Mechanisms of Ageing and Development
122 (2001) 1221– 1255
www.elsevier.com/locate/mechagedev

Review

Mutant and genetically modiﬁed mice as
models for studying the relationship between
aging and carcinogenesis
Vladimir N. Anisimov *
Department of Carcinogenesis and Oncogerontology, N.N. Petro6 Research Institute of Oncology,
Pesochny-2, St Petersburg 197758, Russia
Received 30 October 2000; received in revised form 23 March 2001; accepted 3 April 2001

Abstract
Increased interest is emerging in using mouse models to assess the genetics of aging and
age-related diseases, including cancer. However, only limited information is available regarding the relationship between aging and spontaneous tumor development in genetically
modiﬁed mice. Analysis of various transgenic and knockout rodent models with either a
shortened or an extended life span, provides a unique opportunity to evaluate interactions of
genes involved in the aging process and carcinogenesis. There are only a few models which
show life span extension. Ames dwarf mutant mice, p66 − / − knockout mice, aMUPA and
MGMT transgenic mice live longer than wild-type strains. The incidence of spontaneous
tumors in these mutant mice was usually similar to those in controls, whereas the latent
period of tumor development was increased. Practically all models of accelerated aging
showed increased incidence and shorter latency of tumors. This phenomenon has been
observed in animals which display a phenotype that more closely resembles natural aging,
and in animals which manifest only some features of the normal aging process. These
observations are in agreement with an earlier established positive correlation between tumor
incidence and the rate of tumor incidence increase associated with aging and the aging rate
in a population. Thus, genetically modiﬁed animals are a valuable tool in unravelling
mechanisms underlying aging and cancer. Systemic evaluation of newly generated models

* Tel.: + 7-812-5968607; fax: +7-812-5968947.
E-mail address: aging@mail.ru (V.N. Anisimov).
0047-6374/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 4 7 - 6 3 7 4 ( 0 1 ) 0 0 2 6 2 - 7

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should include onco-gerontological studies. © 2001 Elsevier Science Ireland Ltd. All rightsreserved.
Keywords: Aging; Cancer; Transgenic animals; Knockout animals; Mutant animals

1. Introduction
The differences in longevity and pathology incidence, including cancer incidence
among rodent strains, provide strong evidence of genetic inﬂuence on these
parameters, although one can argue that the differences between strains are also
inﬂuenced by epigenetic mechanisms such as imprinting. Genes may affect the
variation in life span within an inbred genetic background (de Haan et al., 1998),
possibly through a response threshold (Jazwinski, 1999a). During the last decade a
number of genetic models with extended or reduced life spans have been generated.
These models offer new approaches towards understanding the aging process. An
overview of transgenic methods to increase the life span of fruit ﬂies has recently
been published (Tower, 2000). Spontaneous and induced genetic modiﬁcations,
including homozygous null mutations (‘knockout’) and transgenic mammalian
animals, have also been introduced in experimental gerontology (Gordon et al.,
1993; Ingram and Jucker, 1999; Morgan et al., 1999; Guarente and Kenyon, 2000).
It is worth noting that, although the effects of some genetic manipulations only
manifest themselves at speciﬁc developmental periods of animal life, these genetic
manipulations are in effect during embryonal development as well as throughout
adult life. Therefore, there are signiﬁcant limitations in the interpretations of data
generated by utilizing these genetic manipulations. Some of them have been
discussed recently (Jazwinski, 1999a; Morgan et al., 1999). On the one hand, the
elimination of a speciﬁc activity or a speciﬁc pathway can lead to an erroneous
conclusion regarding gene function because the compensatory mechanism may
markedly alter the animal physiology. On the other hand, overexpression of a
transgene may readily yield no effect on the life span, or on any other aging
parameter of the animal for that matter. Jazwinski (1999b) has noted that overexpression of a transgene will likely create interactions with other genes and with the
environment, both external and internal.
A relationship between aging and carcinogenesis has been intensively discussed
(Dix et al., 1980; Anisimov, 1983, 1987, 1998a,b; Miller, 1991; Campisi, 1997, 2000;
Dix and Cohen, 1999; DePinho, 2000). The increased incidence of cancer as a
function of aging has long been interpreted to suggest that multiple genetic changes
are required for carcinogenesis. Cancer cells differ from normal cells in many
characteristics, including loss of differentiation, phenotypic changes, increased
invasiveness, and decreased drug sensitivity (Kinzler and Vogelstein, 1997;
Lengauer et al., 1998; Hanahan and Weinberg, 2000). Although recent advances in
molecular biology have helped to clarify the possible relationships between carcinogenesis and aging, it remains unclear whether genetic markers may be common to
all cancer types or which markers may be associated with the increased age of
cancer patients. Transgenic animal technology has resulted in a plethora of murine

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models for cancer research, providing insight into the complex carcinogenic events
contributing to the loss of cell cycle control and tumor development. Transgenic
and null mutant animal models also offer an important opportunity to identify and
study both carcinogen and chemopreventive agents (Alexander, 2000; Gulezian et
al., 2000). Genetically modiﬁed animal models which are characterized by a
shortening or extension of life spans, give a unique possibility to evaluate the role
of aging genes involved in the mechanisms of carcinogenesis.

2. Ames dwarf mice
Ames dwarf mice, which typically have a longer life than other inbred mice, is
one of several novel models in the investigation of aging (Mattison, 2000; Bartke et
al., 2001). These mice are homozygous autosomal recessive mutants (single point
mutation in the Prophet gene) in a line of extreme non-agouti mice derived from a
cross with descendants from an irradiation experiment. The dwarfs, which live from
50 to 64% longer (males and females, respectively) than wild-type siblings (BrownBorg et al., 1996; Brown-Borg and Rakoczy, 2000), are one of the ﬁrst mammalian
examples of a single gene’s ability to signiﬁcantly extend individual life spans. The
autosomal recessive mutation results in the developmental failure of the pituitary to
initiate synthesis and secretion of the growth hormone (GH) and the prolactin and
thyroid stimulating hormone (TSH). These dwarf mice also have a low IGF-1 and
blood insulin level, high sensitivity to insulin and decreased body temperature. Both
male and female Ames dwarf mice are hypogonadal and infertile (Mattison, 2000;
Bartke et al., 2001). Ames dwarf mice show signs of immunodeﬁciency, which is
evident in the lymphocyte depletion in peripheral lymphoid tissue and by the
involution of thymus, a decreased natural killer activity of splenic lymphocytes,
although they have normal antibody production in response to tetanus toxoid
(Duquesnoy, 1972; Esquiﬁno et al., 1991; Mattison, 2000). There is evidence that
the tissues of the Ames dwarf mice have lower liver glutathione and ascorbate levels
and a higher catalase activity compared to normal controls. They are also less
vulnerable to oxidative damage (Brown-Borg et al., 1999; Mattison, 2000; BrownBorg and Rakoczy, 2000). Spontaneous tumor incidence in aging dwarf and normal
mice does not differ. However, dwarfs live signiﬁcantly longer than normal mice,
therefore, it is possible that the tumors develop later in dwarfs, or that the tumors
grow more slowly (Mattison, 2000; Mattison et al., 2000).

3. Senescence accelerated mice (SAM)
The SAM strain was generated by selective inbreeding of AKR/J mice (Takeda
et al., 1997; Takeda, 1999). There are several senescence-prone strains (SAMP),
which live 12– 15 months, and several senescence-resistant (SAMR) strains, which
are normal controls for accelerated aging mice and have a life span of 24–30
months. It has been shown that SAMP mice develop normally until the age of 4

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months and then they reveal signs of accelerated aging such as loss of hair, skin
ulceration, decrease in locomotor activity, deﬁciency in learning and memory,
emotional disorders, abnormal circadian rhythms, brain atrophy, hearing impairment, cataracts, increased production of reactive oxidation specimens (ROS) and
8-hydroxyguanine levels in all organs (Takeda et al., 1997; Bulygina et al., 1999;
Choi et al., 1999; Takeda, 1999; Yuneva et al., 2000). The amount of Cu, Zn-SOD
in the mitochondrial fraction of the SAMP-1 was only half that of the SAMR-1
(Park et al., 1996). The reproductive life span of SAMP was shorter than that of the
SAMR and the reproductive senescence of the SAMP strain was more accelerated
than that of the SAMR strain (Miyamoto et al., 1995). It is worth noting that
O6-methylguanune-DNA methyltransferase, which repairs alkylated DNA, shows
no difference in its activity in SAMP1 when compared with the SAMR1 strain
(Choi et al., 1999).
The accelerated senescent-prone strain, SAMP-1, shows a striking increase in the
frequency of chromosome aberrations from the age of 3–8 months, whereas the
SAMR-1 strain shows only a slight increase in chromosome aberrations at the same
age (Nisitani et al., 1990). Uryvaeva et al. (1999) have shown an accelerated
accumulation of micronuclear aberrations with age in the liver cells of SAMP mice
compared to the SAMR strain. The age-associated incidence of somatic Hprt
mutations in splenic lymphocytes, as well as DNA damage (mainly DNA single
strand breaks) in six organs, are also accelerated in SAMP1 mice compared to the
SAMR1 (Odagiri et al., 1998; Hosokawa et al., 2000).
The incidence of spontaneous lymphomas is 17.5% in SAMP strains (from 0% in
SAMP11 and SAMP6 to 60.2% in SAMP7) and 13.7% in SAMR strains (from
2.7% in SAMR5 and 23.1% in SAMR4). The incidence of other malignancies varies
from 0 to 4.8% in SAMP and from 3.8 to 4.1% in SAMR strains (Takeda et al.,
1997). The levels of murine leukaemia virus titres are found to be higher in the
blood and spleen and much higher in the brain of SAMP-8 than in the same tissues
of the SAMR1 strain (Meeker and Carp, 1997). Sugimura et al. (1994) revealed a
high incidence of stromal hyperplasia with ﬁbrosis and inﬂammation in the dorsal
lobe of the prostate gland in SAMP mice. Atypical glandular epithelial cells and
cribriform glandular deformity were observed in the dorsal and lateral lobes of the
prostate gland of the SAMP strain.

4. Mutation in the mouse klotho gene
Kuro-o et al. (1997) established a novel mouse autosomal recessive mutant,
klotho, that exhibits multiple phenotypes very similar to those observed in human
aging, including a short life span (less than 100 days), decreased body weight,
infertility, arteriosclerosis, skin and thymus atrophy, osteoporosis and emphysema.
These mice are hypoglycaemic and have decreased levels of insulin in the pancreas.
Glucose tolerance and sensitivity to insulin are increased in klotho mice, compared
to these parameters in wild-type mice (Mori et al., 2000; Utsugi et al., 2000).
Uncoupling protein-1 gene expression of the brown adipose tissue and body

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temperature in klotho mice were lower than those in wild-type mice, suggesting that
klotho mice have less energy expenditure than wild-type mice (Mori et al., 2000).
Immunohistochemistry of the pituitary glands of kl/kl mice conﬁrmed a decrease in
the growth hormone, as well as the luteinizing hormone and follicle-stimulating
hormone production. The gene has homology with the membrane sparring region
and with the b-glucosidase enzymes. Recently the i-klotho (ikl) gene, which
encodes a type I membrane protein, has been cloned (Ito et al., 2000). Kuro-o et al.
(1997) suggested that the klotho gene product may function as part of a signalling
pathway regulating aging in vivo and morbidity in age-related diseases. The authors
also concluded that kl/kl mice were not a model for mouse aging, but rather for
human progeroid syndromes. There are no data on tumor pathology in these mice.
The klotho mouse differs from SAM in several aspects: (a) the multiple aging-associated phenotypes in kl/kl mice are autosomal recessive and are not inﬂuenced by
the genetic background, whereas the conditions of inheritance in SAM are more
complex; (b) the multiple aging-associated phenotypes occur in kl/kl mice, whereas
speciﬁc aging-associated phenotypes are typical for various SAM substrains; and (c)
the aging-associated phenotypes in kl/kl mice manifest themselves much earlier
than in SAM (Kuro-o et al., 1997). It is worth noting that the klotho mouse is the
ﬁrst laboratory animal model with multiple phenotypes resembling human aging
caused by a single gene mutation. The study, focused on osteopenia in kl/kl mice,
has shown that a defect in the klotho gene expression causes the independent
impairment of both osteoblast and osteoclast differentiation, leading to low cell
turnover and osteopenia (Kawaguchi et al., 1999).

5. DNA repair gene transgenic and knockout models
DNA repair plays an important role in genome stability. The DNA damage
theory assumes that aging in mammals is due to the accumulation of DNA damage
in somatic cells, being preceded by the somatic mutation theory of aging (Anisimov,
1987; Bernstein and Bernstein, 1991; Vijg, 2000). Taking into consideration the
relationship between DNA damage, defective DNA repair, and carcinogenesis, it
could be suggested that age-related changes both in efﬁcacy and the rate of DNA
repair in an individual might modify the susceptibility to exogenous or endogenous
carcinogens (Anisimov, 1987). There are several types of DNA damage that occur
in nature: spontaneous depurination and depyrimidination, cytosine deaminations,
single-strand breaks, O6-methylguanine, glucose and glucose-6-phosphate adducts,
oxidative damage (thymine and thymidine glycols, hydroxymethyluracil, 8-hydroxideoxyguanosine, methyl adducts, cross-links and double-strand breaks (Bernstein
and Bernstein, 1991)). All of them could play a role in aging and carcinogenesis
(Anisimov, 1987, 1998a; Bernstein and Bernstein, 1991).
Xeroderma pigmentosum, characterized by a deﬁciency in the nucleotide excision
repair and an over 1000-fold increased risk of skin cancer, represent a paradigm to
understand the role of unrepaired lesion in the development of cancer (Benhamou
and Sarasin, 2000). Recently, two mouse models were generated with a defect in

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one of the nucleotide excision DNA repair genes (XPD and CSB), displaying
distinctive symptoms of premature aging (de Boer et al., 1999). No data on tumor
incidence in these mice have been reported yet.
The primary embryonic ﬁbroblasts isolated from the xeroderma pigmentosum
group G (XP-G) gene-deﬁcient mice underwent premature senescence and exhibited
the early onset of immortalization and accumulation of p53 (Harada et al., 1999).
Xeroderma pigmentosum group A (XPA) gene-deﬁcient mice have an almost
complete deﬁciency in DNA nucleotide excision repair, and only 15% of the mice
develop spontaneous tumors (hepatocellular adenomas) after 1.5 years (Van Steeg
et al., 1998). However, XPA − / − mice are very susceptible to ultraviolet B radiation
and to different chemical carcinogens (Van Steeg et al., 1998, 2000).
Failure of DNA repair and the ﬁxation of DNA damage as mutation, eventually
leads to cellular transformation and carcinogenesis. In response to DNA damage,
a nuclear enzyme, poly (ADP-ribose) polymerase (Parp), is activated, and poly
(ADP-ribosyl) activates various nuclear proteins using NAD as a substrate. Parp is
involved in the base-excision repair process and in DNA strand break repair
(Ruscetti et al., 1998; Dantzer et al., 1999) and the induction of cell death (Berger
et al., 1983). Parp knockout mice were established by disrupting Parp exon 1, 2 or
4 in the genetic background of 129Sv/C57BL6 or 129Sv/ICR mice (Parp − / − )
(Masutani et al., 2000). It has been shown that these mice are very susceptible to
the effects of alkylating agents and ionizing radiation. Parp − / − mice show severe
myelosuppression (Masutani et al., 2000). It is very important to investigate the
survival and spontaneous tumor incidence in Parp − / − mice. However, these data
have not yet been reported.
Ku80 is important for the repair of DNA double-strand breaks by the nonhomologous end-joining protein, Ku70. The Ku80–Ku70 heterodimer (Ku) binds to
DNA ends, nicks, gaps, and hairpins. Ku80-mutant mice (ku80 − / − ), when compared with wild-type littermates, prematurely exhibited age-speciﬁc changes characteristic of senescence that include osteopenia, atrophic skin and hair follicles,
hepatocellular degeneration, hepatic hyperplastic foci, and age-speciﬁc mortality
(Vogel et al., 1999). The cancer and likely sepsis (suggested by reactive immune
responses) were partly responsible for age-speciﬁc mortality in both cohorts. But
diseases occurred earlier in ku80 − / − mice. It is worth noting that the onset of
age-related mortality in ku80 − / − mice begins shortly after sexual maturity, possibly
accounting for their reduced fecundity. It was observed that mouse cells deﬁcient in
Ku80 display a marked increase in chromosomal aberrations, including breakage,
translocations and aneuploidy (Diﬁlippantonio et al., 2000). Cancer incidence was
reduced by 13-fold in ku80 − / − mice in comparison to the control, however cancers
were observed earlier in mutant mice (Vogel et al., 1999). At the same time,
knockout ku70 − / − mice with the same genetic background (129Sv x C57BL/5)
showed a reduction in life span and had a high incidence of CD4+ CD8+ T-cell
lymphomas at a mean age of 6 months (Gu et al., 1997; Li et al., 1998) which
suggests that one or both of these proteins work independently. It was shown that
p53 monitors chromosome damage and either arrests the cell-cycle progression or
triggers apoptosis in cells with unrepaired lesion (Levine, 1997). To determine

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whether p53 is involved in the growth arrest of Ku8 − / − mice, double-mutant mice
Ku80 − / − p53 − / − were generated (Diﬁlippantonio et al., 2000). Although the mice
developed normally all of them died within 12 weeks after birth from disseminated
B-cell lymphoma. In contrast, p53 − / − mice are predisposed to thymic lymphomas
(Jacks et al., 1997) which develop at a slower rate than Ku80 − / − p53 − / − pro-B-cell
lymphomas, and Ku80 − / − mice only occasionally develop T-cell lymphoma after 7
months (Vogel et al., 1999). It was concluded that Ku80 is a caretaker gene that
maintains the integrity of the genome by a mechanism involving the suppression of
chromosomal rearrangement (Diﬁlippantonio et al., 2000).
The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is
a suicide acceptor protein, which removes alkyl groups from the O6 position of
guanine alkylated by potent carcinogen nitroso compounds (Pegg, 2000). The
activity of this enzyme decreases with aging (Anisimov, 1987). Several transgenic
mouse strains that overexpressed MGMT in the brain (150-fold increase) and liver
(25-fold increase) were produced (Walter et al., 1997). In a pilot study it was shown
that overexpression of MGMT in the liver reduced the frequency of spontaneous
hepatocellular carcinomas in these mice compared to the wild-type (Walter et al.,
1997). The authors have claimed that life span studies were initiated to determine
whether an increased MGT activity affected rodent lifespan. They have suggested
that these mice could demonstrate an increased life span and decreased spontaneous
tumor incidence. However, the ﬁnal results have still not been published. Recently
it has been reported that transgenic mice, which overexpress the MGMT gene, have
fewer malignant tumors and survive longer, indicating that MGMT plays a
protective role against malignant transformation (Qin et al., 2000). When overexpressed in the thymus, MGMT protects mice from N-nitrosomethylurea (NMU)-induced thymic lymphomas (Allay et al., 1999), whereas MGMT − / − knockout mice
are more sensitive to the toxic effect of NMU and other alkylating agents (Glassner
et al., 1999).

6. Overexpression of Cu, Zn-SOD or catalase
Several studies have shown that aging cells and organisms accumulate oxidantdamaged nuclear DNA, whereas superoxide dismutase, catalase and some other
enzymes and scavengers of free radicals protect a cell and an organism from
oxidative stress (Finkel and Holbrook, 2000). The human Cu, Zn superoxide
dismutase (hSOD-1) gene, catalyses the dismutation of O2 to H2O2 and O2. It is
located in chromosome 21 in q22.1 and is overexpressed in Down’s syndrome (DS)
patients. These patients show various abnormalities including mental retardation,
congenital heart disease, immunological deﬁcits, premature aging and increased
cancer risk. In order to explore the potential role of SOD-1 overexpression in DS,
two lineages of transgenic mice for the hSOD-1 gene have been generated and
studied, at the ultrastructural level, to evaluate the effect of hSOD-1 overexpression
on the thymic microenvironment (Nabarra et al., 1997). Modiﬁcation of the cellular

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architecture and morphology associated with a lipidic invasion, signs of a premature involution of the thymus, were observed in both lineages. A rupture of the
ﬁlamentous network in the extracellular and probably also in the intracellular
matrix was observed ﬁrst. These results correlate the thymic alterations visualized
in light microscopy, on the thymus of DS patients, and raise the question of the
relationship between SOD-1 overexpression and the different morphological alterations associated with the premature thymic involution observed in SOD-1 transgenic mice. It was suggested that thymic and immunological impairments present
in DS patients may be related to the SOD-1 gene dosage effect. Overexpression
of the human Cu, Zn-SOD gene was not beneﬁcial to transgenic mice and caused
increased lipid peroxidation in the brains of the animals (Cebalos Picot, 1993).
At the same time, in hSOD-1 transgenic mice, age-related accumulation in the
brainstem and the striatum of a marker of oxidative DNA damage, 8-hydroxy-2%deoxyguanosine (8OHdG), and carbonyl oxidation products were signiﬁcantly
attenuated compared to the wild-type control (Cardozo Pelaez et al., 1998).
Twenty-four male mice transgenic with human Cu, Zn-SO, resulting in an overexpression of the cytosolic enzyme, were compared with 11 matched controls
over 19 months. There was no difference in longevity, locomotor activity or
dopamine uptake sites in the brain regions between the two groups (Gallagher et
al., 2000). Five of the 24 transgenic mice died over the age of 19 months
together with ﬁve of the 11 controls (not statistically signiﬁcant). It is very
important to evaluate the rate of spontaneous tumor development in these mice.
Two types of transgenic mice were generated to evaluate the role of hydrogen
peroxide in the formation of nuclear DNA damage. One set of lines overexpresses wild-type human catalase cDNA, which is localized to peroxisomes,
whereas the other set overexpresses a human catalase construct that is targeted to
the nucleus (Schriner et al., 2000). Both types of transgenic animals had signiﬁcant increases of catalase activities compared to littermate controls. Despite enhanced activities of catalase, there were no changes in the levels of 8OHdG, a
marker of oxidative damage to DNA. No data were reported on the survival rate
and cancer incidence in these mice.
A speciﬁc DNA glycosylase, a product of the OGG1 gene, excises 8OHdG
from DNA in eukaryotic cells. Homozygous ogg1 − / − null mice were viable but
accumulated abnormal levels of 8OHdG in their genome (Klungland et al.,
1999). Despite this increase in potentially miscoding DNA lesions, OGG1-deﬁcient mice exhibited only a moderately, but signiﬁcantly, elevated spontaneous
mutation rate in nonproliferating tissues, did not develop malignancies, and
showed no marked pathological changes (Klungland et al., 1999). It is important
to note that the last two conclusions have been made on the background of
results gained during a histopathological examination of two such animals sacriﬁced at 8 and 11 months of age (!) This evidence is totally insufﬁcient for the
evaluation of tumor incidence in mice and the examination was carried out at a
stage which was too early for the development of spontaneous tumors in the
mouse strain 129 (Anisimov, 1987).

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7. Growth hormone (GH) transgenic and knockout mice
Homozygous growth hormone-receptor (GHR/BP) mice, knockout mice, were
generated through gene targeting (Zhou et al., 1997). GHR − / − mice showed severe
postnatal growth retardation, proportionate dwarﬁsm, decreased lengths of bones
and bone mineral content, absence of the GHR and GH binding protein, signiﬁcantly decreased serum insulin-like growth factor I, IGFBP-3 and elevated serum
GH concentrations (Zhou et al., 1997; Sjorgen et al., 2000), and lived signiﬁcantly
longer than heterozygous (+/ −) and wild-type mice (Kopchik and Laron, 1999;
Coschigano et al., 2000).
On the other hand, in a number of experiments with transgenic animals expressing genes determining hyperproduction of human or animal GH, it was shown that
these mice exhibit signs of premature aging and live only half as long as wild-type
siblings (Pendergrass et al., 1993; Steger et al., 1993). Mice overexpressing GH
exhibit increased indices of free radical production (Rollo et al., 1996), signiﬁcant
reduction of catalase activity in the liver and kidney (Brown-Borg and Rakoczy,
2000), signs of premature central nervous system aging including reduced catecholamine turnover, increased astrogliosis, and impaired learning and memory
(Steger et al., 1993; Miller et al., 1995; Meliska et al., 1997). These animals reach
sexual maturation earlier and cessation of reproduction sooner than wild-type
controls (Naar et al., 1991; Steger et al., 1994). This effect in GH-transgenic mice
is related to accelerated degenerative processes in the ovaries, not present in the
wild-type control (Mayerhofer et al., 1990). Most importantly, GH overexpressed
mice have a high incidence of tumor development (Wolf et al., 1993; Bartke, 1998;
Snibson et al., 1999). It is worth noting that old (16–24 months of age) mice
transgenic with human growth hormone-releasing hormone developed pituitary
adenomas, and produced both GH and prolactin (Asa et al., 1990).
Age-related disturbances in the regulation of the insulin-like growth factor II
activity plays an important role in the development of some metabolic disorders
and diseases, including cancer in the elderly (Anisimov, 1987; Dilman, 1994). By
using a construct in which the coding region of the mouse insulin-like growth factor
II gene (Igf-2) was placed under the control of a keratin gene promoter, four
transgenic lines were established, all of which displayed overgrowth of the skin as
judged by wrinkling (Ward et al., 1994). Transgene expression was high in the skin,
in the gastrointestinal tract and uterus. Adult total body weight was slightly
increased and there was no macroscopic evidence of tumor formation. However,
the increase in cell proliferation was observed in the sites of IGF-II expression.

8. Mutant and transgenic models of immunosenescence
During aging in mice and humans, a gradual decline in thymus integrity and
function occurs (thymic involution) (Remarque, 1998; Anisimov and Soloviev,
1999; Miller, 1999). To determine whether T-cell reactivity or development affects
thymic involution, the thymic phenotype in old (12 months) and young (2 months)

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mice transgenic with rearranged a/b or b-2B4 T-cell receptor (TCR) genes, mice
made deﬁcient for CD4 by gene targeting (CD4 − / − ), mice made deﬁcient for major
histocompatibility complex (MHC) class I (b2M − / − ) or class II genes (Ab − / − )
have been compared (Lau and Spain, 2000). The expected aging-related reduction
in thymic weights was observed for all strains except those bearing disruption of
both class I and class II MHC genes. Therefore, disruption of MHC class I and
class II appeared to reverse or delay aging-related thymic atrophy at the age of 12
months. Immunohistochemical analysis of aging-associated alterations in thymic
morphology revealed that TCR a/b transgenes, D4 disruption, and MHC class II
disruption, all reduced or eliminated these changes. All strains examined at 12
months showed alterations in the distribution of immature thymocyte populations
relative to young controls. These observations show that aging-associated thymic
alterations can be separated and are therefore causally unrelated.
Mutant immunosuppressed NMRI mice (nu/nu), even when kept under germ-reduced conditions and fed with a germ-reduced diet, have an extremely short life
span — the last mouse died at the age of 5 months (Freisleben et al., 1994, 1997).
Systematic observation of 1141 nude mice (Swiss background strain) that received
human tumor grafts revealed 24 spontaneous tumors, 18 of lymphoreticular origin
and six lung adenomas (Sharkey and Fogh, 1979). Spontaneous tumors were seen
at an average age of 9.1 months, and 22 of the tumors were seen only in that
fraction of the group (324 mice) surviving for 5 months or more (6.8%). Nevertheless, the incidence of spontaneous tumors in these nude mice was similar to the
thymus-bearing background strain (Sharkey and Fogh, 1979). The incidence and
type of spontaneous tumors in athymic nude (nu/nu) mice partially inbred (CBA/H)
background and which were also carrying the viable yellow gene (A6y, derived from
C57BL/6JAvy mice), were comparable to those observed in the phenotypically
normal nu/ + and +/ + control crosses carrying the A6y gene (Stutman, 1979).
The A6y gene increases the incidence of spontaneous tumors in most mouse strains.
The effect of the nude gene heterozygocity on spontaneous AKR thymic
lymphomagenesis was studied by comparing female littermates of AKR/Ms nu − / +
and + / + (Shisa et al., 1986). Overall incidences of thymic lymphomas were
comparable in the two genotypes, but the mean latent period for lymphoma
development was signiﬁcantly shorter in (nu/ +) mice (266911.6 days) than in the
(+ / +) mice (31997.9 days).
T-cell dysfunction and thymic involution are major immunologic abnormalities
associated with aging (Remarque, 1998; Miller, 1999). Fas (CD95) is a bifunctional
molecule that is critical for apoptosis and stimulation during T-cell development.
Using fas-transgenic mice, it was shown that T-cell senescence is associated with
defective apoptosis, and that the CD2-fas transgene allows for maintenance of the
Fas apoptosis function and T-cell function in aged mice (Zhou et al., 1995). In
transgenic mice overexpressing the bcl-2 gene in thymocytes, a resistance of
immature thymocytes to apoptosis mediated by corticosteroids and calcium
ionophores was observed (Siegel et al., 1992). It was also shown that overexpression
of bcl-2 enabled a proportion of thymocytes and peripheral T cells to escape the
process of clonal deletion, which normally eliminates self-reactive T cells during

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thymocyte maturation. These ﬁndings implicate the Bcl-2 protein in regulating the
life span of maturing thymocytes and in the antigen-selection process. The evaluation of a risk of spontaneous tumor development in Bcl-2 transgenic mice is of
critical interest.
Transgenic mice that contained constructs of the L-myc gene under the transcriptional control of the immunoglobulin heavy chain enhancer (E mu) developed
thymic hyperplasia and were predisposed to T-cell lymphomas and to highly
malignant mesenchymal neoplasms that closely resemble human ﬁbrous histiocytoma (Moroy et al., 1992).

9. Transgenic and knockout models of age-related neurodegenerative diseases
Increased interest is emerging in the use of mouse models to assess the genetics
of brain aging and age-related neurodegenerative disease (Ingram and Jucker,
1999). A mutant amyloid precursor protein (APP/RK), designed to interfere with
processing by a -secretase, caused a severe phenotype in transgenic mice including
behavioural abnormalities, e.g. neophobia, aggression, hypersensitivity to kainic
acid, and premature death (Moechars et al., 1996). The major and consistent
ﬁnding in these mice that died prematurely was extensive neurodegeneration and
apoptosis, mainly in the hippocampus and cortex, accompanied by astrocytosis
throughout the brain (Moechars et al., 1999).
The formation of ﬁbrillar deposits of amyloid-b protein in the brain is a
pathological hallmark of Alzheimer’s disease (AD). It was shown, however, that
mice transgenic with the amyloid-b precursor protein that developed amyloid
deposits in the brain do not show the degree of neuronal loss or tau phosphorylation found in AD (Geula et al., 1998). Shoji et al. (2000) observed an age-related
amyloid beta protein accumulation in transgenic mice, which expressed a gene
encoding 18 residues of signal peptide and 99 residues of the carboxyl-terminal
fragment of the amyloid-b precursor, under the control of the cytomegalovirus
enhancer/chicken beta-actin promoter. The authors concluded that overproduction
of amyloid b protein causes accumulation of the amyloid b ﬁbrils, with accompanying cellular degeneration and macrophage activation in vivo.
In another study it was reported that transgenic FVB/N mice overexpressing
human or mouse Alzheimer amyloid precursor protein (APP695) died early and
developed a CNS disorder that included neophobia and impaired spatial alteration,
with diminished glucose utilization and astrogliosis mainly in the cerebrum (Hsiao
et al., 1995; Chapman et al., 1999). Age at the onset of neophobia and age at death
decreased with increasing levels of brain APP. No extracellular amyloid was
detected, indicating that some deleterious processes related to APP overexpression
were dissociated from the formation of amyloid. It is worth noting that a similar
clinical syndrome occurs spontaneously in 20% of wild-type mice when they reach
mid- to late-adult age, suggesting that APP overexpression may accelerate naturally
occurring age-related CNS disorders in FVBN mice (Hsiao et al., 1995). Aged
Tg2576 transgenic mice overexpressing human bAPP695 have limited neuron loss

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and tau pathology, but frequent ubiquitin- and a-synuclein-positive, tau-negative
neurites, resembling those seen in the Lewy body variant of Alzheimer’s disease
(Yang, F. et al., 2000).
There is another model of Alzheimer disease in transgenic mice harbouring the
human gene S-100b. This gene is a neurotrophic factor realised by astroglial cells
and localised to chromosome 21 within the region, which is considered obligate for
Down’s syndrome. S-100b is increased in the post mortem brains of both Down’s
syndrome and Alzheimer’s disease. By 1 year of age, the transgenic animals have
signiﬁcant loss of dendrites compared to controls and the number of cells showing
cell body staining was further increased. Behaviourally, younger transgenic animals
could not perform in learning tasks as well as controls (Whytaker Azmitia et al.,
1997). The authors suggest that the increased S-100b in the brain may lead to
accelerated development, followed by increased aging.
It was shown that some cases of amyotrophic lateral sclerosis (a fatal disease in
which spinal cord motor neurons degenerate resulting in progressive paralysis) are
caused by mutations in the antioxidant enzyme Cu, Zn-SOD. Transgenic mice
expressing amyotrophic lateral sclerosis-linked Cu, Zn-SOD mutation (SODMutM)
exhibit a phenotype similar to that of human patients. The onset of the disease
occurred in mice placed at 6 weeks of age (Pedersen and Mattson, 1999). Dietary
restriction failed to delay the onset of the disease or shorten its duration.
It should be noted that data on the development of spontaneous tumors are
practically absent in all the reviews included in this section of papers on genetically
modiﬁed animal models of neurodegenerative diseases. However, this aspect is
important and should also be studied in depth.
a-MUPA is a line of transgenic mice that, compared with their wild-type
counterparts, spontaneously eat less (approximately 20%) and live longer (also
approximately 20%), thus resembling dietary-restricted mice (Miskin et al., 1999).
a-MUPA produced mRNA in the brain which encoded the extracellular protease
urokinase plasminogen activator. These transgenic mice have signiﬁcantly reduced
food consumption, body weight and size, body temperature and decreased plasma
corticosterone at old age compared to the wild strain (Miskin and Masos, 1997).
a-MUPA mice also showed a high frequency of leg muscle tremor seen only in
unstable body states (Miskin et al., 1999). It is unfortunate that the authors did not
study the occurrence of spontaneous tumor incidence, since caloric restriction
inhibits spontaneous tumor development in a variety of mouse and rat strains
(Weindruch and Walford, 1988; Anisimov, 2001).
In order to explore the role of bcl-2 protooncogene, which protects various cell
types from apoptotic cell death and which is expressed in the developing and adult
nervous system, transgenic mice expressing Bcl-2 under the control of the neuronspeciﬁc enolase promoter have been generated (Farlie et al., 1995). It was shown
that these mice had an increased number of neurons; they learned faster, were more
accurate in the Hebb– Williams maze and committed fewer errors (Coleman et al.,
1999). It was also shown that the Bcl-2 transgenic mice were faster than the
wild-type mice, in particular the older mice.

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A murine model of ataxia telangiectasia was created by disrupting the Atm locus
via gene targeting (Barlow et al., 1996; Elson et al., 1996; Eilam et al., 1998).
Homozygous Atm − / − mice displayed growth retardation, neurologic dysfunction,
male and female infertility secondary to the absence of mature gametes and defects
in T-lymphocyte maturation. The majority of animals developed malignant thymic
lymphomas between 2 and 4 months of age.

10. p53 knockout mice
The cancer suppressor p53 is a phosphoprotein barely detectable in the nucleus
of normal cells. Upon cellular stress, particularly that induced by DNA damage,
p53 can arrest cell cycle progression thus allowing DNA to be repaired or it can
lead to apoptosis. These functions are achieved, in part, by the transactivational
properties of p53, which activate a series of genes involved in cell cycle regulation.
In cancer cells bearing a mutant p53, this protein is no longer able to control cell
proliferation, resulting in inefﬁcient DNA repair and the emergence of genetically
unstable cells. Downstream to p53, p21 is responsible for growth arrest in G1, but
other p53 target genes are responsible for the G2 cell-cycle arrest. The transcriptional activity of p53 is progressively activated with the accumulation of cell
doubling in vitro (Bond et al., 1996). Since senescence is characterized by a
permanent cell-cycle block, signiﬁcant emphasis has been placed on the p53 targets
that mediate cell-cycle arrest (Bringold and Serrano, 2000). At the same time it is
worth noting that in some cancers (e.g. cervical carcinoma) the senescence signaling
pathway may be p53-independent (Goodwin et al., 2000).
In response to genotoxic insult, p53-induced apoptosis results from overlapping
downstream pathways that both suppress mutagenic and survival signaling and
promote pro-apoptotic signaling. The frequency of observed mutations in p53
predict that its inactivation is a requisite step in tumorigenesis (Colman et al.,
2000). However, no signiﬁcant differences were found in the mutation spectra and
the mutation incidence in the liver, spleen and brain between p53 − / − and p53 + / +
mice with a lambda shuttle vector harboring the LacI gene (Nishino et al., 1995;
Buettner et al., 1997). These ﬁndings suggest a need to reconsider the role of the
p53 gene as ‘guardian of the genome’.
Transgenic mice with both alleles of the p53 tumor suppressive gene product
‘knocked out’ by gene targeting are susceptible to the early development of tumors,
mainly lymphomas, malignant teratomas and hemangiosarcomas and are characterized by a reduced life span (Donehower et al., 1992, 1995; Hursting et al., 1995;
Perkins et al., 1997; Atardi, and Jacks, 1999; Artandi et al., 2000). Finch et al.
(1998) observed a reduction in the survival of the heterozygous p53 + / − knockout
mice compared to the p53 + / + wild-type mice.
A great deal of evidence shows that there is an age-related gradual decrease in
thymus integrity and function (thymic involution) in humans and in animals
(Remarque, 1998; Miller, 1999). The development and aging of the immune system
was accelerated in p53-deﬁcient (p53 − / − ) mice; the accumulation of memory T

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cells was spontaneously accelerated, and a strong T-cell-dependent Ab response and
Th2 cytokine expression (IL-4, IL-6, and IL-10) was induced by Ag stimulation in
young p53 − / − mice at developmental stage (Ohkusu Tsukada et al., 1999). The
authors showed that the high T-cell proliferative response in the young mice rapidly
progressed to a depressed proliferative response in adult mice. It was suggested that
the loss of regulation of the cell cycle, DNA repair, and apoptosis by p53
deﬁciency, potentially leads to immunosenescence with the accumulation of memory T cells (Ohkusu Tsukada et al., 1999). However, there are no other data on
premature aging phenotype features in p53 − / − mice. This aspect is under consideration in our current research on biomarkers of aging in p53 − / − mice.
In order to examine whether cooperation exists between inherited p53 and Rb
deﬁciency in carcinogenesis, crosses were made between p53- and Rb-deﬁcient mice
and these animals were monitored for subsequent tumor incidence (Harvey et al.,
1995). It was shown that Rb + / − or p53 − / − developed pituitary adenomas or
lymphomas and sarcomas, respectively, whereas mice deﬁcient in both Rb and p53
showed a faster rate of tumorigenesis and a wider array of tumors than animals
deﬁcient only in Rb or p53. It is worth noting that heterozygous p53 knockout
(p53 + / − ) mice do not respond to many carcinogenic chemicals that show strain- or
species-speciﬁc responses in conventional bioassays (Dass et al., 1999; Spadling et
al., 2000; Sukata et al., 2000).
It was shown that mice functionally deﬁcient in all isoforms of p73 which has
high homology with the tumor suppressor p53, as well as with p63, a gene
implicated in the maintenance of epithelial stem cells, exhibit profound defects
including hypocampal dysgenesia, hydrocephalus, chronic infections and inﬂammation, as well as abnormalities in pheromone sensory pathways and a greatly reduced
life span (Yang, A. et al., 2000). In contrast to p53-deﬁcient mice, however, p73 − / −
mice showed no increased incidence of spontaneous tumors. Thus, after an autopsy
of over 100 p73 − / − mice ranging in ages from 2 to 15 months, the authors failed
to observe an increased tumor incidence. However, due to the reduction in survival,
it is impossible to conclude that the maximal age of the autopsied mice was
sufﬁcient for tumor development. The mean life span of the background mouse
strain 129 is approximately 22–24 months and total incidence of spontaneous
tumor reaches up to 21% (Storer, 1966).

11. Regulation of cell-to-cell communication and knockout mouse models
In multicellular organisms, the role of gap junction intercellular communication
in the regulation of cell proliferation, cell differentiation and apoptosis is becoming
increasingly recognized as one of the major cellular functions through normal
development to aging (Trosko et al., 2000). The lost of cell-to-cell communication
is one of the important characteristics of the malignancy (Yamasaki et al., 1999;
Trosko et al., 2000). Connexins are subunits of gap junction channels, which
mediate the direct transfer of ions, second messenger molecules and other metabolites between contacting cells. In vitro studies with endothelial cells have shown that

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connexins play a role in the aging process (Xie and Hu, 1994). Deletion of different
connexin genes from the mice results in various disorders, including cancer, heart
malformation or conduction abnormality, cataract, etc. (Yamasaki et al., 1999). It
was shown that Cx − / − mice develop a progressive demyelinating peripheral
neuropathy beginning at 3 months of age with a prevalence of motor ﬁbers (Scherer
et al., 1998).
Modjanova et al. (1983) observed an age-related decrease in intercellular coherence strength in the lungs of the mice strain A, predisposed to spontaneous
adenoma development, and in the livers of the CBA, C3H and C3HA mice
predisposed to the development of hepatomas. Male and female 1-year-old mice
deﬁcient for connexin-32 (Cx32) had 25-fold and eightfold more spontaneous liver
tumors than wild-type mice, respectively (Temme et al., 1997). Transfection of
connexin genes into tumor cells restores normal cell growth, supporting the idea
that connexins form a family of tumor-suppressor genes (Yamasaki et al., 1999).

12. Knockout p66shc gene mice
An adaptor protein p66shc becomes tyrosine phosphorylated upon activation of
growth factor receptors and forms stable complexes with Grb2, another adaptor
protein for the ras exchange factor SOS. However, it does not affect mitogen-activated protein kinase (MAPK) and does not inhibit c-fos promoter activation. p66shc
is a splice variant of p52shc/p46shc, a cytoplasmatic signal transducer involved in the
transmission of mitogenic signals from activated receptors to Ras. The Sch protein
complexity increased during evolution from one locus in Drosophila, to at least
three loci in mammalian (Luzi et al., 2000). Genetic and biological evidence
indicates that the mammalian Sch isoforms regulate functions as diverse as growth
(p52/p46Sch), apoptosis (p66Sch) and life-span (p66Sch) (Luzi et al., 2000). Targeted
mutation of the mouse p66shc gene induces stress resistance to paraquat, which
generates superoxide anions upon cellular intake, and increases life span by 30%
(Migliaccio et al., 1999). The mean survival of homozygous p66shc − / − mice was
973 9 37.3 days, whereas for wild-type mice it was 7619 19.0 days, and for
heterozygous p66shc 9 mice 815937.5 days. After 28 months, when all 14 wild-type
animals had died, three of the eight heterozygous (37%) and 11 of the 15
homozygous mice (73%) were still alive. No statistically signiﬁcant differences were
found in body weight and food consumption between the knockout and wild-type
mice. There were no obvious abnormalities in the p66shc − / − mice. However,
spontaneous tumorigenesis in these mice has not been adequately investigated.
A hypothesis was put forward in which p66shc is assumed to be involved in
phenoptosis, i.e. programmed death of an organism, mediated by the reactive
oxygen species-dependent massive apoptosis in an organ of vital importance
(Skulachev, 2000). The reactive oxygen species are suggested to oxidize phosphatidyl serine in the inner leaﬂet of the cell plasma membrane, resulting in the
appearance of this phospholipid in the outer membrane leaﬂet, an effect recognized
by a special receptor and causing the p66shc phosphorylation as a serine residue.

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Serine-phosphorylated p66shc is proposed to block mitosis and initiate apoptosis.
The large-scale apoptosis leads to phenoptosis and, hence, shortens the life span of
the organism. The study of spontaneous tumor incidence, localization and type, as
well as susceptibility of the p66shc − / − mice to carcinogens, would be very
intriguing.

13. Telomerase transfected and knockout mice
Telomeres are repetitive DNA sequences at the end of linear chromosomes. Each
time a cell divides, telomeres shorten leading to an irreversible growth arrest state
called replicative senescence. Telomere maintenance is thought to play a role in
signaling cellular senescence. In most instances cells become senescent before they
can become cancer cells. However, almost all cancer cells are immortal having
overcome cellular senescence. Maintenance of telomere stability is required for cells
to escape from replicative senescence and proliferate indeﬁnitely. Telomerase, a
cellular reverse transcriptase, is upregulated and reactivated in most human cancers
and helps to stabilize telomere length by adding TTAGGG repeats onto the
telomeres (Cerni, 2000; Goyns and Lavery, 2000; Ishikawa, 2000). However, the
link between telomerase activity, telomere length and the aging processes in an
organism has not been established.
Expression of the catalytic component of human telomerase, human telomerase
reverse transcriptase (hTERT), extends the life span of human ﬁbroblasts, retinal
pigment epithelial cells, large vessel and microvascular endothelial cells and keratinocytes beyond senescence without causing neoplastic transformation (Bodnar et
al., 1998; Jiang et al., 1999; Morales et al., 1999; Yang et al., 1999; Dickson et al.,
2000).
However, it is worth noting that ectopic expression of hTERT is not sufﬁcient to
immortalize normal human keratinocytes and mammary epithelial cells (Kiyono et
al., 1998). Ectopic hTERT expression immortalized normal mesothelial cells and a
premalignant, p16 (INK4a)-negative keratinocyte line (Dickson et al., 2000). Thus,
telomere length stabilization alone is unable to permit keratinocytes to bypass
senescence, but the subsequent slow, indeﬁnite continued growth permitted by
telomerase expression permits immortalized variants to arise. Human mammary
epithelial cells (HMEC), cultures which normally stop dividing at 55–60 population
doubling, being infected with a hTERT retrovirus at the 40th passage, were
maintained until population doubling 250 (Wang et al., 1998). The increase in the
expression of c-myc in HMEC-hTERT has been observed at 107th to 135th
population doublings (Wang et al., 2000). The authors concluded that, although
telomerase activation extends the life span of HMECs, it is also associated with the
overexpression of c-myc and therefore is not ultimately genoprotective. The extension of life span that is conferred by TERT causes c-myc activation and this
immortalizes cells, in part by activating TERT expression. These ﬁndings indicate
that the use of hTERT for expansion of normal human cells for therapeutic
purposes must be approached with caution (Wang et al., 2000). Although it was

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demonstrated that hTERT-immortalized cells can retain normal growth and differentiation control mechanisms, it is possible that the loss of the p16-mediated
growth arrest mechanism (loss of the pRB/p16INK4a) and unlimited replicative
potential predisposes such cells to further changes that may result in malignant
transformation. It has been shown that expression of hTERT cooperates with the
simian virus 40 large T oncoprotein and oncogenic ras to transform human
ﬁbroblasts and kidney epithelial cells to tumorigenicity (Hahn et al., 1999). These
observations clearly provide evidence supporting recent proposals (Reddel, 1998;
Wynford-Thomas, 1999) that multiple ‘clocks’ function to limit the proliferation
capacity of human cells.
The telomerase knockout mice provide an opportunity to understand the effects
associated with critical telomere shortening at the level of the organism (Kipling
and Faragher, 1999). C57BL6 mTR − / − mutants have been generated, which
showed shorter telomeres than the original mixed genetic background C57BL6/
129Sv mice (Herrera et al., 1999). These mice could be bred for only four
generations and the survival of the late generation mTR − / − mice decreased
dramatically with age compared to their wild-type counterparts. Fifty percent of the
4th generation of these mice died at only 5 months of age. This decreased viability
with age in the late generation mice was coincident with telomere shortening,
sterility, atrophy of the spleen, reduced proliferative capacity of B and T cells,
abnormal hematology and atrophy of the small intestine. The loss of telomere
function in mTR − / − mice did not elicit a full spectrum of classical pathophysiological symptoms of aging (Lee et al., 1998; Rudolph et al., 1999). However, age-dependent telomere shortening and accompanying genetic instability were associated
with a shortened life span, as well as a reduced capacity to respond to stresses such
as wound healing and hematopoietic ablation. It is interesting that an increased
incidence of spontaneous malignancies (mainly lymphomas, teratocarcinomas) have
been observed in mTR − / − mice (Rudolph et al., 1999). The appearance of these
tumors is thought to be a consequence of chromosomal instability in these mice
(Blasco et al., 1997; Chin et al., 1999; Greenberg et al., 1999). Recently it was
shown that late-generation Terc − / − mice, which have short telomere and are
telomerase-deﬁcient, are resistant to the two-stage skin tumorigenesis (GonzalezSuarez et al., 2000). Early generation telomerase-deﬁcient INK4A − / − mice retain
long telomeres and remain highly cancer prone, whereas the late generation of these
mice have short dysfunctional telomeres and are more cancer-resistant (Greenberg
et al., 1999). Moreover, these in vivo observations parallel those obtained from in
vitro-based transformation assays (Greenberg et al., 1999). Experiments in the
telomerase-deﬁcient mice have shown that in the setting of a compromised p53
pathway, telomere-based crisis can facilitate carcinogenesis by promoting chromosomal instability (Artandi and DePinho, 2000). Moreover, the unbalanced chromosomal rearrangements caused by telomere compromise, closely resemble the
regional losses of chromosomes in human carcinoma (Artandi and DePinho, 2000;
DePinho, 2000).
A recent study using telomerase-deﬁcient mice has shown that differences in
telomere length and regulation might impact dramatically on both the spectrum

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and cytogenetics of tumors during aging (Artandi et al., 2000). It is important that
the aging telomerase-knockout mice, heterozygous for mutant p53, exhibited a
pronounced shift in their tumor spectrum to epithelial neoplasia, including mammary, colon and skin carcinomas (Artandi et al., 2000). DePinho (2000) suggests
that these data are sufﬁcient for understanding the role of telomere-induced genome
instability in mechanisms of development of epithelial cancers in humans.

14. ErbB-2/neu transgenic mice
The human c-erbB-2/neu protooncogene is a member of the epidermal growth
factor receptor (EGFR), family to receptor tyrosine kinases (Andrechek et al.,
2000). The erbB-2/neu harboring transgenic mice revealed a high incidence of
mammary carcinomas and died within 4 months after birth (Stocklin et al., 1993).
In our laboratory it was shown that the mean life span of virgin female erbB-2/neu
transgenic mice (FVB background) was 3119 56 days and maximum life span was
431 days. Eighty percent of the mice developed mammary adenocarcinomas.
Premature age-related alterations in the estrus function were also observed in these
mice (Alimova, 2000). The survival of wild-type FVB/N female mice at 24 months
of age was 62% and spontaneous tumor incidence was 66% (Mahler et al., 1996). In
these mice, lung adenomas, pituitary adenomas, ovarian tumors, lymphomas,
histiocytic sarcomas, Harderian gland adenomas and pheochromocytomas were
observed, however no mammary adenocarcinomas have been observed. It is worth
noting that reduced p66Shc expression may play a role in erbB-2-positive breast
cancer development (Stevenson and Fracjelton, 1998).

15. Conclusion
Because DNA damage accumulates with aging (Bernstein and Bernstein, 1991)
and plays a signiﬁcant role in carcinogenesis (Anisimov, 1987), it would be
reasonable to suggest that the risk of spontaneous tumor development should
increase in long-living murine strains in comparison to short-living strains. However, no signiﬁcant positive correlation between life span and tumor incidence was
found in the different strains of the inbred mice (Storer, 1966; Smith et al., 1973;
Anisimov, 1976, 1987). Table 1 shows that the incidence of spontaneous tumors is
determined by genetic background and sex rather than by the duration of life span.
It is well known that in some long-living and short-living mouse strains, the
incidence of spontaneous tumors is low, whereas in other mouse strains, characterised by different life spans, spontaneous tumor incidence is high (from 80 up to
100% of cases) (Storer, 1966; Staats, 1980; Anisimov, 1987). It is worth noting that
there is a close positive correlation between DNA repairs of adducts of the
carcinogen benzo[a]pyrene in different organs and the life span of C57BL/6
(long-living) and BALB/c (shorter living) strains (Boerrigter et al., 1995).

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Pour et al. (1979) stressed that genetic factors were much more responsible for
the variation in hamster spontaneous tumor incidence, localization, and histological
type, than the length of life span of these animals in various populations. Similar
results were obtained in the analysis of spontaneous tumor incidence in rats of
different strains or stocks (Anisimov, 1976). Generally speaking, the available data
show no positive correlation between spontaneous incidence and the life span of
some strains or stocks of a species (Anisimov, 1987, 1998a). However, in the latter
case we are dealing with genetically different animals. Some of them are infected
with oncornaviruses (e.g. murine mammary tumor virus, MuMTV, in C3H mice).
Much more important is the correlation between the life span and spontaneous
tumor incidence in different groups (populations) of animals of one strain or stock.
Our data have shown that more ‘rectangular’ patterns of survival curves are directly
associated with an increased rate in the development of fatal tumors in rats of the
same strain. In contrast, increased frailty in the animals at a younger age was
followed by decreased mortality in older age, and correspondingly, by a decreased
rate of fatal tumor development (Anisimov, 1987, 1998a).
Taking into account the exponential character of the relationship between the
incidence of the majority of tumors and age, we calculated the correlation coefﬁcient between the parameters of the aging process and tumor development in 10
Table 1
Life span and spontaneous tumor incidence in 20 mouse strains (Storer, 1966; Anisimov, 1987)
Strain

Males

Females

Mean life span
(weeks)
C57BL/10
129
C3H
CBA
C3H.K.
DBA/2J
C57BR/odJ
129/J
C57BL/6J
RF/J
DBA/2
BALB/c
CBA/J
A/J
DBA/1J
ST/bJ
BDP/J
PL/J
C58/J
AKR/J

Tumor
incidence (%)

Mean life span
(weeks)

Tumor incidence
(%)

118
117
113
107
104
101
100
97
96
93
82
77
75
70
62
62
60
55
53
46

33
7
28
19
44
18
25
12
7
49
15
0
72
13
2
2
5
0
73
81

99
104
70
91
–
102
99
93
99
65
101
82
75
84
107
73
67
64
50
39

31
21
67
55
–
37
34
20
14
43
49
29
87
30
22
8
24
71
88
92

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groups of intact rats, kept under identical conditions and which were used as
control animals in our experiments (Anisimov, 1987). For each group, a life table
was created, and the level of mortality (R), mean life span, and parameter h
according to the Gompertz equation (R= R0e ht, where R0 = mortality at time
(t)= 0; h = constant) were calculated. The cumulative incidence for each group of
animals was found by day 1000 of their life according to the actuarial method and
by the equation: Q= Q0e st, where Q= cumulative tumor incidence, t= time,
Q0 =Q at t =0, and s =constant, the value of this constant (s) was found. We
observed a positive correlation between the population aging rate (evaluated as h)
and tumor incidence Q (r =0.70, PB 0.05) and the constant s (r= 0.77, PB 0.05).
It seems that the constant s might be considered as an index of the age-related
increase in tumor incidence in the population (tumor increase rate). Therefore, our
data suggest a positive correlation between tumor incidence and tumor rate and the
aging rate of a population (Anisimov, 1987). A similar conclusion has been made
on the basis of the analysis of the effect of different geroprotectors on mortality
curves in mice and rats (Anisimov, 1987, 1998a, 2001).
The analysis of the available data on transgenic and mutant mice has shown that
only a few models represent examples of life span extension. Ames dwarf mutant
mice, p66 − / − knocked out mice, a-MUPA and MGMT transgenic mice live longer
than wild-type strains. As usual, the incidence of spontaneous tumors in these mice
was similar to those in controls, whereas the latent period of tumor development
was increased. Practically all models of accelerated aging show increased tumor
incidence and a shortening of tumor latency (Tables 2–4). It is worth noting that
this phenomenon has been observed both in mice which display phenotype resembling the more natural aging process, and in mice which show only some features
of the normal aging process. Why is this the case? It is a fact that the aging
processes predisposes cells to accumulate mutations, some of which are necessary
for initiation of tumorigenesis in target tissues (Lengauer et al., 1998; Turker, 1998).
Recent ﬁndings suggest that certain types of DNA damage and inappropriate
mitogenic signals can also cause cells to acquire a senescent phenotype (Campisi,
1997, 2000). Thus, the cells respond to a number of potentially oncogenic stimuli by
adopting a senescent phenotype. These ﬁndings suggest that the senescence response
is a fail-safe mechanism that protects cells against malignant transformation.
Despite the protection from cancer conveyed by cellular senescence and other
mechanisms that suppress tumorigenesis, the development of cancer is almost
inevitable as mammalian organisms age. It is certain that aging predisposes cells to
the accumulation of mutations (Holliday, 2000; Vijg, 2000), several of which are
necessary before malignant transformation occurs.
It was shown that there was an increase in tumor incidence, as well as an
age-related accumulation of chromosome aberrations in the liver of the short-living
mouse strain A, compared to long-living C57L/6 mice (Crowley and Curtis, 1963).
Short-living BDF1, SAMP6/Tan and A/J mice showed a signiﬁcant age-related
increase in spontaneous frequencies of micronucleated reticulocytes, whereas the
long-living ddY, CD-1, B6C3F1, SAMR1, and MS/Ae did not show signiﬁcant
age-related differences in the mean frequencies of spontaneous micronuclei (Sato et

Strain, mutation

Effect on signs
of aging

Effect on
longevity

Effect on tumor development

Incidence
Ames dwarf mice
SAMP (senescence-accelerated
mouse)
Klotho (kl−/−)
nu/nu (athymic mice)

Postponed aging
Accelerates
Accelerates

+50–64%
Decreases

Less than 100
days
Immunosenescence Decreases

Latency

No effect
No effect

Increases
Decreases

References

No data
No effect

Mattison, 2000
Takeda et al., 1997
Kuro-o et al., 1997

Decreases

Sharkey and Fogh, 1979; Shisa et al., 1986

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Table 2
Effect of mutations on longevity and tumor development in mice

1241

1242

Gene, function

Effect on signs of
aging

Effect on
longevity

Effect on tumor development

Incidence

References

Latency

hGH, bGH (growth hormone)

Accelerates

Decreases

Increases

Decreases

hGH-releasing factor
MGMT (DNA repair)
hSOD-1 (Cu,Zn-superoxide dismutase)
erbB-2 /neu (oncogene, EGF receptor)
L-myc (oncogene, DNA binding)
h-MUPA (urokinase plasminogen
activator)

Accelerates
No data
Accelerates
Accelerates
Accelerates
Postponed aging

Decreases
Increases
Decreases
Decreases
Decreases
Increases

Increases
Decreases
No data
Increases
Increases
No data

Decreases

Decreases
Decreases

Wolf et al., 1993; Bartke,
1998
Asa et al., 1990
Qin et al., 2000
Cebalos Picot, 1993
Alimova, 2000
Moroy et al., 1992
Miskin et al., 1999

V.N. Anisimo6 / Mechanisms of Ageing and De6elopment 122 (2001) 1221–1255

Table 3
Effect of gene overexpression on longevity and tumor development in transgenic mice

Gene, function

Effect on signs of
aging

Effect on
longevity

Effect on tumor development

Incidence

References

Latency

ghr−/− (growth hormone receptor)

Postponed aging

Increases

No data

XPA−/− (DNA nucleotide excision repair)

Accelerates

Decreases

No effect

Parp−/− (base-excision and DNA strand break
repair)
Ku80−/− (DNA double-strand break repair)
Ku70−/− (DNA double-strand break repair)
p66 shc−/− (adaptor protein)

Accelerates

Decreases

Accelerates
Accelerates
No effect

Decreases
Decreases
+30%

Increased susceptibility to alkylating
agent and ionizing radiation
Decreases
Decreases
Increases
Decreases
No effect

Atm−/− (ataxia telangiectasia)

Accelerates

Decreases

Increases

Decreases

p53−/− (anti-oncogene, apoptosis)

Accelerates

Decreases

Increases

Decreases

Cx32−/ (connexin32, gap junction gene)

Accelerates

Decreases

Increases

Decreases

mTR−/− (telomerase)

Accelerates

Decreases

Increases

Decreases

Increases

Coschigano et al.,
2000
Van Steeg et al.,
1998
Masutani et al.,
2000
Vogel et al., 1999
Gu et al., 1997
Migliaccio et al.,
1999
Barlow et al.,
1996
Donehower et al.,
1992
Temme et al.,
1997
Rudolph et al.,
1999

V.N. Anisimo6 / Mechanisms of Ageing and De6elopment 122 (2001) 1221–1255

Table 4
Effect of gene knockouts on longevity and tumor development in mice

1243

1244

V.N. Anisimo6 / Mechanisms of Ageing and De6elopment 122 (2001) 1221–1255

al., 1995). Long-living mutant Ames dwarf mice and knockout p66shc − / − mice
were less vulnerable to oxidative damage than wild-type controls (Migliaccio et al.,
1999; Brown-Borg and Rakoczy, 2000), whereas the senescence-prone strain,
SAMP, had increased production of ROS (Takeda, 1999), DNA damage and
somatic mutation compared to the senescence-resistant SAMR strain (Hosokawa et
al., 2000). MGMT-overexpressed mice are more resistant to alkylating agents
(Walter et al., 1997; Allay et al., 1999), whereas MGMT − / − and Parp − / − mice,
deﬁcient in DNA repair, are more susceptible to the effects of alkylating chemicals
and ionizing radiation (Glassner et al., 1999; Masutani et al., 2000). No signiﬁcant
differences were found in the mutation spectra and the mutation incidence between
p53 − / − and p53 + / + mice (Nishino et al., 1995; Buettner et al., 1997); however, the
incidence of spontaneous tumors in p53 − / − mice was increased compared to the
wild-type control (Hursting et al., 1995; Jacks et al., 1997; Atardi and Jacks, 1999).
Gap junction-deﬁcient mice (Cx32 − / − ) have an extremely increased susceptibility
to spontaneous and chemically-induced carcinogenesis (Temme et al., 1997). Mice
with a defect in the xeroderma pigmentosum group A (XPA) gene, have a complete
deﬁciency in nucleotide excision repair and have a more than 1000-fold higher risk
of developing UV-induced skin cancer, as well as increased susceptibility of internal
organs to mutagenesis and development of cancer after exposure to chemical
carcinogens (Van Steeg et al., 1998, 2000). However, the incidence of spontaneous
tumors in these mice is relatively low — only 15% and even then these tumors only
develop after the age of 18 months (Van Steeg et al., 1998). It is very important to
note that the rate of accumulation of somatic mutations with age signiﬁcantly
varies in the different tissues in mice (Dolle et al., 1997, 2000; Ono et al., 2000;
´
Stuart et al., 2000; Vijg, 2000).
Numerous benign or relatively well controlled malignant tumors may also
harbour many potentially oncogenic mutations, suggesting that the tissue microenvironment can suppress the expression of many malignant phenotypes (Campisi,
2000; DePinho, 2000). Cellular senescence has been proposed to contribute to the
aging of an organism. Senescent cells have recently been shown to accumulate in
human tissues with aging (Campisi, 2000). It has been proposed that the accumulation of dysfunctional senescent cells disrupts tissue microenvironment (Rinehart
and Torti, 1997; Campisi, 2000). Thus, the accumulation of mutations may
synergize with the accumulation of senescent cells, leading to an increased risk of
developing cancer which is a hallmark of mammalian aging. However, in discussing
the differences in human and mouse telomere biology, it has been suggested that,
unlike human cells, mouse cells do not undergo replicative senescence (Wright and
Shay, 2000).
According to the multistage model of carcinogenesis, the proportion of partially
transformed cells that have progressed through some stages will increase with age
(Vainio et al., 1992; Anisimov, 1998a). The evidence supporting age-related accumulation of ‘premalignant’ cells in several tissues (skin, lymph node, thymus,
spleen, liver, ovary, mammary gland) has been summarized and discussed elsewhere
(Anisimov, 1998b).

V.N. Anisimo6 / Mechanisms of Ageing and De6elopment 122 (2001) 1221–1255

1245

Most cancer susceptibility genes were originally thought to directly control cell
proliferation and death, acting as ‘gatekeepers’. During the last few years it has
become clear that genes which maintain the integrity of the genome (DNA repair
genes) are ‘caretakers’ and may even be more frequent causes of predisposition to
cancer. Gatekeepers are genes that directly regulate (typically, inhibit) tumor
growth. Inactivation of a given gatekeeper gene leads to a very speciﬁc tissue
distribution of cancer. In contrast, inactivation of a caretaker gene leads to genetic
instabilities, which result in increased mutation of all genes, including gatekeepers
(Kinzler and Vogelstein, 1997). However, it seems that this classiﬁcation is an
oversimpliﬁcation of the real situation. For example, defects of the DNA repair
gene MSH2 result in a limited subset of colon cancer and do not affect other types
of cancer in humans. At the same time, defects in p53- and Rb-pathways are
present in 80– 90% of tumors. Genes involved in metabolism, tissue growth
pathways and immune signaling genes, e.g. GH, IGF-1, APO E, TCR, etc. also play
an important role in tumor promotion and progression. These genes act as
‘homeostatic’ genes. Available data have shown that all types of genes are also
involved in the control of aging (Fig. 1). It is clear that both aging and carcinogenesis are a complex multifactor process that can have many causes. In this sense, new
transgenic and knockout mouse models with prolonged or reduced longevity will be
an important instrument to evaluate the role of genes involved in aging in the
mechanisms of carcinogenesis.
It has to be stressed that the number of animals used in the studies on the effect
of either overexpression or suppression of individual genes on longevity, is usually
too small to draw an ultimate conclusion concerning the relationship between the
aging rate and tumorigenesis in genetically modiﬁed and wild-type animals. Typically, only a few biomarkers of aging have been evaluated in the reviewed studies.
Sometimes it is also difﬁcult to distinguish a true acceleration of normal aging from
a progeroid syndrome. In a number of studies, the information on the cause of
animal death, as well as on tumor incidence, is omitted. A program for the
adequate testing of biological interventions to promote healthy aging has recently
been discussed (Warner et al., 2000). There are standardized protocols for longterm and short-term assays for the evaluation of cancer risk from chemicals and
other interventions in experimental rodents (Montesano et al., 1986). Gerontological studies have now been included in the protocols of systemic evaluation of newly
generated mouse strains. Researchers will frequently wait up to long periods for
cancer to develop in the life span of mice; however, little attention is paid to the
aging process of these mice. It appears that researchers need to introduce approaches that involve both the study of cancer and the aging.

Acknowledgements
This paper was supported in part by grant c 00-04-48481 from the Russian
Foundation for Basic Research. The authors thank James W. Vaupel for the
opportunity to use the facilities of the Max Planck Institute for Demographic

1246

V.N. Anisimo6 / Mechanisms of Ageing and De6elopment 122 (2001) 1221–1255

Fig. 1. Cellular targets of gene effects on aging and carcinogenesis.

Research, Rostock, Germany, to complete this paper, Professor Anatoli I. Yashin,
(Max Planck Institute for Demographic Research), Dr Alexander Yu. Nikitin
(Cornell University, Itaca, NY) and Dr Sergey Yu. Revskoy (North-Western
University, Chicago, IL) for the criticism and valuable recommendations, and Karl
Brehmer for excellent editorial assistance.
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