Experimental Gerontology, Vol. 33, Nos. 1/2, pp. 81–94, 1998
Copyright © 1998 Elsevier Science Inc.
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PII S0531-5565(97)00086-7

MOLECULAR ASPECTS OF THE RELATIONSHIP BETWEEN
CANCER AND AGING: TUMOR SUPPRESSOR ACTIVITY DURING
CELLULAR SENESCENCE

IGOR GARKAVTSEV, CHRISTOPHER HULL, and KARL RIABOWOL
Departments of Medical Biochemistry and Oncology and Southern Alberta CancerResearch Centre,
The University of Calgary, Calgary, Alberta T2N 4N1, Canada

Abstract—Normal cells cultured in vitro lose their proliferative potential after a finite
number of doublings in a process termed replicative cellular senescence (Hayflick, 1965).
The roles that growth inhibitory tumor suppressors play in the establishment and maintainence of cellular senescence have been reported in many different systems. The Rb and p53
tumor suppressors are examples of growth inhibitors that lose the ability to be regulated and
are constantly activated during senescence. Other proteins that inhibit the initiation of DNA
synthesis in early passage fibroblasts and that link the action of tumor suppressors with the
cell cycle machinery, are also expressed at higher levels in senescent cells. For example, the
increased expression of the cyclin-dependent kinase inhibitor p16 may contribute to arresting
the growth of senescent cells. Identification and characterization of additional genes encoding
growth inhibitors that are upregulated in senescent cells, such as the recently isolated p33ING1
protein, should provide a better understanding of the “aging program” that ceases to operate
in the generation of immortal cancer cells. © 1998 Elsevier Science Inc.
Key Words: cell cycle, senescence, inhibitors of DNA synthesis

INTRODUCTION
IN ALMOST all cases, normal diploid cells have a finite replicative potential. At the end of this
replicative life span, cells cease to divide in a process known as replicative or cellular
senescence (Hayflick, 1965). By observing the number of mean population doublings (MPDs)
a cell strain can undergo before reaching senescence in vitro, an inverse correlation has been
observed between the age of the donor and the maximum number of MPDs that cells can
undergo before reaching senescence (Bierman, 1978; Rohme, 1981; Effros and Walford, 1984).
¨
Similarly, the chronological life span of a species influences the number of MPDs isolated cell
strains can undergo, with strains isolated from shorter lived species senescing after fewer MPDs

Correspondence to: Karl Riabowol. E-mail: kriabowo@acs.ucalgary.ca
(Accepted 5 May 1997)
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than those isolated from longer lived species. These data suggest that senescence is an in vitro
manifestation of an in vivo phenomenon. More recently, morphological and molecular analysis
of tissue in vivo suggests the in vivo relevance of the in vitro characterized process of
senescence (Goldstein, 1990; Dimri et al., 1995; Campisi, 1996).
Several hypotheses have been put forward to explain this process of cellular senescence. The
first suggests that senescence results from an accumulation of errors or damage in a variety of
macromolecules that occur naturally as an organism ages. A second hypothesis proposes that there
is a genetic basis for aging and that a cellular or genetic “clock” records, and perhaps contributes, to
the molecular changes that characterize senescence. Although a variety of observations support the
idea of a genetic clock mechanism (Goldstein, 1990), the identity of such a senescence mechanism(s)
is not yet clear. However, normal human cells undergo a progressive shortening of their
chromosomal telomeres due to loss of terminal repeat elements with each round of chromosomal
replication throughout their proliferative lifespan (Harley et al., 1990; Kim et al., 1994). This
loss of telomeric DNA repeats may therefore provide a genetic clock or counting mechanism
that halts cell proliferation when the loss of telomeric DNA interferes with the expression of
genes at chromosomal termini (Shay et al., 1994). Consistent with this idea, the majority of
immortalized cells do not undergo additional loss of their terminal telomeric repeat elements, in
part because of the activation of the enzyme telomerase (Counter et al., 1992).
Study of the “escape from senescence” that is necessary for the immortalization of cancer cell
lines is useful in developing an understanding of the mechanisms through which cells normally
senesce (see Fig. 1). Cells initially follow a first “mortality phase” (M1) that usually culminates
in entry into a state of senescence after a certain number of MPDs (Wright et al., 1989).
Treatment of primary cells with viral oncogenes that have the ability to inhibit or block the
function of the retinoblastoma and p53 tumor suppressors can bypass M1 and extend the number
of MPDs cells undergo in an “M2” pathway before arresting. This arrest, termed crisis, is
distinct from senescence and results in the death of cells in a process that shows some
characteristics that are typical of apoptosis, the programmed cell death pathway responsible for
actively removing cells during embryonic development. Some cells can escape from both the
M1 and the M2 pathways and become immortal (Bryan and Reddel, 1994), but this occurs with
a variable, species-specific frequency that may be related to the expression of telomerase (Kim
et al., 1994; Bestilny et al., 1996).
Although the mechanisms of the theoretical M1 and M2 pathways are not fully understood,
it is becoming clearer that senescence is a function of at least some of the normal cell cycle
control mechanisms that are abrogated during spontaneous immortalization and malignant
transformation. This review will cover some aspects of cellular senescence and will focus on the
relationship between cancer-related mechanisms of growth regulation (that include tumor
suppressors) and cell cycle regulatory proteins relevant to the processes of senescence and
immortalization.
Senescent cells arrest in the G1 phase of the cell cycle
Flow cytometric analysis of senescent cells has demonstrated that the majority of senescent
cells, even in the presence of growth promoting mitogens (such as insulin, epidermal growth
factor, platelet-derived growth factor, transferrin, and other factors found in fetal bovine serum),
are arrested with a DNA content similar to that in cells that are in the G0/G1 phase of the cell
cycle. Normally, cells reside in a quiescent (G0) state in which they do not proliferate, but upon
addition of mitogens enter the first “gap” phase (G1) where they synthesize the components

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83

FIG. 1. Senescence vs. “escape from senescence” in human diploid fibroblasts. Fibroblasts from
normal embryonic sources are capable of undergoing 40 – 80 mean population doublings (MPDs)
before becoming senescent, a metabolically active but not-proliferative state in which changes occur
in gene expression and activity indicated in the figure. The “Mortality 1” (M1) program can be
abrogated by certain transforming oncoproteins (such as SV40 large T antigen, Adenovirus E1a and
E1b, or Human Papilloma Virus E6 and E7), allowing an extra 10 –30 MPDs before cells enter
“crisis,” a state where the death of cells, possibly because of chromosome instability, exceeds their
ability to replicate. With a low frequency (ϳ10Ϫ7), cells can escape this “M2 program,” which leads
to crisis, acquire telomerase activity and become transformed, corresponding to progression to a
tumorigenic state in vivo. Escape from M2 is often accompanied by the acquisition of telomerase
activity.

necessary for the duplication of their DNA during the subsequent synthesis (S) phase. The
S-phase is followed by a second gap phase (G2) that precedes mitosis (M), during which the
duplicated genetic material is segregated into two compartments that go on to physically divide
into two daughter cells. The G0/G1 DNA content suggests that senescent cells express an
inhibitor(s) of DNA synthesis that blocks entry into S-phase (Schneider and Fowlkes, 1976;

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Sherwood et al., 1988). Consistent with this suggestion, fusion of senescent cells with young,
proliferation-competent cells results in a hybrid cell that does not enter the S-phase under
conditions that are normally growth promoting. Also, microinjection of poly (A)ϩ RNA
isolated from senescent human diploid fibroblasts (HDFs) into young HDF’s inhibits DNA
synthesis, while microinjection of poly(A)ϩ RNA from young HDFs does not (Lumpkin et al.,
1986). These experiments provide evidence that senescent HDFs express gene products that can
dominantly block the initiation of DNA synthesis in early-passage HDFs.
To understand the mechanisms of growth arrest characteristic of senescence, it is useful to
compare the characteristics of cells entering senescence with those seen in cells undergoing
other forms of growth arrest. For example, similar levels of expression are seen in the growth
inhibitory genes such as p21, p16, and p33, and the activity of several tumor suppressors such
as p53 and Rb (Meyyappan et al., 1996) in senescent and in quiescent cells. However, one of
the best markers of quiescence, the growth arrest specific (gas) genes that were defined by their
elevated expression during quiescence, do not show elevated levels of expression during
senescence in mouse fibroblasts (Cowled et al., 1994). Thus, the states of senescence and
quiescence appear to share similar expression levels of many, but not all, mediators of growth
arrest. For the remainder of this review we shall focus on the potential roles of tumor
suppressors in blocking the passage of senescent cells through the cell cycle.
Rb during cellular senescence
The Rb gene was the first tumor suppressor gene identified and isolated. Mutation of Rb was
found to predispose carriers to the inherited eye cancer retinoblastoma (Dryja et al., 1986;
Friend et al., 1986; reviewed in Levine, 1993), and the Rb protein (pRb) has been suggested to
play a central role in senescence. The Rb gene is located on human chromosome 13q14, spans
ϳ200 kbp of DNA and encodes a 105 kDa protein that localizes in the nucleus (Lee et al., 1987).
Inactivation of the Rb gene has been associated with multiple cancers in a transgenic mouse
model (Friend et al., 1986; reviewed in Levine, 1993) and with human cancers including
retinoblastoma, small-cell lung carcinomas, breast carcinomas, osteosarcomas, bladder carcinoma, prostate carcinoma and cervical carcinomas (Friend et al., 1986; Harbour et al., 1988).
The Rb protein affects progression through the cell cycle by directly or indirectly regulating
the transcription of growth-related genes. Rb appears to inhibit transcription by RNA polymerase 1 (Cavanaugh et al., 1995) and to block the activity of members of the E2F family of
transcription factors. By blocking E2F, Rb is thought to prevent the transcription of many genes
necessary for the initiation of DNA synthesis to occur in many cell types, thereby maintaining
cells in G0/G1 (Hollingsworth et al., 1993). The retinoblastoma protein is one of the substrates
of the cyclin D-CDK4 and cyclin E-CDK2 complexes that are catalytically active during the G1
phase of the cell cycle and that contribute to the regulation of G1-to-S-phase progression. In
quiescent (G0) cells and in cells in G1 of the cell cycle, the Rb protein is in an active,
hypophosphorylated form that blocks the transcription of growth-related genes by binding to
members of the E2F family of transcription factors. In the late G1, S, G2, and M phases the
protein is progressively phosphorylated at multiple sites (Lin et al., 1991) by cyclin D and E
kinase complexes, which results in the liberation of active E2F. Active E2F transcription factor
then induces the expression of several genes required for cell growth by binding to specific sites
in the promoters of growth-associated genes. This phosphorylation of Rb appears to be necessary for traversing the G1/S boundary in some cell types and is not observed in senescent cells
(Stein et al., 1990; Futreal and Barrett, 1991; Riabowol, 1993).

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Compelling evidence that Rb plays a role in senescence of certain cell types has come from
experiments in which dominant viral oncoproteins have been used to inactivate Rb through
direct binding. For example, the large T antigen (Tag) from simian virus 40 contains a site that
binds to and inactivates Rb as well as a functionally distinguishable site that inactivates the p53
tumor suppressor (Linzer and Levine, 1979). Additionally, overexpression of the products of the
human papillomavirus E6 and E7 genes that bind and inactivate p53 and Rb, respectively,
indicate that E6 activity may be enough to overcome the M1 mechanism in human mammary
epithelial cells, but E7 also appears to be required to overcome M1 in human fibroblasts (Shay
et al., 1993). This implies that in human fibroblasts, at least, Rb plays a role in senescence.
Experiments in which antisense RNA constructs were used to selectively block Rb expression
also suggest that such blocking of Rb function is sufficient to extend the proliferative lifespan
of normal fibroblasts (Hara et al., 1991). However, subsequent studies have reported that the
inactivation of both p53 and Rb are required for this effect (Shay et al., 1991).
A role for p53 in senescing human fibroblasts?
Another gene that has been suggested to play a role in senescence encodes the p53 tumor
suppressor. The p53 gene is located on human chromosome 17p13 and germ-line mutations
result in the Li-Fraumeni syndrome, a genetic cancer disorder. Similar to Rb, p53 is expressed
in all tissues of the body, with the highest levels of p53 mRNA found in the spleen and thymus.
Somatic mutations of p53 are found in 50 – 60% of spontaneous human cancers, including
carcinomas of the breast, esophagus, stomach, liver, ovary, and lung (Carson and Lois, 1995).
p53 is believed to be a central regulator of cell growth and has been implicated both in
promoting apoptosis and in imposing growth arrest in response to DNA damage (Ko and Prives,
1996). Overexpression of p53 in the absence of DNA damage also leads to the arrest of cell
growth, most likely by affecting the transcription of a broad spectrum of genes. The p53 protein
has been shown to act both as a general inhibitor of transcription (Ginsberg et al., 1991;
Santhanum et al., 1991; Chin et al., 1992; Seto et al., 1992) and as a site-specific transcription
factor that can drive the expression of target genes (Kern et al., 1991; Funk et al., 1992;
Zambetti et al., 1992). One of the genes that has been suggested to be vital for establishing
growth arrest via p53 is the p21 gene (El-Deiry et al., 1993; Brugarolas et al., 1995) as described
below.
Several groups have attempted to test whether p53 plays a role in senescence. Although the
levels of p53 protein were reported to increase (Kulju and Lehman, 1995), or to remain
unchanged during cellular senescence (Rittling et al., 1986; Afshari et al., 1993; Atadja et al.,
1995a), it appears clear that p53 activity increases as cells age in culture (Atadja et al., 1995a).
Consistent with this observation, expression of many of the genes induced by p53 including
IGF-BP3 (Buckbinder et al., 1995), cyclin D1 (Chen et al., 1995; Del Sal et al., 1996) and p21
(El-Diery et al., 1993), are upregulated in senescent cells (Goldstein et al., 1991; Murano et al.,
1991; Dulic et al., 1993; Lucibello et al., 1993; Noda et al., 1994; Atadja et al., 1995b).
Increased expression of these p53-inducible genes has the potential to contribute to growth
suppression during senescence because, when overexpressed in diverse experimental systems,
cyclin D1 (Atadja et al., 1995b; Kyu-Hu Han et al., 1995; Wilhide et al., 1995), p21 (El-Diery
et al., 1993; Noda et al., 1994), and IGF-BP3 (Buckbinder et al., 1995) are all growth inhibitory.
Experiments aimed at blocking p53 activity have also provided evidence of a role for p53 in
senescence. p53 activity can be reduced or eliminated through the use of dominant negative
mutants, antisense mRNA approaches, or through the use of a number of viral oncoproteins that

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bind to, and inactivate, p53. Blocking p53 activity by these methods has been reported to extend
the number of MPDs that some types of cells can undergo (Bond et al., 1994, 1995). It,
therefore, appears that blocking p53 activity is enough, at least in some cell types, to temporarily
bypass the M1 mechanism that may limit the growth of normal diploid cells, but is not sufficient
to immortalize cells. In addition, cells containing mutant p53 are more easily immortalized by
the introduction of oncogenes, supporting the thought that by itself, blocking p53 activity does
not efficiently immortalize primary cells in culture (Ulrich et al., 1992; Rittling and Denhardt,
1992; Harvey et al., 1994; Conzen and Cole, 1995; Metz et al., 1995). Consistent with the idea
that p53 plays an important role in senescence but is only one component of a complex senescent
state, a study performed on fibroblasts from heterozygous Li-Fraumeni carriers showed that loss
of p53 correlates both with an extension of in vitro lifespan and immortalization, but that
immortalization requires at least one more additional molecular event. In this study, cells with
an extended in vitro life span were all found to have lost or mutated their remaining p53 gene
(Rogan et al., 1995). Three out of four cultures examined, however, did not continue to divide
indefinitely, implying that they were blocked by an M2-type mechanism. The fourth culture
continued to proliferate and this “escape” from M2 was correlated with a loss of expression of
p16, the product of the multiple tumor suppressor locus (MTS-1), which encodes an inhibitor of
D-type cyclins (Serrano et al., 1993). These observations suggest that p16 may play an
important role in a second “senescence pathway” independent of p53 and that immortalization
of normal cells requires perturbation of multiple pathways.
Cyclin-dependent kinase inhibitors
Control of entry into, and progression through, the cell cycle is regulated by the synthesis and
degradation of a family of proteins known as cyclins that associate with their catalytic subunits,
the cyclin dependent kinases (cdks). Catalytic activity is controlled through numerous mechanisms including cyclin binding, phosphorylation, and by the binding of cyclin dependent kinase
inhibitors (cdis; Sherr and Roberts, 1995). Cdis are a group of growth inhibitors that are believed
to block cdk activity by directly binding to cdks, preventing their interaction with activating
subunits such as cyclins, subsequently halting progression through the cell cycle. For example,
cdis may function to halt, or prevent, entry into the cell cycle as a response to DNA damage, or
contribute to regulating the stepwise deactivation of cyclin– cdk complexes required for passage
through specific points within the cell cycle. A characteristic seen for all cdis tested to date is
that ectopic overexpression results in growth arrest, although not always in the same phase of
the cell cycle (Sherr and Roberts, 1995).
Mammalian cdis can be grouped into two major classes: p21 and related proteins, and the p16
family of cdk inhibitors (Morgan, 1995). Presently, three members of the p21 family are known,
p21 (also referred to as SDI1, WAF1, CIP1, and PIC1), p27 (KIP1), and p57(KIP2). These proteins
contain a conserved domain that is necessary for both cyclin-cdk interaction and for inhibition of
kinase activity. Members of this family do not target specific cyclin– cdk complexes but are
capable of binding to and deactivating complexes containing several different cyclins that are
known to function in different points in the cell cycle (cyclins A, B, D, or E; Xiong et al., 1993;
Harper et al., 1995). Because p21 is also known to bind to and inactivate the proliferating cell
nuclear antigen (PCNA) component of the DNA replicative machinery (Xiong et al., 1992; Waga
et al., 1994), this cdi has the potential to block proliferation at several points of the cell cycle.
Increased expression of p21 has implicated this cdk inhibitor in contributing to growth arrest
in senescent cells (Noda et al., 1994), in cells subjected to DNA damage (Di Leonardo et al.,

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1994), and in response to agents that induce differentiation (Jiang et al., 1994; Halevy et al.,
1995; Liu et al., 1996). Consistent with a role in responding to DNA damage, mice in which the
p21 gene has been genetically inactivated by homologous recombination (p21 knockout mice)
are defective in their ability to arrest their growth in G1 in response to DNA damage and to
nucleotide pool perturbations, although they undergo normal development (Deng et al., 1995;
Brugarolas et al., 1995). The related p27 protein has been implicated as a mediator of growth
arrest in response to TGF␤, serum deprivation, or contact inhibition in an established epithelial
cell line (Polyak, 1994), although it does not appear to mediate these responses in primary cells
including HDFs (Nakayama et al., 1996). However, a role in negatively regulating the growth
of several cell types is supported by observations of p27 knockout mice that show larger total
body size, elevated cdk2 activity, overdevelopment of several organ systems, female sterility,
and the development of pituitary tumors (Nayakama et al., 1996; Kiyokawa et al., 1996; Fero
et al., 1996). The role of p57 in regulating the cell cycle has yet to be established, although
overexpression of either p27 or p57 from exogenous synthetic DNA expression constructs
blocks cell proliferation (Sherr and Roberts, 1995).
The p16 family includes several members (p15, 16, 18, and 19), all of which are small
polypeptides containing four ankyrin repeats (protein sequences that are known to promote
protein:protein interactions) that preferentially bind to free cdk4 and cdk6 and inhibit the
activity of cyclin D/cdk complexes (Serrano et al., 1993; Guan et al., 1994; Hannon and Beach,
1994). Experiments in which p16 was ectopically expressed from an engineered plasmid
suggested a potential mechanism by which members of the p16 family inhibit cell growth by
preventing the binding of the activating cyclin subunit to cdk4 and cdk6 (Serrano et al., 1993).
Unlike members of the p21 family of cdk inhibitors that are not mutated in known cancer types,
two members of the p16 family (p15 and p16) are mutated at a high frequency in a wide variety
of human cancer cell lines (Kamb et al., 1994; Nobori et al., 1994; Okamoto et al., 1994), as
well as in several types of primary tumors (Hussussian et al., 1994), suggesting a role for these
genes in tumor suppression.
Compared with young, proliferation-competent cells, senescent cells express several-fold
higher levels of both p16 (Hara et al., 1996; Wong and Riabowol, 1996) and p21 (Noda et al.,
1994; Atadja et al., 1995b), implying potential growth-inhibitory roles for these cdk inhibitors
in senescence. However, it is not known whether this increased expression plays a causal role
in establishing or maintaining the growth arrested phenotype of senescence or whether this
increased expression is a consequence of the activation of “growth-arrest” pathways.
p33ING1, a novel growth regulator
When isolating genes of unknown function, assays with positive or with negative selection
parameters can be used. For example, if the gene to be isolated normally functions to inhibit cell
growth, then isolation of the gene based on its normal function would require a negative
selection procedure where cells blocked for growth would survive and cells continuing to grow
would be selected against. Such a procedure is, for practical considerations, difficult to implement because cells carrying the gene of interest cannot be grown and expanded into colonies that
can be manipulated experimentally. To circumvent this difficulty we have utilized a new
positive selection procedure that combines subtractive hybridization with an in vivo selection
assay to clone a new potential tumor suppressor gene that we have named ING1 for inhibitor of
growth. Subtractive hybridization enabled the removal of mRNA sequences that were expressed
at similar levels in different normal and cancer cells and allowed the isolation of mRNAs

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expressed at higher levels in normal human cells grown in vitro. Suppression of ING1 levels by
expression of antisense mRNA is associated with tumor formation in nude mice and with focus
formation and growth in soft agar in vitro (Garkavtsev et al., 1996). Consistent with a growth
inhibitory role, overexpression of ING1 following the introduction of DNA expression constructs by transfection or by needle microinjection resulted in an accumulation of cells in G0/G1.
These observations suggested that the 33 kDa protein encoded by ING1 (p33ING1) could fulfill
the criteria of a tumor suppressor, a possibility strengthened by the localization of the gene to
chromosome band 13p33–34, which has been implicated in the progression of a number of
different naturally occurring cancers (Garkavtsev et al., 1997a).
The increased expression or activity of several well-characterized tumor suppressors during
senescence led us to analyze ING1 expression in aging diploid fibroblasts. We found that the
expression of ING1 RNA and protein increases 8 –10-fold in late-passage HDFs compared with
early-passage HDFs, suggesting that p33ING1 plays a role in controlling cell growth as fibroblasts reach the end of their in vitro lifespan. Additionally, expression of antisense ING1 RNA
in presenescent fibroblasts led to a limited extension of their in vitro lifespan in several
independent trials, again implying an important role for p33ING1 in at least some aspects of
senescence (Garkavtsev and Riabowol, 1997b).
Figure 2 provides a summary of the changes in tumor suppressor expression and activity that
occur during cellular aging. The maintenance of Rb in a hypophosphorylated state is believed
to prevent the liberation of E2F, thereby blocking the expression of genes required for cell cycle
progression. However, this interpretation has been brought into question by recent observations
that E2F-1 knockout mice (mice in which the E2F-1 gene has been inactivated by homologous
genetic recombination) show aberrant cell proliferation and the development of several tumor
types (Field et al., 1996; Yamasaki et al., 1996). In addition to affecting E2F activity, active Rb
might contribute to downregulating cell growth in a more global manner through the inhibition
of RNA polymerase I (Cavanaugh et al., 1995) and/or RNA polymerase III (White et al., 1996).
The inability of senescent cells to inactivate Rb is believed to be due to a lack of cyclin– cdk
complexes that are active in G1 of the cell cycle. This is consistent with the observations that
the activities of the G1 cyclin, cyclin E (Ohtsubo et al., 1995), and its catalytic subunit cdk2 (van
den Heuvel and Harlow, 1993; Tsai et al., 1993) are required for entry into the S phase in
response to mitogens, and are known to efficiently phosphorylate Rb in vivo (Hinds et al.,
1992). Senescent cells are known to express much higher levels of the p21 and p16 cdk
inhibitors, which would be expected to efficiently inhibit the activity of G1 cyclins, preventing
the inactivation of Rb. Despite similar or lower levels of all other cyclins examined (Wong and
Riabowol, 1996), senescent cells express much higher levels of some of the D-type cyclins than
young diploid or immortalized cell types from a variety of tissues (Noda et al., 1994; Atadja et
al., 1995b; Wong et al., 1996). The three representatives of the D-type cyclin family, cyclins D1,
D2, and D3 are thought to function in the early phases (G and/or G1) of the cell cycle. Although
results differ among different laboratories regarding the effects of increasing and decreasing
cyclin D1 levels, it will be interesting to determine if the D-type cyclin complexes that are
expressed at high levels in senescent cells are active as kinases.
The isolation and characterization of additional growth inhibitory genes such as p33ING1
should add to our knowledge of how tumor suppressors and senescence factors work and will
help to clarify the links between aging and cancer. For example, p33ING1 levels increase in
senescent cells and p33ING1 appears to associate with p53, suggesting that p33ING1 may be
responsible for the activation of p53 seen in senescent cells (Atadja et al., 1995a). The
relationship between aging and tumor suppression is perhaps one of the most fascinating in

TUMOR SUPPRESSORS AND SENESCENCE

FIG. 2. The role of tumor suppressors and cell cycle regulators during cellular senescence. Activities
of the cyclin-CDK complexes are required for progression through G1 of the cell cycle but appear to
be downregulated in senescent cells. (A) In young, proliferation-competent cells cyclins and cdks
form complexes that are capable of phosphorylating Rb, releasing members of the E2F family of
transcription factors that can then induce the expression of genes required for progression through G1,
and entry into the S phase of the cell cycle. Inactivation of Rb through phosphorylation is also thought
to prevent Rb from inhibiting transcription through RNA polymerase I and III. (B) In senescent cells
the expression of D-type cyclins is increased, which may be growth inhibitory due to sequestering
factors required for cell cycle progression (Atadja et al., 1995b). In addition, the increased expression
of cdk inhibitors such as p16 (Hara et al., 1996; Wong and Riabowol, 1996), p21 (Noda et al., 1994;
Atadja et al., 1995b) and possibly the novel growth inhibitor p33ING1 (Garkavtsev et al., 1996)
contribute to the inactivation of G1 cyclin-cdk activity. Loss of this activity, which is believed to be
required for the inactivation of Rb, leads to continued inhibition of RNA polymerases I and III, and
an inability to induce the expression of genes regulated by E2F, resulting in continued G0 or G1
arrest. The physiologic inducers of p16 and p33 have yet to be established although p21 expression
is induced by the tumor suppressor p53 among other factors. The box indicates changes in the
expression or activity of the tumor suppressors p53 and Rb, and the cyclins, cdks, and cdis, that have
been reported to occur during cellular aging.

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cellular biology. These processes seem to share many components, modification of some of
which could prove useful in treating both cancer and other age-related diseases. We hope that
such a thought might appeal to Dr. Alex Comfort, who has contributed so much to the biology,
philosophy, and psychology of aging through the publication of numerous scientific articles,
poems, plays, and textbooks. We dedicate this review to this truly expansive thinker.
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