Deletion of Ku86 causes early onset of senescence in mice.

Abstract

DNA double-strand breaks formed during the assembly of antigen receptors or after exposure to ionizing radiation are repaired by proteins important for nonhomologous end joining that include Ku86, Ku70, DNA-PK(CS), Xrcc4, and DNA ligase IV. Here we show that ku86-mutant mice, compared with control littermates, prematurely exhibited age-specific changes characteristic of senescence that include osteopenia, atrophic skin, hepatocellular degeneration, hepatocellular inclusions, hepatic hyperplastic foci, and age-specific mortality. Cancer and likely sepsis (indicated by reactive immune responses) partly contributed to age-specific mortality for both cohorts, and both conditions occurred earlier in ku86(-/-) mice. These data indicate that Ku86-dependent chromosomal metabolism is important for determining the onset of age-specific changes characteristic of senescence in mice.

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Proc. Natl. Acad. Sci. USA
Vol. 96, pp. 10770–10775, September 1999
Genetics
Deletion of Ku86 causes early onset of senescence in mice
HANNES VOGEL*, DAE-SIK LIM†‡, GERARD KARSENTY§, MILTON FINEGOLD*, AND PAUL HASTY†¶
*Department of Pathology and§Department of Human and Molecular Genetics, Baylor College of Medicine, Houston, TX 77030; and†Lexicon Genetics, 4000
Research Forest Drive, The Woodlands, TX 77381-4287
Edited by Philip Leder, Harvard Medical School, Boston, MA, and approved July 7, 1999 (received for review May 24, 1999)
ABSTRACT
the assembly of antigen receptors or after exposure to ionizing
radiation are repaired by proteins important for nonhomolo-
gous end joining that include Ku86, Ku70, DNA-PKCS, Xrcc4,
and DNA ligase IV. Here we show that ku86-mutant mice,
compared with control littermates, prematurely exhibited
age-specific changes characteristic of senescence that include
osteopenia, atrophic skin, hepatocellular degeneration, hep-
atocellular inclusions, hepatic hyperplastic foci, and age-
specific mortality. Cancer and likely sepsis (indicated by
reactive immune responses) partly contributed to age-specific
mortality for both cohorts, and both conditions occurred
earlier in ku86?/?mice. These data indicate that Ku86-
dependent chromosomal metabolism is important for deter-
mining the onset of age-specific changes characteristic of
senescence in mice.
DNA double-strand breaks formed during
Some segmental progeroid syndromes suggest that chromo-
somal metabolism plays an important role during senescence
(1). Werner’s syndrome (WS), an inherited autosomal reces-
sive disease caused by a mutation in WRN, is of particular
interest because of its similarity to ordinary senescence (2).
WRN is homologous to the RecQ family of DNA helicases (3)
and catalyzes DNA unwinding (4) and 3?–5? exonuclease
activity (5). WS patients prematurely exhibit signs of senes-
cence, including atrophic skin, graying and hair loss, osteopo-
rosis, malignant neoplasms, and shortened lifespan. Cells
derived from WS patients prematurely undergo replicative
senescence (6), which describes the limited lifespan of cells
grown in tissue culture (7). Even though there is some discor-
dance between WS and ordinary senescence, the WS pheno-
type suggests chromosomal metabolism is a part of a genetic
process of senescence.
Chromosomal metabolism appears to be associated with
senescence in mice. Chromosomal aberrations progressively
increase in bone marrow cells as some strains of senescence
accelerated mice age (8) and in liver cells as C57BL?6J mice
age (9). Interestingly, cells deleted for some chromosomal
metabolism proteins exhibit premature replicative senescence.
For example, murine cells deleted for a segment of the murine
WRN homologue exhibit hypersensitivity to topoisomerase
inhibitors and premature replicative senescence (10). Addi-
tionally, murine cells deficient for the recombinational repair
proteins,Atm(11)andBrca2(12–14),andthenonhomologous
end-joining (NHEJ) proteins, Ku70 (15), Ku86 (16), Xrcc4
(17), and DNA ligase IV (18), undergo premature replicative
senescence. Yet an early onset of senescence has not been
reported for mice harboring mutations in any of these genes.
Here, we investigate the role of Ku86 during senescence in
the whole mouse. Ku86 is important for the repair of DNA
double-strandbreaks(DSB)byNHEJinassociationwithKu70
(19, 20). The Ku86–Ku70 heterodimer (Ku) binds to DNA
ends, nicks, gaps, and hairpins. In vitro, Ku forms a complex
called DNA-dependent protein kinase by associating with a
450-kDa catalytic subunit (DNA-PKCS). These subunits, to-
gether with Xrcc4 and DNA ligase IV, are important for
repairing DNA DSB formed during the assembly of antigen
receptors and after exposure to ionizing radiation (15–18,
21–23).
Further analysis of ku86-mutant mice shows early onset of
age-specific changes. Compared with control littermates,
ku86-mutant mice prematurely exhibit osteopenia, epiphyses
closure, atrophic skin and hair follicles, hepatocellular degen-
eration, and age-specific mortality. The age-specific diseases:
cancer and likely sepsis (suggested by reactive immune re-
sponses), are partly responsible for age-specific mortality in
both cohorts. Both diseases occur earlier in ku86-mutant mice.
These data suggest that Ku86 influences the process of senes-
cence.
MATERIALS AND METHODS
Ku86 Genotypic Analysis by PCR. The wild-type allele was
detected with the sense primer (5?-GAGAGTCTACGA-
CAACTGTGC-3?) and the antisense primer (5?-AGAGG-
GACTGCAGCCATATTA-3?) located in sequences deleted
in the mutant Ku86 allele described as xrcc5M1(21). The
mutant allele was detected with a sense primer (5?-
GGTTGCCAGTCATGCTACGGT-3?), which anneals to in-
tronic sequences upstream of the positive selection cassette,
and an antisense primer (5?-CCAAAGGCCTACCCGCTTC-
CATT-3?), which anneals to the PGK promoter in the positive
selection cassette. PCR reactions were preincubated at 94°C
for 5 min and then 30 cycles of amplification at 94°C for 30 sec,
59°C for 1 min (with 1-min ramp), and 72°C for 30 sec in a
Perkin–Elmer DNA Thermal Cycler 480. Wild-type (0.3-kb)
and mutant (0.4-kb) fragments were separated on a 1.2%
ethidium bromide stained gel by electrophoresis. PCR prod-
ucts were sequenced to prove they were not artifacts, and
results were confirmed by Southern analysis (21).
Histological Analysis. Tissues were fixed in 10% buffered
formalin phosphate, paraffin embedded, cut into 4 ?M sec-
tions, and stained with haematoxylin and eosin by standard
procedures.
Immunohistochemistry for Liver Sections. Immunohisto-
chemistry was performed by using 3-?m paraffin sections that
were deparaffinized, hydrated, blocked in 3% methanol?
hydrogen peroxide, then incubated with the primary antibody
against glutamine synthetase (GS) (gift from James W. Camp-
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
PNAS is available online at www.pnas.org.
This paper was submitted directly (Track II) to the Proceedings office.
Abbreviations: WS, Werner’s syndrome; DSB, double-strand break;
NHEJ, nonhomologous end joining; Ku, the Ku86–Ku70 heterodimer;
GS, glutamine synthetase; FAH, fumarylacetoacetatehydrolase;
MAFP, mouse ? fetoprotein; ALT, serum alanine aminotransferase
concentration.
‡Present address: Department of Hematology—Oncology, St. Jude
Children’s Research Hospital, 332 North Lauderdale Street/D1034,
Memphis, TN 38105-2794.
¶To whom reprint requests should be addressed. E-mail: phasty@
lexgen.com.
10770

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bell, Rice University); fumarylacetoacetatehydrolase (FAH)
(gift from Robert Tanguay, University of Laval, Quebec); and
mouse ? fetoprotein (MAFP) (ICN catalogue no. 645611),
Ki-67 [Immunotech (Westbrook, ME) Mib-1 clone]. All pri-
mary antibodies are rabbit polyclonal. Secondary anti-rabbit
antibody was applied as SuperSensitive MultiLink Anti-rabbit
(BioGenex?ABN no. HK326-UR) followed by Super Sensitive
Label (BioGenex?ABN no. HK330–9K) for FAH and MAFP,
and with secondary anti-rabbit and strepavidin-alkaline phos-
phatase for GS and Ki-67, followed by 3-amino-9-ethylcarbo-
zole (BioGenex?ABN no. HK129–5K) for colorimetric detec-
tion, then hydrated, counterstained with hematoxylin, and
covered with coverslip.
Serum Alanine Aminotransferase Concentration (ALT).
Blood was taken from anesthetized mice by intracardiac
puncture. Serum was isolated by centrifugation and analyzed
by using a standard assay of ALT activity (Vitrosslides,
Johnson and Johnson, New Brunswick, NJ).
RESULTS
Ku86wasdeletedinmicebymutatingthegeneXrcc5(21).This
mutation was previously designated xrcc5M1; however, for
clarity, it is called ku86?/?or Ku86?/?in the homozygous or
heterozygous condition, respectively. A cohort of 47 control
mice (wild type and Ku86?/?) and 89 ku86?/?mice, in a
C57BL?6 ? 129Sv crossbred background, were observed for
age-specific changes associated with advanced age.
Shortened Lifespan for ku86?/?Mice. The relationship
between chronological age and mortality is an important
indicator of senescence (24); therefore, a survival curve was
established starting with 3-wk-old mice. The lifespan of
ku86?/?mice is shorter than that of control mice, primarily
because of an early onset of age-specific mortality, which is
about 8 and 56 wk, respectively (Fig. 1A). About 50% of
ku86?/?and control mice die by 36 and 102 wk, respectively.
The average lifespan is 38 ??? 14 wk for ku86?/?mice and 97
??? 17 wk for control mice. Within the cohorts observed, the
most long-lived ku86?/?and control mice died at 87 and 127
wk, respectively. By comparing survival curves, there is a
difference in the rate of age-specific mortality (P ? 0.005). The
first 60% of the ku86?/?population died at a slightly faster rate
than controls, whereas the remaining 40% died at a progres-
sively slower rate (Fig. 1A).
Mortality is not necessarily caused by senescence; however,
age-specific illnesses like sepsis likely result from a progressive
deteriorative process associated with advanced age (25). Age-
specific acute and chronic immune reactions were observed in
a variety of organs that include the liver (Fig. 1 B and C),
kidney, spleen, urogenital tract, oropharynx, skin, and sub-
mandibular glands for both cohorts. These reactive immune
responseswereobservedatayoungerageinku86?/?micethan
control mice (observation of reactive immune responses for
the liver are described in Table 1). Sepsis is implicated through
the activation of reactive immune responses, especially in the
liver and kidney, and could progress into a lethal condition.
It is possible that exogenous opportunistic infection caused
the reactive immune responses in ku86?/?mice because they
are immune deficient because of defective V(D)J recombina-
tion (21). However, this is not likely for the following reasons.
First, fatal systemic opportunistic infection derived from nor-
mal flora increase with advanced age (25). Second, ku86?/?
mice were housed in a specific pathogen-free environment
(tests negative for all known pathogens). These ku86?/?mice
lived much longer than ku86?/?mice housed in a conventional
colony contaminated with mouse hepatitis virus (MHV) and
Taielers encephalomyelitis (50% die by 17 wk, 57 ku86?/?mice
observed). MHV likely caused morbidity in at least two
ku86?/?mice housed in the conventional colony, because they
suffered from necrosis and inflammation of the liver (not
shown). Third, reactive immune responses were commonly
observed in the immune-competent control mice housed in the
same cages. Therefore, the etiology of reactive immune re-
sponsesinku86?/?miceislikelytobesimilartothatforcontrol
mice. Even though the reactive immune responses are similar
for both cohorts, it is possible that some differences exist, such
as predisposition to certain pathological agents. A more
detailedanalysisofthepathogensintheseanimalsiswarranted
in future studies.
Cancer incidence progressively increases with age in both
mice (26) and humans (27) and causes morbidity in both
cohorts. Compared with control mice, ku86?/?mice exhibited
an early onset of cancer. Both cohorts exhibited malignant
lymphoma (Fig. 1 D and E): eight of 47 control mice (75–119
wk) and two of 89 ku86?/?mice (37 and 54 wk). In addition to
lymphoma, the following cancers were found in one of the 47
control mice: harderian gland adenocarcinoma (86 wk), squa-
mous-cell carcinoma (90 wk), angiosarcoma (94 wk), adeno-
carcinoma of the lung (100 wk), mammary adenocarcinoma
(102 wk), germ-cell neoplasm (110 wk), and hemangiopericy-
FIG. 1.
Survival curve (100%? number of mice alive after each week?total
number of mice at beginning of study). The survival curve begins after
weaning (3 wk) because ku86?/?pups are less fit to compete for
resources than control littermates, and about 50% die before weaning
unless control pups are removed soon after birth (16). Control mice,
blue squares; ku86?/?mice, green triangles. A dashed blue line
representing the control survival curve is superimposed onto the
ku86?/?curve, and a dashed green line representing the ku86?/?
survival curve is superimposed onto the control curve, to illustrate
their differences. The percent of population that dies each week is
displayed to the left of the ku86?/?survival curve and the right of the
control survival curve for each interval of 20% starting at the onset of
age-specificmortality.Numberofmiceobserved:control,47;ku86?/?,
89. (B and C) Section of liver with reactive immune response from (B)
89-wk-old control mouse and (C) 61-wk-old ku86?/?mouse. Note
infiltration of mononuclear cells for B and neutrophils for C. (D and
E) Section of malignant lymphoma from (D) 71-wk-old control mouse
(infiltrating intestine) and (E) 37-wk-old ku86?/?mouse (subjacent to
bronchial epithelium). (B–E, ?205.)
Lifespan and potential causes of age-specific death. (A)
Genetics: Vogel et al.Proc. Natl. Acad. Sci. USA 96 (1999)10771

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toma (119 wk). The mice with adenocarcinoma and germ-cell
neoplasm also had lymphoma (included in the eight mice).
These forms of cancer were not observed in the ku86?/?
cohort. Thus, 13 of 47 control mice had cancer (27.6%),
whereas two of 89 ku86?/?mice had cancer (2.2%).
Early Onset of Age-Specific Changes Observed in ku86?/?
Mice. Control and ku86?/?mice were observed for outward
changes associated with age. Both cohorts exhibited kyphosis
(abnormally increased convexity in the curvature of the tho-
racic spine from a lateral view) and decreased skin thickness
as they aged with an earlier onset for ku86?/?mice (Fig. 2
A–D). These outward changes occurred, by varying degrees,
for the most long-lived 30% of the population of both cohorts.
Bone, epiphyses, skin, and hair follicles were observed for
age-specific histological changes as both cohorts aged (Table
1). Bone was observed for osteopenia (28), epiphyses for
closure, and skin and follicles for atrophy (29). No difference
was observed for bone, epiphyses, skin, or hair follicles be-
tween 1- to 15-wk-old control and ku86?/?mice. However,
ku86?/?mice, but not control mice, exhibited osteopenia by 37
wk (Fig. 2 E and F), epiphyseal closure by 22 wk (Fig. 2 G and
H) and skin and follicular atrophy by 37 wk (Fig. 2 I–L).
Control mice, greater than 70 wk of age, exhibited osteopenia,
epiphyses closure, and skin and follicular atrophy (Table 1).
Osteopenia and skin and follicular atrophy are commonly
associated with advanced age in both mice and humans
(28–30). Even though epiphyses closure is not considered to be
characteristic of senescence, it is an age-specific change that
results in cellular decline of function in reproductively-mature
animals; therefore, the genetic process of epiphyses closure
may be similar to, or overlap with, that of senescence.
The liver was examined for signs of senescence because
genomic rearrangements accumulate in the liver as mice age
(9).ALTwasperformedtoassesshepatocellularnecrosisasan
indicator of liver damage. ALT concentration was not elevated
for control and ku86?/?mice between 1–15 wk (Table 1).
However, ku86?/?mice, but not control mice, exhibited mildly
elevated ALT concentrations by 33 wk of age. Control mice
greater than 70 wk of age exhibited mildly elevated ALT
concentrations (Table 1).
Livers were examined grossly and by histology for age-
specificchanges.Nodulesappearedintheliversofbothcontrol
and ku86?/?mice. These lesions are best observed microscop-
ically, represent a response to unspecified hepatocellular in-
jury, and represent proliferative activity that is potentially
preneoplastic; however, there is no evidence that these lesions
impact mortality. Hyperplastic foci were observed at a younger
age for ku86?/?mice than control mice (Table 1). Histology
shows the nodules compress normal liver for control (Fig. 3A)
and ku86?/?mice (Fig. 3B). Special stains performed on a
section at the border of normal liver and liver nodule from a
ku86?/?mouse showed dysregulation, proliferation, and ded-
ifferentiation of hepatocytes. Abnormalities were detected
only in hepatocytes within the nodule. Improper expression of
GS and FAH demonstrates hepatocellular dysregulation. GS is
normally expressed in hepatocytes that are two deep from the
portal veins (Fig. 3C); however, GS was expressed in nodular
hepatocytes not bordering the portal veins (Fig. 3D). FAH is
normally expressed in a diffuse pattern; however, FAH was
expressed in a mosaic pattern for nodular hepatocytes (Fig.
3E).ProliferationofnodularhepatocyteswasshownbyaKi-67
stain (stains cells in late G1, S, G2, and M; Fig. 3F). Addition-
ally, dedifferentiation of nodular hepatocytes was shown by
expression of MAFP, which is normally expressed in fetal, but
not adult liver (Fig. 3G). Thus, these changes demonstrate this
liver nodule is adenomatous.
In addition to hyperplastic foci, histology revealed another
age-specific change in the liver. Both control (Fig. 3H) and
ku86?/?(Fig. 3I) mice exhibit hepatocellular inclusions as they
age with an earlier onset for ku86?/?mice (Table1). Cyto-
plasmic and nuclear inclusions in hepatocytes are character-
istic of age-specific changes in mice (31).
DISCUSSION
Do ku86?/?mice truly exhibit an early onset of senescence? To
answer this question, it is important to understand the distinc-
tion between aging and senescence. Aging encompasses all
time-related changes that may have positive, neutral, or dete-
riorative effects. Senescence, as defined by Edward Masoro,
describesonlythe‘‘deteriorativechanges[thatoccur]withtime
during postmaturational life that underlie an increasing vul-
nerability to challenges, thereby decreasing the ability of an
organism to survive’’ (24). By this definition, ku86?/?mice
exhibit an early onset of senescence.
Proposed theories for a genetic basis of evolutionary aging
generally agree that senescence is not subject to natural
selection (24). Data presented here do not dispute these
theories; however, they suggest that a genetic process influ-
enced by Ku86 determines the onset of senescence that would
be subject to natural selection. It is interesting to note that for
ku86?/?mice, the onset of age-specific mortality begins shortly
after sexual maturity, possibly accounting for their reduced
fecundity (P.H., unpublished results). Therefore, Ku86 in-
Table 1. Summary for ku86???(mt) and control (con) mice
Genotype
mt
con
mt
con
mt
con
mt
con
mt
con
The number of mice affected (left) compared to the total number observed (right). nd, not done.
*Number observed: for ku86???at 1–15 wk, 3; 31–50 wk, 3; 51–70 wk, 6 and for control at 1–15 wk, 6; 31–50 wk, 1; 50–70 wk, 6; ?70 wk, 11.
†Cytoplasmic hepatocellular inclusions observed for all ku86???mice and four control mice, nuclear hepatocellular inclusions observed for two
control mice.
‡Includes microabscesses (acute response with neutrophils) and granulamatous inflamation (chronic response with mononuclear cells) observed
in the liver.
§Nodules were grossly observed in seven livers from control mice and in two livers from ku86???mice, the remainder could only be observed by
histology (micronodular).
Age, wk
1–15
Osteopenia
0?3
0?5
nd
nd
3?3
0?4
nd
nd
nd
4?5
Epiphysis
closure
0?6
0?7
2?2
0?2
1?1
0?1
nd
nd
nd
4?4
Skin and follicular
atrophy
0?4
0?7
nd
nd
3?3
0?4
nd
nd
nd
8?8
ALT,*
units/liter
57 ? 1
56 ? 17
nd
nd
81 ? 4
40
93 ? 30
38 ? 6
nd
126 ? 109
Inclusions†
0?7
0?12
nd
nd
2?4
0?4
3?8
0?9
1?1
6?23
Reactive immune
response‡
0?7
0?12
nd
nd
1?5
0?4
2?7
0?9
0?1
19?23
Nodular
hyperplasia§
0?7
0?12
nd
nd
2?4
0?4
5?8
1?9
1?1
9?23
22
31–50
51–70
?70
10772Genetics: Vogel et al.Proc. Natl. Acad. Sci. USA 96 (1999)

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FIG. 2.
visualization of kyphosis. (A) Control (??? and ???) and ku86?/?(???) mice at 2.5 wk. No kyphosis observed. (B) Control and ku86?/?mice
at 31 wk. Kyphosis observed in only ku86?/?mouse. (C) Control and ku86?/?mice at 75 and 79 wk, respectively. Kyphosis observed in only ku86?/?
mouse. (D) Control mouse at 120 wk. Kyphosis observed. (E and F) Section of vertebral articular processes from (E) 45-wk-old control and (F)
49-wk-old ku86?/?mouse. For ku86?/?bone, compared with control, the cortical wall (cw) and trabeculae (t) is thinner, the number of trabeculae
are reduced and the medullary cavity (mc) is expanded. (G and H) Section of epiphysis from 22-wk-old (G) control and (H) ku86?/?mouse. For
ku86?/?epiphysis, compared with control, the number of chondrocytes is reduced and the columnar organization of chondrocytes is lost. (I–L)
Section of skin (dorsal region over cranial to mid-thorax) from (I and J) 45-wk-old control and (K and L) 49-wk-old ku86?/?mouse. For ku86?/?
skin, compared with control, all subcutaneous elements, including superficial collagen (sc), subcutaneous adipose (sa), and skeletal muscle (sm)
are reduced and hair follicles and sebaceous glands (arrow in J and L) atrophied. (Bar ? A–D 1 cm.) (E and F, ?65; G and H, ?200; I and K,
?35; J and L, ?175.)
Age-specific changes in control and ku86?/?mice. (A–D) Outward signs of senescence. The dorsal region was shaved to enhance
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Page 5

creases fitness (ability to survive and reproduce) and would be
subject to selective pressure.
Ku86, Chromosomal Metabolism, and Senescence. Data
presented here support models that propose senescence is
influenced by chromosomal metabolism. These models in-
clude accumulation of genetic damage induced by reactive
oxygen species (32) and maintenance of telomeres (33) and?or
ribosomal DNA (34, 35).
Analyses of senescence in the budding yeast, Saccharomyces
cerevisiae offer intriguing possibilities for the relationship of
DNA DSB repair and the onset of senescence. Yeast cells,
deficient for either NHEJ or recombinational repair, have
shortened lifespans. Sir proteins (Sir2, Sir3, Sir4), important
for NHEJ (36), delay the onset of senescence by translocating
from telomeres to the nucleolus (37) to reduce the accumu-
lation of extrachromosomal ribosomal DNA (rDNA) circles
(ERCs) (38). yKu may participate with the Sir complex by
virtue of the Sir4–yKu70 association (36). Recent publications
describe the yKu and Sir proteins translocating from telomeres
to an induced DSB in DNA as a part of a Mec1, Rad9 response
(39, 40). Thus, onset of senescence may be delayed by a
cell-cycle response to DSB in rDNA. By comparison, a defi-
ciency of recombinational repair prematurely translocates Sir3
from telomeres, however without accumulation of ERCs.
Therefore, a general response to DSB and not just DSB in
rDNA may regulate onset of senescence.
It will be interesting to determine whether a similar model
applies to mammals. Perhaps the wide variance in lifespan
within a species is partly caused by subtle differences or
requirements within the population to repair DSB by NHEJ
and recombinational repair. If this were true, then ku86?/?
mice would be particularly sensitive to subtle differences or
requirements in recombinational repair because of absence of
NHEJ, which could contribute to their altered lifespan curve.
Ku86 and Cancer Incidence. Cancer is a cause of mortality
for both ku86?/?and control cohorts. Even though cancer was
observed earlier in the ku86?/?cohort, cancer incidence was
reduced by 13-fold. This reduction may be simply a conse-
quence of insufficient time for cancer to develop in ku86?/?
mice because of their shortened lifespan. Alternatively, it is
possible that deletion of Ku86 reduces cancer incidence in
accord with the theory that senescence is antioncogenic (41),
perhaps because of increased cell-cycle responses to inefficient
repair of DSB. Several observations support the latter view.
First, ku86?/?mice exhibit an early onset of a variety of
age-specific conditions (including cancer) that are not usually
observed in control mice until their age exceeds that of the
longer-lived ku86?/?mice. Second, the mortality rate signifi-
cantly declines for the most long lived 40% of the ku86?/?
cohort compared with control, indicating diminished rate or
occurrence of some lethal condition. Third, ku86?/?mice have
a sufficiently long lifespan for a large fraction to develop
cancer, exemplified by mice deleted for p53 (42), Atm (43),
Brca2 (12, 44), Ku70 (15, 45), and some mismatch repair
proteins (46).
Regardless whether deletion of Ku86 reduces cancer inci-
dence, its deletion does not significantly increase cancer
incidence. This observation is surprising given that deletion of
DNA repair proteins frequently increase the risk of cancer
(47). Most surprising is that mutation of Ku86 affects cancer
riskdifferentlythanmutationofKu70(highincidenceofCD4?
CD8?T cell lymphoma) and suggests that one or both of these
proteins work independently of the other for certain functions
(15, 45). Independent function is possible because Ku86 is
present at low levels in ku70?/?mice (15) and vice versa (16).
T cell lymphoma may be caused by leaky T cell development
that occurs exclusively in ku70?/?mice (15, 45). Alternatively,
the difference in cancer incidence may be because of different
genetic backgrounds; however, this seems unlikely because the
genetic backgrounds are very similar (129Sv ? C57BL?6).
We thank Drs. Mariana Yaneva and Greg Donoho for helpful
discussions; Drs. Molly A. Bogue, David B. Roth, Mariana Yaneva,
Greg Donoho, Brian Zambrowicz for critical review of the manuscript;
Mrs. Shirley Jackson and Mr. Darrin Shiver for technical assistance;
and Drs. James W. Campbell and Robert Tanguay for the generous
gifts of anti-GS and anti-FAH, respectively. Lexicon Genetics Inc. and
the National Cancer Institute (1RO1CA76317-01, to P.H.) supported
this work.
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FIG. 3.
(A) 110-wk-old control mouse and (B) 61-wk-old ku86?/?mouse.
Normal part of liver (Upper Left) is compressed by nodule (Lower
Right), separated by dashed line. (C) Expression of GS in normal part
of liver. GS present in cells only two deep from portal vein (pv). (D–G)
Dysregulation, proliferation, and dedifferentiation of cells in a liver
nodule from a 61-wk-old ku86?/?mouse. (D) GS stain. Diffuse
hepatocellular staining in liver nodule, but not normal liver. (E) FAH
stain. Loss of FAH diffuse positivity resulting in mosaic expression
pattern in liver nodule but not normal liver. (F) Anti-Ki-67 stain.
Cellularproliferationinnodularhepatocytes,butnotnormalliver.(G)
MAFPstain.MAFPisnotnormallyexpressedinadulthepatocytes.(H
and I) Hepatocellular cytoplasmic inclusions (arrows) from: (H)
111-wk-old control mouse and (I) 65-wk-old ku86?/?mouse. (A–
F ?115; G, ?210; H and I, ?420.)
Age-specific changes in liver. (A and B) Liver nodules from
10774Genetics: Vogel et al.Proc. Natl. Acad. Sci. USA 96 (1999)

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