. Molinaro1, Kate E. Kim1, Eva Tolosa3, Jonathan D. Ashwell3, Michael P. Rosenberg2, Qimin Zhan1, Pedro M. Fernández-Salguero4, William F. Morgan5,6, Chu-Xia Deng7 & Albert J. Fornace Jr1 Gadd45a-null mice generated by gene targeting exhibited several of the phenotypes characteristic of p53-deficient mice, including genomic instability, increased radiation carcinogenesis and a low frequency of exencephaly. Genomic instability was exemplified by aneuploidy, chromosome aberrations, gene amplification and centrosome amplification, and was accompanied by abnormalities in mitosis, cytokinesis and growth control. Unequal segre© 1999 Nature America Inc. • http://genetics.nature.com

gation of chromosomes due to multiple spindle poles during mitosis occurred in several Gadd45a–/– cell lineages and may contribute to the aneuploidy. Our results indicate that Gadd45a is one component of the p53 pathway that contributes to the maintenance of genomic stability.

Introduction

Results

A complex interconnecting network of key regulatory factors is involved in normal growth control processes and the maintenance of genomic stability. Some of these factors include the tumour suppressors p53 (encoded by Trp53), Wt1, Brca1 and Brca2, as well as the product of the oncogene Myc, all of which are involved in the regulation1–4 of Gadd45a (also known as gadd45 and gadd45α). Loss of p53 has many effects, including genomic instability, cell cycle checkpoint disturbances, gene amplification, centrosome amplification, abnormalities in cytokinesis, hypercarcinogenesis, decreased apoptosis, reduced DNA repair and a low frequency of exencephaly3,5–7. Athough p21Cip1/Waf1 (encoded by Cdkn1a) appears to be the major effector of the p53mediated G1 checkpoint and has also been implicated in other checkpoints, Cdkn1a–/– mice do not exhibit features of genomic instability seen in Trp53–/– mice8,9, and the p53-effector genes involved in genomic stability are unknown. Gadd45a expression, which is stress inducible in all mammalian cells examined to date, has been frequently associated with growth arrest and in some cases apoptosis3,4. It is the only member of this three-gene family that is regulated by p53, and it encodes a primarily nuclear protein that can associate with PCNA (ref. 10), p21 (ref. 11), MTK1 (ref. 12), Cdc2 (ref. 13) and core histones14. There is not a convincing role for Gadd45 in the G1 checkpoint, but recent findings indicate a role in the control of G2 cell-cycle progression13,15. Disturbances in progression through G2 and mitosis are probably relevant in the genesis of genomic instability, as exemplified by chromosome aberrations, aneuploidy, centrosome disturbances and gene amplification3,7,16. We report here that disruption of this gene is associated with genomic instability and disturbances in growth control.

Generation and phenotype of Gadd45a–/– mice We replaced a portion of Gadd45a, including exons 1–3, with a PGK-neo cassette (Fig. 1a). Only 19 amino acids are encoded by the remaining fourth exon, which was not expressed due to the deletion of regulatory elements. Genomic Southern-blot analysis of embryonic stem (ES) cell clones exhibited two possible bands for the mutated allele depending on the orientation of the PGK-neo cassette (Fig. 1b). To facilitate genotyping, PCR was used to detect the presence of the endogenous and mutant genes in mice after confirmation by genomic Southern-blot analysis (Fig. 1c). As expected, cells from Gadd45a–/– animals did not express any Gadd45a transcript detected by RNAse protection (Fig. 1d). Matings between Gadd45a heterozygotes yielded the expected frequency of wild-type, nullizygous (Gadd45a–/–) and heterozygous (Gadd45a+/–) offspring, indicating that Gadd45a is not required for normal mouse development. Gadd45a–/– mice grew to adulthood without obvious differences from their wild-type littermates, except that Gadd45a–/– newborns were on average 15% heavier than wild-type newborns, 1.65±0.031 g (s.e., n=71) for null versus 1.43±0.020 g (s.e., n=93 determinations) for wild type. Both male and female Gadd45a–/– mice are fertile, but approximately 50% of null females failed to deliver their first litter, resulting in death of the fetuses and subsequently the dams. Gadd45a–/– mice exhibited a low frequency of exencephaly in crosses between null females and null males; 8% of pups obtained by Caesarean section exhibited this phenotype, usually with more than one affected pup per litter. A similar frequency of exencephaly has been reported in Trp53–/– mice6, comparable with results observed in our laboratory for Trp53–/– mice using a line described previously5.

1Gene Response Section, DBS, National Cancer Institute, Bethesda, Maryland 20892-4255, USA. 2Glaxo Wellcome Research and Development, Inc., Research Triangle Park, North Carolina 27709, USA. Laboratories of 3Immune Cell Biology and 4Metabolism, DBS, National Cancer Institute, Bethesda, Maryland 20892, USA. 5Department of Radiation Oncology, University of California, San Francisco, California 94143-0750, USA. 6Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road MS 934, Berkeley, California 94720, USA. 7Genetics of Development and Disease Branch, NIDDKD, Bethesda, Maryland 20892, USA. Correspondence should be addressed to A.J.F. ([email protected]).

176

nature genetics • volume 23 • october 1999



Fig. 1 Targeting of Gadd45a. a, Design of the Gadd45a targeting constructs. The endogenous Gadd45a contains four exons, shown as filled boxes on the first line and transcribed in the direction of the long arrow. A 3.65-kb EcoRI (R1) fragment of Gadd45a including exons 1–3 was replaced with a PGK-neo cassette (in either the same (line 2) or opposite orientation of Gadd45a transcription), and flanked by HSV thymidine kinase genes. A BglII (Bg) site introduced in the PGK terminator and two genomic BglII sites external to the targeting construct were used for determination of homologous recombination by Southern-blot analysis using a flanking probe as indicated on the third line. Primers used for PCR genotyping are indicated as small arrows below the first and second lines. b, Southernblot analysis of targeted ES cells. Two possible bands were seen for mutant Gadd45a based on the orientation of the PGK-neo cassette in (b) and (c). These two independent clones were designated 45C (derived from ESVJ1183 cells) and 45G (derived from HM1 ES cells). c, PCR genotyping of mouse tail DNA. d, Absence of Gadd45a mRNA in Gadd45a–/– MEFs. RNAse protection analysis was carried out with RNA prepared from wild-type and Gadd45a–/– MEFs 4 h after treatment with the indicated dose of IR.

article

a

b

+/+

c

+/– +/–

+/+

d

–/– +/– –/– +/– +/+

–/–

Gy Gadd45a β-actin

Increased growth and loss of normal senescence in MEFs Gadd45a–/– mouse embryo fibroblasts (MEFs) showed two growth patterns in both 45C and 45G mice. Initially, 4 MEF strains (passage 2) had a similar growth rate and saturation density as wild-type cells, but by passage 5 (Fig. 2a), or passage 7 for a second line tested (data not shown), Gadd45a–/– cells grew more rapidly and attained a higher saturation density, whereas wild-type cells grew more slowly and gradually became senescent. We observed accelerated growth in three other Gadd45a–/– MEF strains, even at early passages as shown for one strain (Fig. 2b). In a standard 3T3 growth protocol, two wild-type MEF strains showed a slowing of their rates by passage 7, whereas two null clones continued to grow at a faster rate through passage 16 with accelerated growth at later passages (data not shown). The plating efficiency, as measured by colony formation of low passage Gadd45a–/– MEFs, was approximately 0.025% compared with 0.005% for wild-type MEFs, whereas the plating efficiency at low passage was nearly 100fold greater for Trp53–/– (1.6%), Cdkn1a–/– (1.2%) and Gadd45a/Cdkn1a–/– (up to 1.4%). Our observations indicate perturbation of growth control with loss of normal senescence in Gadd45a–/– cells, but conservation of some control mechanisms involved in growth at low density. nature genetics • volume 23 • october 1999

Single oncogene transformation of Gadd45a–/– cells While primary mouse cells usually require introduction of two ‘activated’ oncogenes for transformation, disruption of certain key growth control genes allows transformation by oncogenic ras alone17. A retroviral vector was used to introduce an activated ras allele into MEFs, and obvious transformed foci were seen in Gadd45a–/– cells, but not in wild-type cells (data not shown). A more stringent in vitro assay for malignant transformation is growth in soft agar. ras-infected Gadd45a–/– MEFs gained the ability to form colonies in soft agar (Fig. 2c), similar to results obtained for Trp53–/– MEFs (Fig. 2d), except the number of colonies was greater for the latter MEFs. Growth in soft agar of wild-type MEFs did not occur with (Fig. 2e) or without (data not shown) introduction of ras. Results for Cdkn1a–/– MEFs were very similar to those for Gadd45a–/– MEFs (data not shown), and indicate that disruption of either Gadd45a or checkpoint control genes such as Trp53, Cdkn1a or Cdkn2darf (ref. 17), allow single oncogene transformation. Deletion of Gadd45a is associated with genomic instability Chromosome analysis was carried out in Gadd45a–/– and wildtype cells. At low passage, even wild-type MEFs showed some loss of diploidy, with approximately 30% of wild-type MEFs 177

article


a cell count (× 105)

b

d


c

Fig. 2 Increased growth rate and ras transformation of Gadd45a–/– MEFs. a,b, Cells at various passages were plated at 105 per 60 mm tissue culture dish. Cells underwent 2.3 to 3.4 doublings per passage. Three plates were counted at each time point and values represent the mean number of cells per dish. Results are representative of at least two separate experiments. a, Wild-type (clone 1) and null (clone 11) were analysed at passages 2 and 5. b, Wild-type (clone 52) and null (clone 46) at passage 2; MEFs in (a) and (b) were prepared from littermates. Results of soft agar cloning for Gadd45a–/– (c), Trp53–/– (d) and wildtype (e) MEFs are shown for representative plates stained three weeks after infection with a retroviral vector containing H-rasV12. The percentage of cells forming small colonies (for example, 3 colonies in c or 5 in d) or large colonies (>twofold diameter of largest colony in d) compared with all cells plated are in the bottom right corner of each panel. Results with vector alone were similar to (e) for Gadd45a–/– and wild-type cells, whereas infrequent small colonies (1.2%) occurred with Trp53–/– cells (data not shown).

e

being tetraploid at passage 4, consistent with previous findings16. Less than 40% of Gadd45a–/– MEFs at the same passage were diploid, however, and the remainder of the cells exhibited tetraploidy and appreciable levels of aneuploidy (data not shown). At passage 11, Gadd45a–/– cultures were devoid of diploid cells with more than 60% of the cells aneuploid and the reminder tetraploid (Fig. 3a). In contrast, wild-type cultures contained mostly tetraploid and diploid cells with relatively few aneuploid cells. In addition, Gadd45a–/– cells exhibited many more chromosomal aberrations, such as double minutes, centromere fusions/end associations and chromatid-type aberrations such as chromatid deletions, complete and incomplete triradials and quadraradials (Fig. 3a, and data not shown). Furthermore, some of the Gadd45a–/– cells failed to fully condense all their chromatin at mitosis and displayed regions of decondensed chromatin interspersed with fully condensed metaphase chromosomes in the same cell. Because even wild-type MEFs show limited genomic instability with passage in culture, chromosome numbers were also determined from freshly isolated splenic lymphoblasts. Gadd45a–/– cells exhibited 2% aneuploidy, whereas none of the 200 cells from 2 wild-type mice were aneuploid; 45C lymphoblasts showed one cell with 63 chromosomes and one with 88 (66 mitoses examined); 45G lymphoblasts 178

showed one cell with 62 chromosomes and one with 80 chromosomes (100 mitoses examined). Because many Gadd45a–/– MEFs showed double minutes, a hallmark of gene amplification and genomic instability, we subjected wild-type, Gadd45a–/–, Cdkn1a–/–, Gadd45a–/–Cdkn1a–/– and Trp53–/– MEFs to stepwise concentration increases in PALA (L-Aspartic acid, N-(phosphonoacetyl)) to promote cad gene amplification. Because wild-type and Gadd45a–/– cells have low plating efficiency, selection was in bulk culture, but killing by PALA probably reduced the cell density below the threshold for appreciable growth and there was negligible survival. PALAresistant cultures were obtained for the other three genotypes, but even after 20 weeks of selection, the Cdkn1a–/– MEFs failed to grow in greater than 25 µM PALA (2.7-fold the LD50); in contrast, Gadd45a–/–Cdkn1a–/– and Trp53–/– MEFs became resistant to 500 µM PALA after 14 weeks of selection (Fig. 3b). cad amplification was evident for these two lines, but no amplification occurred in the low PALA-resistant Cdkn1a–/– cells (Fig. 3b). A one-step selection for PALA-resistant colonies was carried out, and the frequency of PALA-resistant colonies was similar to that reported previously for Trp53-null MEFs (ref. 16) and varied by less than twofold between Gadd45a–/–Cdkn1a–/– and early or late passage Trp53–/– MEFs nature genetics • volume 23 • october 1999


article

a

chromosomes per cell

chromosomes per cell

Fig. 3 Gadd45a–/– MEFs exhibit aneuploidy and gene amplification. Trp53–/– Cdkn1a–/– Gadd45a–/–Cdkn1a–/– a, Chromosome numbers from 100 MEF cells per genotype at passage 11. Gadd45a–/– In the inset, ‘aberrant cells’ refers to Trp53–/– Cdkn1a–/– MEFs containing one or more gross structural chromosomal rearrangement such as chromatid or isochromatid deletions, or complete or incomplete chromosome or chromatid Cdkn1a–/– exchanges. b, Gadd45a deficiency facilitates carbamoyl-P synthase/aspartate transcarbamylase/dihydroorotase (cad) gene amplification in the presence of N-(phosphonoacetyl)-L-aspartate (PALA). Cell cultures from Trp53–/–, Gadd45a–/–Cdkn1a–/– and Cdkn1a–/– were subjected to stepwise increases in PALA concentration. DNA was prepared from non-resistant cultures (0) and those resistant to 25 µM PALA, which is only double the LD50 dose, to a maximum concentration of 500 µM, and then digested with EcoRI. Genomic Southern blots were incubated with a cad cDNA to detect gene amplification. Equal loading was determined by hybridization to a Gadd34/Myd116 probe as a non-amplified control. Relative cad is indicated as the ratio between cad signal and Gadd34/Myd116 signal determined by phosphorimager analysis. Right, the time course of resistance is shown to increasing concentrations of PALA; the null genotype is shown for the three lines used. Cdkn1a–/– MEFs did not grow in greater than 25 µM PALA.


b

(data not shown). Although loss of Cdkn1a facilitated growth at quency of cells containing four or more centrosomes was more low density, it is concluded that deletion of Gadd45a was also than double that seen in wild-type cells (data not shown). required for gene amplification and that the rate of amplification was similar to that for Trp53–/– MEFs. Increased radiation carcinogenesis To investigate induced carcinogenesis in Gadd45a–/– mice, 106 littermates from heterozygote crosses were γ-irradiated with a Centrosome abnormalities Trp53–/– MEFs and tumour cells show an abnormal increase or sublethal schedule. One year after treatment, there was a three‘amplification’ of centrosomes (for review, see ref. 18), which are fold increase in frequency of Gadd45a–/– mice succumbing to known to interact with Cdc2 (ref. 19), a Gadd45-associated pro- tumours compared with wild type (Fig. 5a). Although some tein13. We rapidly isolated splenic lymphocytes from mice, and wild-type mice did develop tumours, the latency was substanmost of these primarily G0/G1 phase cells contained one centro- tially longer than that for Gadd45a–/– mice. Tumour incidence some (Fig. 4a), because duplication occurs near the G1/S interface for Gadd45a+/– mice was intermediate between that of wild-type with separation into two distinct centrosomes in G2. More than and null mice. This suggests haploinsufficiency because the twice as many Gadd45a–/– cells (∼20% of all cells) contained three remaining wild-type allele was not lost in tumours from or more centrosomes. We next examined rapidly growing ker- Gadd45a+/– mice (data not shown), although point mutations, atinocytes (Fig. 4b), and more Gadd45a–/– cells exhibited four or which would be relatively infrequent with the dose of IR more centrosomes per cell than their wild-type counterparts. employed, have not been ruled out in the remaining allele. Our Representative photomicrographs depict centrosome amplifica- results are similar to those for IR carcinogenesis in Cdkn1b+/– tion and nuclear morphology for Gadd45a–/– cells (Fig. 4c–i). mice lacking a single copy of the gene encoding p27Kip1 (ref. 20), Mitotic morphology for some Gadd45a–/– keratinocytes appeared but differ from those for Trp53+/– mice21. By 12 months of age, normal, but many cells showed a variety of abnormalities in all 22% of wild-type mice developed tumours, compared with 45% phases of mitosis with frequent asymmetric distributions of cen- and 59%, respectively, of Gadd45a+/– and Gadd45a–/– mice. Histrosomes. Considering the centrosome’s role in spindle assembly, tologic examination demonstrated that most of the tumours such events might result in unbalanced chromosomal segregation were lymphomas and necropsy often revealed large thymic (Fig. 4c,f, arrows). It is notable that a number of telophase cells masses, consistent with a thymic origin. In unirradiated animals, with multiple centrosomes were unable to completely separate only 1 of 22 null animals developed a thymus tumour at 12 (for example, the telophase cell in Fig. 4g undergoing unbalanced months of age, whereas none occurred in 21 wild-type mice; segregation is still attached via bridging chromatin). It is also pos- additional spontaneous tumours did not appear before euthanasible that due to defective cytokinesis (Fig. 4h) some cells unable sia at 18 months. to undergo a complete segregation following telophase may continue to replicate and ultimately give rise to polyploidy. In the case Proficient apoptosis and thymic hyperplasia of MEFs, most Gadd45a–/– cells with abnormally amplified cen- Because apoptosis can contribute to genomic stability by trosomes (Fig. 4f) had large, partially cleaved, bilobed nuclei, a removal of damaged cells, we considered the possibility that feature highly suggestive of aberrant cytokinesis, and the fre- Gadd45a–/– mice might be deficient in mounting a full apoptotic nature genetics • volume 23 • october 1999

179


a

c

response. Our initial focus was on the thymus because of our finding of thymic hyperplasia in four-week-old Gadd45a–/– mice. Thymus weight as a per cent of body weight was on average more than 70% higher for null animals compared f with wild type (109.2±3.3 versus 55.7±3.4 mg per 11-g mouse), and there were significantly more thymocytes in each null thymus (9.0±0.38×107 versus 6.0±0.41×107 cells per 11 g mouse). The distribution of CD4+CD8+, CD4+CD8– and CD4–CD8+ thymocytes, however, did not differ between genotypes (data not shown). To determine if the increased thymic cellularity might be i due to deficient programmed cell death –/– (as observed in Trp53 mice), Gadd45a–/–, Trp53–/– and wild-type mice were treated with IR (Fig. 5b). As expected, Trp53–/– thymocytes did not undergo apoptosis, as shown by lack of characteristic internucleosomal DNA laddering on agarose gels. Thymocytes from both wild-type and Gadd45a–/– animals exhibited typical DNA laddering, indicating that like Cdkn1a (ref. 8), Gadd45a is not required for IR-inducted thymocyte apoptosis. Annexin V staining for apoptotic cells revealed only background cell death (