(A) The macroscopic hyperpigmentation and fur phenotype of Acd^acd/acd mice were completely rescued by p53 ablation. Macroscopic appearance of 6 week old mice: Acd^acd/acd p53^−/− mice shown on top of the picture showed normal fur growth and completely lacked hyperpigmentation. As a comparison an Acd^acd/acd p53^+/+ mouse is shown below.
(B) Hyperpigmentation is always present on the footpads of adrenocortical dysplasia mice. In Acd^acd/acd p53^−/− mice the hyperpigmentation completely disappeared at the macroscopic level. Eosin-stained sections showed a striking reduction of pigment in the epidermis and dermis of Acd^acd/acd p53^−/− mice. The same results are shown for tail skin sections (scale bars for foot pads 200μm, for tails 100μm).
(C) Genotype analysis of 162 male pups at weaning age shows an increased survival for Acd^acd/acd p53^−/− mice. Acd^acd/acd mice with either the p53^+/− or the p53^+/+ genotype were not observed in the expected ratio (p=0.03 and p=0.03 as compared to p=0.6 for p53^−/−, Chi-square test).
(D) Acd^acd/acd p53^−/− mice are still smaller than their Acd^+/+ p53^+/+ littermates (p<10⁻⁷). Body weight did not differ from Acd^acd/acd p53^+/+ mice (p=0.4 at 3 weeks and p=0.1 at 6 weeks, 1-way ANOVA and F-test, data shown as mean +/− SD). Post-weaning weight gain was comparable in both Acd^acd/acd groups to their wt littermates..

(A) The adrenal cortex of Acd^acd/acd mice appears highly disorganized and consists of scattered cells with a high grade of nuclear pleomorphy. This phenotype was partially rescued in Acd^acd/acd p53^−/− mice, where adrenal glands appeared more clearly zonated and organized, while increased nuclear size and pleomorphy partially persisted. This became obvious using differentiation markers for the adrenal cortex (Sf1, steroidogenic factor 1) and for the adrenal medulla (Th, tyrosine hydroxylase). Two examples of adrenal glands from Acd^acd/acd p53^+/+ and Acd^acd/acd p53^−/− and, as a comparison, one wt (Acd^+/+ p53^+/+) adrenal, are shown. Acd^acd/acd p53^+/+ adrenal cortices stained positive for p21, a downstream mediator of p53-mediated senescence induction (scale bars for different levels of magnification: high 100μm, low 200μm).
(B) Relative adrenal gland weight is normalized in Acd^acd/acd p53^−/− mice. Adrenal glands from Acd^acd/acd p53^+/+ animals were about 66% the relative weight of Acd^+/+ p53^+/+ adrenal glands (Acd^acd/acd p53^+/+ vs. Acd^+/+ p53^+/+ p=0.008, Acd^acd/acd p53^+/+ vs. Acd^acd/acd p53^−/− p=0.005, Acd^acd/acd p53^−/− vs. Acd^acd/acd p53^+/+ p=0.95, 1-way ANOVA and F-test, data shown as mean +/− SD).
(C) In Acd^acd/acd p53^+/+ mice telomere deprotection induces p21 expression as a marker of senescence. Ablation of p53 strikingly reduced p21 expression (Acd^acd/acd p53^+/+ vs. Acd^+/+ p53^+/+ p=0.02, Acd^acd/acd p53^+/+ vs. Acd^acd/acd p53^−/− p=0.03, Acd^acd/acd p53^−/− vs. Acd^+/+ p53^+/+ p=0.99, 1-way ANOVA and F-test, data shown as mean +/− SD).

(A) Some Acd^acd/acd p53^+/− carcinomas show an intermediate adenosquamous differentiation type with elements of squamous cell as well adenocarcinoma-like elements. Skin associated carcinomas stained highly positive for pankeratin (scale bars 200μm).
(B) Most carcinomas arise in the cutaneous or subcutaneous regions and typically show central necrosis.
(C) 5% of Acd^acd/acd p53^+/− develop adrenocortical cancers. Their origin could readily be identified by positive Sf1 staining. Adrenocortical cancers were p21 negative indicating that they evaded senescence (scale bars 100μm).
(D) Macroscopically, adrenocortical cancer expanded within the peritoneal cavity adhering to adjacent organs. Two examples of ACC from Acd^acd/acd p53^+/− mice are shown.

(A) Table summarizing characteristics of Acd^acd/acd tumors as analyzed by CGH. Acd^acd/acd tumors have significantly more transition points compared to Acd^+/+ tumors as would be expected with repeated BFBs and significantly more chromosomes exhibited copy number alterations.
(B) CGH example of tumor DNA normalized to same animal liver DNA. Multiple amplifications sometimes presenting in a stepwise manner of increasing amplification values on the same chromosome (e.g. chr. 1 and chr. 3 of two samples) were observed in Acd^acd/acd (lower panel) but not in Acd^+/+ tumors (upper panel) (y-axis: log₂ of tumor/liver).
(C) Spectral karyotyping of a cell line derived from a Acd^acd/acd p53^−/− rhabdomyosarcoma revealed genomic rearrangements in the form of hybrid chromosomes consisting of genomic stretches of original chromosome 16 and chromosome 19 (see magnification lower right)

(A) Testes of Acd^acd/acd p53^+/+ as well as Acd^acd/acd p53^−/− mice show focal spermatogenesis. Seminiferous tubules with an intact germinal cell epithelium harbored Gcna-positive germ cells. The Sf1-positive interstitial Leydig (black arrows) and Sertoli cell (red arrows) populations did not appear altered in either genotype compared to Acd^+/+ p53^+/+ littermates (scale bars for different levels of magnification: high 100μm, medium 200μm, low 400μm).
(B) Some Acd^acd/acd p53^+/+ and Acd^acd/acd p53^−/− testes completely lack spermatogenesis and germ cells. Only empty seminiferous tubules were present in these testes (scale bars for different levels of magnification: medium 200μm, low 400μm).
(C) Testis weight relative to body weight is severely reduced in both Acd^acd/acd p53^+/+ and Acd^acd/acd p53^−/− mice and therefore independent of their genetic p53 status (Acd^+/+ p53^+/+ vs. Acd^acd/acd p53^−/− p=1×10⁻¹¹, Acd^+/+ p53^+/+ vs. Acd^acd/acd p53^+/+ p=4×10⁻¹², Acd^acd/acd p53^+/+ vs. Acd^acd/acd p53^−/−, p=0.19, 1-way ANOVA and F-test, data shown as mean +/− SD)

(A) Tumor free survival is shown for the different genotype cohorts as a Kaplan-Meier plot. On average in both groups, p53^−/− and p53^+/−, mice with the Acd^acd/acd genotype developed tumors in approximately half the amount of time compared to their Acd^+/+ littermates (Acd^acd/acd p53^−/− vs. Acd^+/+ p53^−/− p=2×10⁻¹¹ and Acd^acd/acd vs. p53^+/− Acd^+/+ p53^+/− p=2×10⁻⁹, log-rank test). In contrast none of the Acd^acd/acd p53^+/+ developed any tumors.
(B) The main categories of solid tumors in Acd^acd/acd p53^−/− and Acd^+/+ p53^−/− are of the sarcoma spectrum (examples of angiosarcoma, osteosarcoma, rhabdomyosarcoma and undifferentiated sarcoma are shown, scale bars 200μm)).
(C) In Acd^acd/acd p53^+/−, but not Acd^+/+ p53^+/−, mainly carcinomas arose with a varying degree of squamous cell carcinoma or adenocarcinoma differentiation (scale bars 200μm).

(A) Acd^acd/acd tumors had significantly more telomere dysfunction induced foci (TIFs) per total telomere signals than Acd^+/+ tumors indicating that telomere decapping is evident in these neoplasms (p=0.047 for Acd^acd/acd p53^−/− vs. Acd^+/+ p53^−/− tumors and p=0.002 for Acd^acd/acd p53^+/− vs. Acd^+/+ p53^+/− tumors, 1-way ANOVA and F-test, data shown as mean +/− SD).
(B) Decapped telomeres recruit factors of the DNA surveillance machinery, such as γH2ax and form TIFs. TIFs, the classical co-localization of telomere FISH signal (red) and γH2ax immunohistochemistry (green), were observed in Acd^acd/acd tumors (scale bars 5μm).
(C) Tumors from Acd^acd/acd mice (p53^−/− or p53^+/−) have a significantly increased anaphase bridge index (Acd^acd/acd p53^−/− vs. Acd^+/+ p53^−/− p=5×10⁻⁵ and Acd^acd/acd vs. p53^+/− Acd^+/+ p53^+/− p=0.001, 1-way ANOVA and F-test, data shown as mean +/− SD).
(D) Classical anaphase bridges, a morphological correlate of breakage-fusion-bridge cycles (BFBs), are present in tumors from Acd^acd/acd mice (black arrows). In some instances chromosomal material (red arrows) appeared in between the two poles without a clear connection to either of the poles of the emerging daughter cells (scale bars 20μm).
(E) Telomere length as measured by TRF gel analysis. No clear differences between normal tissues (liver) of different genotypes were observed. The Acd^acd/acd genotype itself did not lead to in vivo telomere length alteration in normal tissues. Some Acd^acd/acd as well as Acd^+/+ tumors showed a moderate shortening of telomere length.