Synthesis of a rigidified bicyclic AAZTA-like ligand and relaxometric characterization of its GdIII complex

Article abstract of DOI:10.1016/j.tetlet.2020.152573

The synthesis of a novel constrained polydentate ligand CpAAZTA derived from the heptadentate polyaminocarboxylic ligand AAZTA (6-amino-6-methylperhydro-1,4-diazepine-N, N’,N’’,N’’-tetraacetic acid) in which the ethylene bridge of the 1,4-diazepine ring i

Full text of DOI:10.1016/j.tetlet.2020.152573

PERSPECTIVE
Keratinocyte Apoptosis in Epidermal Development and
Disease
Deepak Raj1.2, Douglas E. Brash3,4 and Douglas Grossman1,2,5
Keratinocyte (KC) apoptosis plays a critical role in regulating epidermal development and restraining carcinogenesis.
Apoptosis balances proliferation to maintain epidermal thickness, contributes to stratum corneum formation and may
eliminate pre-malignant cells. Apart from the normal developmental program, KC apoptosis can be triggered by UV light
and other stimuli. Dysfunctional apoptosis occurs in some skin diseases, such as psoriasis and skin cancer. Here we review
the current state of knowledge of KC apoptosis, with particular focus on apoptotic signaling pathways and molecular
mechanisms of apoptosis control, and discuss new insights into the complex role of apoptosis in skin carcinogenesis that
are emerging from mouse models.
Journal of Investigative Dermatology (2006) 126, 243–257. doi:10.1038/sj.jid.5700008
Perhaps the greatest irony in biology removal of infected or damaged cells first described apoptosis in normal skin,
is that life depends on death — pro- and autoreactive lymphocytes. It is shortly after the term was coined in the
grammed cell death, that is. Cell estimated that each day, in an average medical literature in 1972 (Kerr et al.,
elimination by apoptosis is essential adult, over 50 billion cells undergo 1972). In the following years, Horvitz,
for organ sculpting in development, apoptosis (Reed, 1999). It has been only Sulston, and Brenner discovered sev-
normal tissue turnover, and efficient 30 years since Olson and Everett (1975) eral Caenorhabditis elegans death (ced)
Editor's Note
An organism's or an organ system's ability to grow are subject to environmental damage, programmed cell
and renew itself requires both cell proliferation and death is critical to their normal function. In this issue
regulated cell death. A regulated form of cell death has of the Journal, the Perspectives series continues with
been noted since at least the mid-1800s, and in 1964 two articles on apoptosis in keratinocytes and in the
Lockshin and Williams coined the term “programmed hair follicle. Raj, Brash, and Grossman offer insight into
cell death” to describe developmental events in insect the process of apoptosis in development and diseases
larvae (J Insect Physiol 10:643–649, 1964). In 1972 Kerr, of the keratinocyte. They provide a special focus on
Wyllie, and Currie used the term apoptosis to describe the regulation of apoptosis in the skin and its potential
morphologic features of dying cells observed in a variety application for our understanding of carcinogenesis in
of organs and conditions (Br J Cancer 26:239–257, 1972). the skin. Botchkareva, Ahluwalia, and Shander extend the
Early in the story of apoptosis, Kerr and Searle found focus to include the hair follicle. Understanding how the
the prominent shrinkage necrosis of the cells that is process of apoptosis applies to the hair cycle provides
characteristic of apoptosis in basal cell carcinomas (J great opportunity for future investigations regarding
Pathol 107:41–44, 1972). In 2002 Brenner, Horvitz, and the pathogenesis of many hair disorders as well as the
Sulston were awarded the Nobel Prize in Medicine hair loss associated with aging. These Perspectives on
for their discoveries concerning “genetic regulation apoptosis will provide a platform for further investigation
of organ development and programmed cell death.” of this exciting and important area of the biology of skin
Because skin and hair constantly renew themselves and and skin diseases.
Russell P. Hall
1Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City, Utah, USA; 2Department of Oncological Sciences, University of Utah
Health Sciences Center, Salt Lake City, Utah, USA; 3Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut, USA;
4Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA; and 5Department of Dermatology, University of Utah Health
Sciences Center, Salt Lake City, Utah, USA;
Correspondence: Douglas Grossman, Huntsman Cancer Institute, Suite 5243, 2000 Circle of Hope, Salt Lake City, Utah 84112, USA.
Email: doug.grossman@hci.utah.edu
Abbreviations: ASK1, apoptosis signal-regulating kinase-1; ced, Caenorhabditis elegans death; DLK, dual leucine zipper-bearing kinase; EGF, epidermal growth
factor; ERK, extracellular signal-regulated kinase; Fas-L, Fas ligand; IAP, inhibitor of apoptosis (gene family); KC, keratinocyte; MAPK, mitogen-activated protein
kinase; SCC, squamous-cell carcinoma; TNF, tumor necrosis factor
Manuscript received 5 July 2005; revised 16 August 2005; accepted for publication 2 September 2005
© 2006 The Society for Investigative Dermatology
www.jidonline.org 243

D Raj et al.
Epidermal apoptosis
Cytokines,
Fas ligand
UV radiation
Cytotoxic drugs
of apoptosis in maintaining epider-
mal structure and homeostasis will be
reviewed, as well as its dysregulation in
skin disease and skin cancer. Finally, we
will discuss how KC apoptosis serves
as a cancer-preventive mechanism by
eliminating potentially pre-malignant
cells and may modulate early steps in
skin carcinogenesis.
Death receptors
Caspase-8
Bax,
Bak
FLIP
Bid
Bcl-2,
Bcl-x L
t-Bid
AIF
General Pathways and Mechanisms of
Apoptotic Control
Early work on apoptosis in worms elu-
cidated a sequential genetic pathway
consisting of an activator molecule
(EGL-1), an inhibitor molecule (ced-9),
an adapter molecule (ced-4), and an
effector molecule (ced-3). This para-
digm of activators, inhibitors, adapt-
ers, and effectors is recapitulated in
multiple apoptotic pathways in mam-
malian cells. Apoptotic pathways and
regulators potentially important in KCs
are depicted in Figure 1, and reviewed
extensively elsewhere (Reed, 1999).
Cells respond to environmental, extra-
cellular, and internal death signals
through multiple sensors that coordi-
nate and integrate apoptotic respon-
ses. The ‘extrinsic’ pathway is stimu-
lated by binding of Fas ligand (Fas-L),
tumor necrosis factor (TNF), or related
cytokines to extracellular membrane
‘death receptors’ that recruit adapter
molecules and lead to activation of
IAP
Smac
Cytochrome c
Apaf-1
Caspase-9
PKC δ
Apoptosis
Caspase-3,7
Cell death substrates
Figure 1. Apoptotic pathways in keratinocytes. The extrinsic pathway is stimulated by UVB or binding of
Fas ligand, tumor necrosis factor, or other cytokines to death receptors that results in activation of caspase-
8. The intrinsic pathway is also stimulated by UVB, as well as various cytotoxic drugs, and is linked to
DNA damage responses. It involves mitochondrial depolarization and release of multiple proapoptotic
factors. In addition, UVB results in formation of DNA photo-products and initiation of DNA damage
responses. Mitochondrial content release may directly result from these stimuli or may occur indirectly
as part of the DNA damage response. Cytochrome c promotes activation of caspase-9, Smac inhibits
inhibitor of apoptosis (IAP) caspase inhibitors, and apoptosis-inducing factor (AIF) translocates to the
nucleus and mediates caspase-independent apoptosis. The extrinsic and intrinsic pathways are potentially
linked by Bid, which is cleaved by caspase-8, and the activated fragment (tBid) causes mitochondrial
content release. Activation of upstream caspases (caspase-8 or -9) leads to activation of downstream
caspases (caspase-3 or -7), resulting in cleavage of intracellular substrates, cellular condensation, and
nuclear fragmentation. Apoptotic inhibitors include caspase inhibitors (FLIP, IAP proteins) and the Bcl-2
family proteins Bcl-2 and Bcl-xL that prevent mitochondrial membrane permeability.
genes and established that apoptosis skin. Apoptotic cell death is critical for caspase-8. Caspases (ced-3 homo-
represents a genetically encoded path- balancing of KC proliferation as well as logues; cysteine aspartic acid-specific
way in worms, in work for which they for formation of the stratum corneum. proteases) are constitutive proenzymes
were recently awarded a Nobel Prize. Apoptosis also represents an important that autoactivate or are activated upon
The discovery of bcl-2 translocation in cancer defense mechanism, as KCs that cleavage by other caspases, resulting
follicular lymphoma in 1984 eventual- may have accumulated mutations or in a proteolytic cascade. Some cells
ly led to the first link between apoptosis sustained other genetic damage as a express FLIP, an inhibitor of caspase-8
and cancer. In the past decade, studies consequence of exposure to UV radia- that blocks death receptor signaling.
in mammalian cells have character- tion or oxidative damage are eliminat- The ‘intrinsic’ pathway, triggered by
ized multiple interconnected apoptotic ed by apoptosis. The development of most cytotoxic drugs and DNA dam-
pathways and identified a multitude of genetic mouse models in which apop- age, involves mitochondrial release of
additional regulatory factors. A review totic regulatory molecules are deleted cytochrome c, which combines with
of our current state of knowledge of or overexpressed in the skin has vali- the cofactor Apaf-1 (ced-4 homologue)
keratinocyte (KC) apoptosis, focusing dated many of these functions of apop- in the formation of an activated cas-
on apoptotic signaling pathways and tosis. Here, we will first summarize pase-9 ‘apoptosome.’ Mitochondria
molecular mechanisms of apoptosis the basic components of the cell death may also promote apoptosis through
control, is particularly timely because machinery and their integrated function release of Smac/DIABLO, which blocks
it has been 12 years since the topic of in apoptotic pathways in KCs. Next, inhibitor of apoptosis (IAP) proteins
apoptosis in epidermal development we will describe KC apoptosis in vivo that function as caspase inhibitors, and
and disease was last reviewed in the and consider the evidence that KC cell apoptosis-inducing factor (AIF), which
Journal (Haake and Polakowska, 1993).
death in epidermal development repre- translocates to the nucleus and medi-
In no other organ system does apop- sents apoptosis rather than an alternate ates caspase-independent apoptosis.
tosis play so many vital roles as in the form of cell death. The essential role Release of these proapoptotic factors
244 Journal of Investigative Dermatology (2006), Volume 126

D Raj et al.
Epidermal apoptosis
is regulated by Bcl-2 family proteins
A variety of agents have been shown ing factor-mediated caspase-indepen-
that form homodimers and heterodi- to induce KC apoptosis in vitro.The most dent apoptosis has not been rigorously
mers that control mitochondrial mem- intensively studied, and perhaps most examined. Although caspase-8 and cas-
brane permeability. Some Bcl-2 family physiologically relevant, is UVB (290– pase-9 appear to be concomitantly acti-
proteins (Bcl-2, ced-9 homologue, and 320nm). Although UVB induces expres- vated in response to UVB, Sitailo et al.
Bcl-xL) may block apoptosis whereas sion of multiple genes in KCs, execution (2002) showed that upstream caspase-9
others (Bax, Bak, and Bid) promote of the apoptotic program requires not de is critical for these responses, by block-
apoptosis by interfering with these novo transcription but rather activation ing with specific peptide or dominant-
interactions. The p53 tumor suppres- of pre-synthesized proteins. Multiple negative caspase-9 inhibitors. Consistent
sor promotes apoptosis through both studies have shown that doses ranging with this observation, UVB-induced KC
transcriptional and non-transcrip- from 200 to 700 J/m2 induce KC apop- apoptosis can be blocked by transfection
tional mechanisms, as detailed below. tosis over a 24- to 48-hour period that is of Bcl-2 (Takahashi et al., 2001b). One
The extrinsic and intrinsic pathways preceded by mitochondrial depolariza- target of activated terminal caspase-3 is
are potentially linked in an amplifica- tion, release of cytochrome c, and acti- protein kinase Cδ, which, upon cleav-
tion loop by Bid, which is cleaved by vation of multiple caspases (caspase-3, age, translocates to the mitochondria.
caspase-8, and the resulting fragment -8, and -9). UVB triggers multimeriza- Blocking protein kinase Cδ activation
(tBid) translocates to the mitochondria tion of Fas death receptors, leading to with a kinase-inactive catalytic domain
and triggers cytochrome c release and activation of caspase-8 (Takahashi et al., (Denning et al., 2002) or caspase-resis-
activation of caspase-9. Activation of 2001a), and caspase-8-mediated cleav- tant mutant (D’Costa and Denning,
either of these upstream caspases leads age of Bid is important for amplifica- 2005) prevents UVB-induced KC apop-
to activation of terminal caspase-3 and tion of the apoptotic response (Assefa tosis. Recent studies by Denning and
caspase-7, which dismantle cells by et al., 2000). In most KC-based systems, colleagues (L Sitailo, SS Tibudan and
cleaving proteins involved in nuclear UVB-induced apoptosis appears to be MF Denning, 2005, J Invest Dermatol
membrane and cytoskeletal structure, caspase-dependent, as responses can 124:A129) indicate that activated pro-
DNA repair, and replication systems.
be blocked with caspase inhibitors, tein kinase Cδ phosphorylates the Bcl-2
although induction of apoptosis-induc- homologue Mcl-1 in vitro and disrupts
The Apoptotic Program in Keratinocytes
Apoptotic stimulation in keratinocytes.
It is important to note that the character-
ization of many of these pathways and
regulatory networks — indeed, much
of our mechanistic knowledge of apop-
tosis in general — comes largely from
in vitro studies in HeLa cells or other
cell lines. Similarly, many mechanistic
studies on KC apoptosis have used the
human HaCaT cell line (which harbors
two mutant p53 alleles) as a model for
normal KCs. We have actually found
HaCaT cells to be more susceptible
to apoptosis than normal KCs in vitro
(Bowen et al., 2003), possibly because
of aberrant signaling pathways result-
ing from long-term culture (Chaturvedi
et al., 2001). In addition, most studies
examining apoptosis in normal KCs
have been performed on cultured cells
in the presence of exogenous growth
factors. Apoptotic responses in vivo,
however, necessarily occur in distinct
cellular and tissue contexts. Therefore,
after a summary of observations made
in cultured HaCaT cells and KCs, the
remainder of this review will focus on
studies using epidermal constructs,
in situ detection methods, and animal
models that may be most relevant to KC
apoptosis in vivo.
Matrix proteins
(laminin 5)
EGF
HGF
NGF
PI3K
Akt
α6β4 α3β1
EGFR
MEK
ERK
Bad
Bcl-2,
Bcl-xL
NFκB
IAP
Caspase-9
Figure 2. Survival (antiapoptotic) pathways in keratinocytes. The two primary survival pathways
involve activation of a mitogen-activated protein kinase (MAPK) cascade and activation of Akt.
Integrin-mediated interactions with matrix proteins, particularly laminin 5, as well as signaling through
epidermal growth factor receptor (EGFR), lead to sequential phosphorylation and activation of MAPKs.
The precise mechanism(s) by which activated MAPK kinases (MEKs) promote cell survival is not known
(dotted arrows). Signaling by epidermal growth factor (EGF) or hepatocyte growth factor (HGF) may
also stimulate a second survival pathway involving phosphatidylinositol 3-kinase (PI3K), leading to
phosphorylation and activation of Akt. Activated Akt promotes survival by activating the transcriptional
regulator NF-κB and counteracts apoptosis by phosphorylating and inhibiting Bad and caspase-9.
Caspase-9 is also subject to inhibition by IAP proteins. Finally, signaling by nerve growth factor (NGF)
stabilizes levels of the antiapoptotic proteins Bcl-2 and Bcl-xL. ERK, extracellular signal-regulated kinase.
www.jidonline.org 245

D Raj et al.
Epidermal apoptosis
its interaction with Bax in HaCaT cells.
Early apoptotic signaling by UVB in KCs
stimulates phosphorylation and stabili-
zation of the p38 mitogen-activated pro-
tein kinase (MAPK), which occur within
2 hours of UVB exposure and precedes
caspase activation (Shimizu et al., 1999).
The function of various MAPK pathways
in KCs has been previously reviewed
by Eckert and colleagues (Eckert et al.,
2002). Inhibition of p38 MAPK activa-
tion prevents UVB-induced apoptosis,
translocation of Bax to mitochondria,
and cytochrome c release (Van Laethem
et al., 2004). Lipid peroxidation and
generation of oxidative radicals occur
in KCs after UVB exposure (Carini et al.,
2000), and antioxidants may be protec-
tive (Kulms et al., 2002; Mu et al., 2003).
Oxidative stress may directly activate
p38 MAPK (Peus et al., 1999). As noted
below, UVB-induced KC apoptosis in
vivo involves both membrane-based sig-
naling and DNA damage. Finally, UVB
can activate apoptotic pathways through
induction of ceramide synthase and de
novo ceramide synthesis (Uchida et al.,
2003).
Hyperkeratosis
Psoriasis
Death receptor activation
Skin cancer
Mitochondrial
depolarization
Akt
EGF
NFκB
Cytochrome c
Smac AIF
Galectin-7
p53 Bax
FLIP IAP
p53 mutations
Bcl-2 Bcl-XL E2F1
p38 MAPK
Caspases
PKC δ
Defective apoptotic signaling
Cell survival
Cell death
Apoptotic balance
Sunburn
TEN
GVHD
Figure 3. Apoptotic balance in keratinocytes is mediated by multiple factors at multiple levels.
Predominance of cell survival factors is evident in psoriasis, skin cancer, and hyperkeratotic diseases,
whereas excessive cell death is characteristic of other diseases, including sunburn, toxic epidermal
necrolysis (TEN), and graft-versus-host disease (GVHD). PKCδ, protein kinase Cδ.
Other UV wavelengths may also
induce KC apoptosis in vitro, with
apoptotic potency corresponding to
increasing energy or decreasing wave-
length (Godar, 1999). Although UVC colleagues (Chaturvedi et al., 2004) pase activation. This cell death pathway
(100–290 nm) damages cellular DNA compared apoptotic responses in cul- is promoted by p53 and Bax, which are
and is a more potent apoptotic stimu- tured human versus mouse KCs and minimally expressed in proliferating
lus than UVB, it is largely absorbed in reported that human KCs were more KCs (Bowen et al., 2003). On the other
the atmosphere and does not reach the susceptible to UV- and drug-induced hand, this pathway may be antagonized
Earth’s surface. On the other hand, UVA apoptosis. The relative apoptotic resis- in normal KCs through expression
(320–400 nm), which is less potent tance of mouse KCs was associated of multiple antiapoptotic regulators
than UVB, appears to trigger apopto- with decreased constitutive levels of including Bcl-xL, FLIP, and various IAP
sis through oxidative damage (Fu et p53 and increased expression of kera- molecules (c-IAP1, c-IAP2, XIAP, and
al., 2000). In addition to UV radiation, tin 1, an early marker of KC differentia- Livin) (Bowen et al., 2003). Two nota-
other apoptotic stimuli reported for tion. Although further determinants of ble cell survival pathways have been
KCs include the death receptor agonist this differential apoptotic susceptibility partially characterized in KCs. The first
TNF-related apoptosis-inducing ligand remain to be defined, these experiments is a MAPK pathway (Figure 2) triggered
(TRAIL), rapid detachment (anoikis), suggest that mouse KCs maintained in by epidermal growth factor (EGF) and
growth factor deprivation, ceramide, vitro may exist in a more differentiated phosphorylation of epidermal growth
cytotoxic drugs, 1,25-dihydroxyvita- state and be less prone to p53-depen- factor receptor (EGFR). Whereas EGF
min D3, and transforming growth fac- dent apoptosis. Nevertheless, p53- can protect KCs against UVB-induced
tor β1 (Benassi et al., 1997). Apoptosis dependent apoptosis of mouse KCs in apoptosis, EGFR inhibitors enhance
induced by these agents generally vivo can readily be stimulated by UVB, UVB-induced apoptosis (Peus et al.,
involves activation of caspases and as detailed below.
mitochondrial pathways, but the mech-
2000). Signaling through EGFR stimu-
lates a MAPK cascade that results in
anisms are less well characterized than Antiapoptotic forces in keratinocytes. sequential phosphorylation and acti-
those involving UVB. As is discussed above, the primary KC vation of MAPK kinases (MEKs) and
The majority of in vitro studies on signaling pathway leading to cell death extracellular signal-regulated kinases
KC apoptosis have been performed on is activation of p38 MAPK, followed by (ERKs). The mechanism by which acti-
human KCs. Recently, Chaturvedi and mitochondrial depolarization and cas- vated ERKs promote cell survival in
246 Journal of Investigative Dermatology (2006), Volume 126

D Raj et al.
Epidermal apoptosis
KCs has not been clearly elucidated. of these are derived from interaction formation requires p53. Expression of
Signaling by EGF may also stimulate with extracellular matrix proteins. p53 is upregulated in UVB-treated KCs
a second survival pathway involving Laminin 5 is the primary ligand for epi- through increased protein stability (Liu
phosphatidylinositol
3-kinase-medi- dermal KC adhesion, binding to the et al., 1994). One transcriptional tar-
ated activation of the serine/threonine α3β1 and α6β4 integrins on KCs. Using get of p53 is galectin-7 (PIG1), which
kinase Akt (protein kinase B; Figure 2). KCs derived from α3 knockout mice, parallels p53 stabilization and is highly
In other cell types, Akt is activated by Manohar et al. (Manohar et al., 2004) expressed in sunburn cells (Bernerd et
growth factors and promotes cell sur- showed that laminin 5 binding of α3β1, al., 1999). Overexpression of galectin-7
vival directly by phosphorylating (and and to a lesser extent α6β4, promotes in KCs is sufficient to cause apoptosis
inactivating) Bad and caspase-9 (Datta KC cell survival through activation of (Bernerd et al., 1999), through caspase
et al., 1999). Given the important role a MEK/ERK signaling pathway. This activation and cytochrome c release
of caspase-9 in KC apoptosis, noted survival pathway can also be activated (Kuwabara et al., 2002). In addition
above, it is not surprising that Akt is a through EGFR, protecting KCs against to upregulation of p53, UVB-irradi-
potent KC survival factor (Wang et al., anoikis (Jost et al., 2001). In addition ated skin reveals upregulation of Bax
2003). Akt also promotes survival in to EGF, other growth factors constitute and downregulation of Bcl-2 (Lee et
KCs by activating NF-κB, a transcription a second class of survival signals that al., 2003). Signaling through the Fas
factor that is repressed by interaction may potentially contribute to apoptosis death receptor, which rapidly clusters
with κB inhibitor (IκB). The role of NF- resistance in vivo through Akt. Nerve following UVB exposure (Bang et al.,
κB in KC survival has been previously growth factor and hepatocyte growth 2003), appears to be involved in sun-
reviewed in the Journal by Nickoloff (scatter) factor protect KCs against UVB- burn-cell formation, which is reduced
and colleagues (Nickoloff et al., 2002), induced apoptosis by maintaining lev- in mice deficient in Fas-L (Hill et al.,
who have shown that inhibition of NF- els of Bcl-2 and Bcl-xL (Marconi et al., 1999). In addition, UVB-induced DNA
κB using proteasome or IκB subunit 1999) and by activating Akt (Mildner et damage is also necessary for sunburn-
inhibitors increases KC susceptibility to al., 2002), respectively. Nuclear peroxi- cell formation, which requires genera-
apoptosis. Activation of NF-κB through some proliferator-activated receptors tion of cyclobutane pyrimidine dimers
Akt signaling may promote KC survival in KCs can also promote Akt signaling (Ley and Applegate, 1985) in actively
by modulating expression of IAPs or through transcriptional effects (Di-Poi transcribed genes (Brash et al., 2001).
TRAF death receptor adapters, as has et al., 2002). In many of these cases, Activation of these apoptotic pathways
been shown in other cell types.
KC apoptotic responses can be blocked by UVB is balanced by activation of
Finally, in discussing cell death and by overexpression of apoptosis inhibi- the E2F1 transcription factor, which
survival pathways in KCs, it is impor- tors such as Bcl-2 and the IAP pro- is upregulated in response to DNA
tant to note that apoptotic stimuli may tein Survivin (Grossman et al., 2001). damage and promotes DNA repair.
activate multiple pathways simultane- Upregulation of endogenous apoptotic Transgenic overexpression of E2F1 is
ously. For example, several studies have inhibitors of the Bcl-2 and IAP families associated with reduced UVB-induced
demonstrated that UVB and TNF-α can can promote KC survival in vivo and, as apoptosis in vivo (Berton et al., 2005),
activate the p38 MAPK death pathway discussed below, may play important and E2F1-deficient mice demonstrate
and the MEK/ERK and Akt/NF-κB sur- roles in carcinogenesis.
enhanced susceptibility to sunburn-cell
formation (Wikonkal et al., 2003).
vival pathways (Nickoloff et al., 2002).
Evidence for Keratinocyte Apoptosis In
Vivo
Products of the MAPK pathways may
intersect via a p38 MAPK/ERK complex
Apoptotic versus non-apoptotic cell
that translocates to the nucleus in KCs Sunburn cells. After acute UVB expo- death. Although it is generally accept-
in response to protein kinase Cδ acti- sure, scattered dyskeratotic KCs with ed that sunburn cells represent KCs
vation (Efimova et al., 2004). Thus KCs condensed or absent nuclei and eosin- induced to undergo apoptosis in vivo,
must coordinate a delicate balance ophilic cytoplasm (termed ‘sunburn some controversy exists over whether
of many antiapoptotic and proapop- cells’) can be seen in mouse skin by elimination of normal KCs in epider-
totic forces in responding to extracel- routine hematoxylin and eosin staining. mal development occurs by spontane-
lular stimuli, shifting their equilibrium Sunburn cells represent KCs undergo- ous apoptosis or a ‘non-apoptotic’ form
toward cell survival or cell death when ing UVB-induced apoptosis and can be of cell death. The term ‘apoptosis’ was
one pathway predominates (Figure 3).
identified by in situ end labeling with originally used to describe particular
Most cell types are more susceptible terminal deoxynucleotidyl transfer- morphologic events observed in iso-
to apoptosis in vitro than in vivo, and ase (TUNEL technique) (Brash, 1997) lated cells of normal tissues and tumors
KCs are no exception. For example, and staining for activated caspase-3 (Kerr et al., 1972). Whereas cells under-
lower doses of UVB are required to (Pentland et al., 2004). Sunburn-cell going pathologic cell death (necrosis)
induce KC apoptosis in cultured cells incidence typically peaks 24 hours after are characterized by organelle swell-
compared with normal skin. Multiple irradiation, then gradually declines over ing and membrane rupture, with leak-
survival signals present in tissues are several days (Okamoto et al., 1999). age of cell contents, that generates
likely to account for this increased KC Using p53-deficient mice, Ziegler et al. marked inflammation, apoptotic cells,
resistance to apoptosis in vivo. Many (1994) demonstrated that sunburn-cell by contrast, exhibit cellular condensa-
www.jidonline.org 247

D Raj et al.
Epidermal apoptosis
to be other granular layer proteins basal to the suprabasal layer (Ganguli and activation of survival factors includ-
involved in stratum corneum forma- et al., 2000). A more complex view ing Bcl-2, Akt, and NF-κB, as discussed
tion. The availability of an inducible of KC apoptotic control in vivo was above. Equally important, the timing of
caspase-14 knockdown or knockout revealed in experiments by Nickoloff differentiation must be controlled so
mouse would be particularly useful in and colleagues (Nickoloff et al., 2002), that apoptosis is not delayed. Is KC dif-
further discriminating the role of cas- who fractionated normal epidermal ferentiation inextricably linked to cell
pase-14 from the roles of other caspas- compartments by density gradient and death? Although forced expression of
es in epidermal differentiation and KC assessed each for expression of various ASK1 or DLK, as noted above, induces
apoptotic responses.
apoptotic components. They found that differentiation but not apoptosis, many
although some death receptors (TNF stimuli that induce KC differentiation
receptor 1, death receptor 4) and cas- in vitro do lead to apoptosis (Benassi et
pase-8 were expressed more intensely al., 1997). Cultured KCs that become
Apoptosis in Epidermal Homeostasis and
Stratum Corneum Formation
A multigenic developmental program. in lower-layer fractions, other mole- senescent, however, are resistant to
Although the rates of KC proliferation cules (death receptor 5, Decoy R2, cas- apoptosis (Qin et al., 2002). Although
and apoptosis are high in newborn skin pase-14, Bcl-xL) were more prevalent in KC differentiation and apoptosis have
and decrease with aging (Haake et al., upper-layer fractions.
been dissociated in vivo with caspase
1998), they remain matched throughout Similarly, apoptotic control is criti- inhibitors as noted above and animal
life to maintain epidermal homeostasis. cal for fetal epidermal development models as discussed below, it would
Epidermal development is coordinated in establishing the final architecture also be interesting to examine the con-
at each stage through sequential expres- of the epidermis and its appendages. verse scenario in which differentiation
sion of multiple gene programs, as typi- Polakowska et al. (1994) showed that (but not apoptosis) was blocked. The
fied by the keratins (Fuchs, 1993) and transglutaminases in fetal periderm availability of genetic mouse models
summarized in Figure 4. In the basal localize to cells with DNA fragmenta- deficient in ASK1 or DLK, and one
layer, epidermal stem cells give rise to tion that lack Bcl-2. The bone morpho- deficient in caspase-14 as suggested
transient amplifying cells that prolifer- genetic protein signaling pathways are above, would be particularly useful
ate and express keratins 5 and 14. In the important promoters of apoptosis dur- in distinguishing the relationship and
spinous and granular layers, differenti- ing skin morphogenesis, as overexpres- individual roles of differentiation and
ating KCs terminate expression of ker- sion of the bone morphogenetic protein apoptosis in epidermal development.
atins 5 and 14 and initiate expression antagonist noggin is associated with
of keratins 1 and 10. Two key signal- retardation of KC differentiation and Altered epidermal development in
ing molecules activated by the MAPK with downregulation of death recep- animal models. The importance of
cascade are expressed in the upper tors and the KC differentiation mark- apoptosis in epidermal development
epidermis and appear to trigger the dif- ers loricrin and involucrin (Sharov et and homeostasis has been substan-
ferentiation program: apoptosis signal- al., 2003). Although we have limited tiated in several genetically modi-
regulating kinase-1 (ASK1) and dual understanding of all the factors control- fied mouse models. Many apoptosis-
leucine zipper-bearing kinase (DLK). ling these gene expression programs impaired mouse strains have been con-
Introduction of activated ASK1 (Sayama and directing KC transition to succes- structed by knockout of various apop-
et al., 2001) or DLK (Robitaille et al., sive stages, these experiments and oth- totic effector and regulatory molecules.
2005) in KCs is sufficient to induce ers noted above suggest that at least Unfortunately, most of these were not
morphologic changes and expression some components of the KC apoptotic constructed as conditional knockouts or
of the differentiation markers transglu- machinery are involved in normal fetal with skin-specific gene deletions. Given
taminase-1, loricrin, and involucrin. and adult epidermal development.
the critical role of apoptosis in embry-
In the transitional layer, expression of Even though apoptotic morphol- onic development, it is not surprising
caspase-14 correlates with the distinct ogy is not apparent until the transi- that many of these animals (particu-
changes seen in KC morphology. The tional layer, it has been suggested larly those deficient in caspases) exhibit
'apoptotic gene program' — unlike that downregulation of β1 integrins prenatal or perinatal lethality that pre-
those associated with proliferation and associated with KC exit from the basal cludes assessment of skin phenotype.
differentiation — is not localized to compartment represents 'anoikis in By contrast, most mouse models based
one region but is manifest throughout vivo'and signals the initiation of apop- on KC-specific transgenic expression or
the epithelium (Figure 4). For example, tosis (Ishida-Yamamoto et al., 1999). deletion of apoptotic regulators have
basal KCs are protected from apoptosis Although it is unclear at what point KCs proven viable and amenable for study.
by expression of Bcl-2, which is absent are irreversibly committed to under-
Mice harboring a Bcl-2 transgene
from suprabasal KCs (Hockenbery et al., go apoptosis, the integration of the driven by the suprabasal keratin 1 pro-
1991), and a gradient of Bak expression apoptotic machinery into the KC devel- moter (HK1.bcl-2) display increased
increases from lower to upper layers to opmental program must be highly resistance to sunburn-cell formation
promote cell death (Krajewski et al., regulated so that apoptosis does not (Rodriguez-Villanueva et al., 1998).
1996). Similarly, expression of the p53 precede differentiation. Premature Although their skin appears clinically
inhibitor MDM2 increases from the apoptosis is prevented by expression normal, HK1.bcl-2 mice have areas of
248 Journal of Investigative Dermatology (2006), Volume 126

D Raj et al.
Epidermal apoptosis
Apoptotic
gradients
Genetic
program
decreased filaggrin staining and reten-
tion of KC nuclei in the upper layers
(Weil et al., 1999) and prevented stra-
tum corneum formation (Allombert-
Blaise et al., 2003). Thus caspase
activation appears to be required for
normal epidermal development.
Markers
Apoptosis
TUNEL,
caspase-14
Differentiation Keratins 1, 10,
ASK1, DLK,
transglutaminase-1,
loricrin, involucrin
Caspase-14,
a
keratinocyte-specific
Proliferation
Keratins 5, 14,
β1 integrins
caspase. Several groups reported
in 1998 a newly identified caspase
— designated caspase-14 — that,
unlike other known caspases that are
expressed ubiquitously, is found only
in skin (Van de Craen et al., 1998).
Caspase-14 expression is detectable
in the basal layer and dramatically
increases in the granular layer, with
both the 28-kilodalton proenzyme
and cleavage fragments of 17 and 11
kilodaltons visualized in extracts from
Figure 4. Genetic programs in epidermal development. Apoptosis maintains epidermal homeostasis,
balancing cell death in the granular layer with proliferation in the lower layers. The proliferative gene
program is characterized by a restricted pattern of keratin gene expression. The differentiation program
directs changes in expression of keratin genes, master differentiation genes (apoptosis signal-regulating
kinase-1 (ASK1), dual leucine zipper-bearing kinase (DLK)), and genes involved in stratum corneum
formation. The apoptotic gene program is manifest throughout the epithelium, maintaining gradients of
antiapoptotic and proapoptotic factors that regulate the timing of apoptosis in epidermal development.
tion and nuclear fragmentation and are from epidermis have not been defined) whole human epidermis and stratum
rapidly cleared by phagocytosis with- may explain the decrease in the num- corneum (Eckhart et al., 2000b). In
out inflammation. Apoptosis is increas- ber of TUNEL-positive cells in basal and mouse skin, a 35-kilodalton precur-
ingly defined, however, at a biochemi- spinous layers, we know that most KCs sor is cleaved into 20- and 18.5-kilo-
cal rather than a cellular level by events undergoing spontaneous cell death in dalton peptides (Lippens et al., 2000).
such as DNA fragmentation and cas- epidermis are not removed but persist Complementary experiments with
pase activation, considered hallmarks to form the structural component of the HaCaT cells and normal human KCs
of apoptosis. Failure in other systems stratum corneum. Therefore, failure to in vitro demonstrated that caspase-
to demonstrate one or more of these identify apoptotic KCs in the upper epi- 14 was only expressed and processed
molecular features in the absence of dermis may reflect a technical limitation under culture conditions mimicking
necrosis has led to the suggestion that of the TUNEL technique in the granular terminal differentiation and could not
some cells may be eliminated in vivo layer and sub-stratum corneum.
be induced by death receptor signal-
by non-apoptotic forms of cell death
(Leist and Jaattela, 2001).
Detection of caspase activation in ing or UVB treatment (Lippens et al.,
situ initially appeared to be similarly 2000). Splice variants encoding trun-
Many morphologic features of problematic, as early studies identi- cated caspase-14 molecules have
apoptosis have been observed in the fied precursor forms of most caspas- been found in both human (Eckhart et
transitional layer of the epidermis. es, but not their activated fragments. al., 2000a) and mouse (Kuechle et al.,
Transitional KCs lose their keratohyalin Subsequent experiments demonstrat- 2001) skin, but their particular func-
granules and develop condensation of ed cytochrome c release and multiple tion is unknown. N-terminal sequenc-
cytoplasm and nuclei, which contain caspase activation in reconstituted ing of caspase-14 cleavage fragments
fragmented DNA (Ishida-Yamamoto et human epidermis (Allombert-Blaise suggests that, unlike processing of
al., 1998). There has been some difficul- et al., 2003) and caspase-3 activity in other caspases, which undergo either
ty, however, in detecting specific mark- human epidermal cytosolic extracts autolytic or caspase-mediated cleav-
ers for apoptosis in the upper layers of (Weil et al., 1999), although other age at aspartate residues, processing of
the epidermis. For example, TUNEL investigators failed to detect caspase- caspase-14 occurs by a novel caspase-
staining often reveals surprisingly few 3 activation in mouse skin (Lippens et independent proteolytic mechanism
positive cells in normal skin (Haake et al., 2000). Recent studies by Nickoloff (Chien et al., 2002). The protease cal-
al., 1998). Studies in other tissues and et al. (BJ Nickoloff, B Bodner, L Sitailo, pain is co-expressed with caspase-14
tumors have found similarly low rates MF Denning, and V Chartuvedi, 2005, in the granular layer and may mediate
of spontaneous apoptosis (Staunton and J Invest Dermatol 124:A59) using its processing, as Kuechle and col-
Gaffney, 1995). One possible explana- human epidermal equivalents dem- leagues (AJ Chien, A Pirrone, and MK
tion for the rare detection of apoptotic onstrated activation of both initiator Kuechle, 2005, J Invest Dermatol 124:
cells in vivo is that they are efficiently caspase-8 and -9 and effector cas- A67) recently reported that caspase-14
removed from tissues. Although rapid pase-3 during terminal differentiation. processing can be blocked in organo-
clearance by phagocytic cells (presum- Moreover, addition of cell-permeable typic skin cultures by a calpain inhibi-
ably Langerhans cells, although cells caspase inhibitors to a developing tor. The targets of activated caspase-14
capable of removing apoptotic cells reconstituted epidermis resulted in have not been defined but are likely
www.jidonline.org 249

D Raj et al.
Epidermal apoptosis
focal epidermal hyperplasia character- mal homeostasis, controlling differenti- cells tend to affect skin phenotype
ized by increased proliferative activ- ation, and mediating UVB responses.
differently from those active in the
ity and aberrant expression of keratin
There are, however, additional suprabasal cell layers.
6. Mice deficient in Bcl-2 also have mouse models in which an altered
normal skin (Veis et al., 1993) (with apoptotic state is not associated with
Dysfunctional apoptosis in skin disease
the exception of premature hair gray- perturbation in epidermal develop- Diseases of increased keratinocyte
ing due to the importance of Bcl-2 for ment. For example, mice transgenic apoptosis. As sunburn cells represent
melanocyte survival) (Nishimura et for Bcl-2 under the control of a keratin the histologic manifestation of iso-
al., 2005), but sunburn-cell responses 14 promoter have a normal-appearing lated KC apoptosis, clinical sunburn
in these mice have not been reported. epidermis despite increased apoptotic is the classic example of generalized
Spontaneous formation of apoptotic resistance in KCs (Rossiter et al., 2001). UVB-induced apoptosis. Although
cells was seen in unirradiated mice Similarly, mice with keratin 14-driven UVB activates both death receptor and
with KC-specific deletion of Bcl-xL, transgenes expressing antiapoptotic mitochondrial apoptotic pathways in
and these mice demonstrated a further Bcl-xL (Pena et al., 1997) and Survivin KCs, several skin diseases have been
increase in susceptibility to sunburn- (Grossman et al., 2001), as well as those ascribed predominantly to excessive
cell formation after UVB exposure deficient in p53 (Grossman et al., 2001; death receptor activation, as reviewed
(Umeda et al., 2003). Mice transgenic Ziegler et al., 1994), exhibit increased previously in the Journal by Wehrli et
for the p53 inhibitor MDM2 (K14- KC resistance to UVB-induced apop- al. (2000). Death receptor signaling
MDM2) driven by the basal keratin tosis yet normal KC differentiation and is triggered in KCs by cytokines and
14 promoter exhibit a more dramatic epidermal maturation. Thus, whereas in direct contact with intraepidermal lym-
phenotype, developing scaly skin in some cases, as noted above, manipula- phocytes, via TNF and Fas receptors,
the first week of life (Ganguli et al., tion of the KC apoptotic program can respectively. These interactions likely
2000). Histologic examination of skin significantly affect KC differentiation play a key role in the pathogenesis of
in K14-MDM2 mice revealed an epi- and epidermal development, in many toxic epidermal necrolysis and cutane-
dermis with increased thickness and of these mouse models apoptotic dys- ous graft-versus-host disease. Apoptotic
KC-cell number, and the presence of regulation (as reflected by altered sun- KCs are also a feature of cutane-
nucleated cells in the upper compart- burn-cell formation) does not. Some of ous viral infections, alopecia areata,
ments. Detection of keratins 5 and 14 the variation in these effects may relate Kindler’s syndrome, incontinentia pig-
in suprabasal KCs provided further evi- to differences in the levels of transgene menti, lichen planus, and other lichen-
dence that the differentiation program expression (that is, promoter effects), as oid reactions, but the role of excessive
was also altered in K14-MDM2 mice. reflected by differences seen between apoptosis in these disorders is poorly
Mice with KC-specific deletion of the hemizygous and homozygous K14- understood and hence will not be dis-
Akt inhibitor PTEN (K5-PTEN-flox/flox) MDM2 mice (Ganguli et al., 2000). cussed further. Although apoptosis-
develop epidermal hyperplasia and Another factor potentially affected based therapies have had limited use
hyperkeratosis (Suzuki et al., 2003). by the particular promoter used is the in patients, many have been demon-
These examples appear to validate the resulting KC compartment targeted, as strated in animal models or are in clini-
role of apoptosis in maintaining epider- promoters active only in stem or basal cal development (Table 1).
Table 1. Summary of skin diseases with dysregulated apoptosis
Skin disease
Apoptosis
Mechanisms
Potential therapies
Sunburn
Increased
UVB-induced apoptosis
Cytokine secretion
—
Toxic epidermal necrolysis
Graft-versus-host disease
Psoriasis
Increased
Increased
Decreased
Upregulation of Fas ligand
TNF production
Antibody blocking
(anti-Fas, anti-TNF,
intravenous Ig1)
Fas-mediated lymphocyte-directed
killing
TNF production
Antibody blocking
(anti-Fas, anti-TNF)
Keratinocyte senescence
Increased TNF (paradoxical)
IL-15
TNF inhibitors1
Anti-IL-15 antibody
Survivin, Bcl-xL expression
Skin cancer (basal-cell and squamous-cell
carcinoma)
Decreased
p53 mutation or deletion
Decreased death receptors
Stat3 activation
Introduction of p53
TRAIL
Bortezomib
Stat3 decoy
Survivin, Bcl-2, Bcl-xL
1Currently used in these patients. Abbreviations: TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand.
250 Journal of Investigative Dermatology (2006), Volume 126

D Raj et al.
Epidermal apoptosis
In toxic epidermal necrolysis, a leukemia reactions that often accom- Survivin, multiple studies consistent-
drug-induced hypersensitivity reaction pany graft-versus-host disease (Tsukada ly demonstrated increased levels of
causes massive KC apoptosis, result- et al., 1999).
ing in widespread epidermal detach-
Bcl-xL, which is downregulated in
response to topical vitamin D3 treat-
ment. French and colleagues demon- Diseases of decreased keratinocyte ment (Fukuya et al., 2002).
strated that Fas-L, which is sequestered apoptosis. Whereas diseases asso-
Non-melanoma skin cancers dem-
by intermediate filaments in normal ciated with increased KC apoptosis onstrate multiple examples of apoptot-
KCs (Viard-Leveugle et al., 2003), is tend to be acute, those associated ic dysregulation in which proapoptotic
upregulated and expressed extracellu- with decreased apoptosis tend to be regulatory molecules are reduced or
larly in involved skin and capable of chronic. In fact, most skin diseases or antiapoptotic molecules are overex-
mediating cytotoxicity against normal cutaneous lesions characterized by pressed. Mutation or deletion of p53
Fas-expressing KCs (Viard et al., 1998). epidermal hyperplasia or hyperkerato- occurs in many skin cancers and is dis-
Soluble Fas-L may also be secreted sis likely involve decreased KC apop- cussed in detail below. Basal-cell car-
by mononuclear cells in patients with tosis. Psoriasis and skin cancer are cinomas tend to have reduced expres-
toxic epidermal necrolysis (Abe et al., the most notable examples and will sion of Bax (Tomkova et al., 1998) and
2003) and is found in lesional blister be discussed here. A common feature increased expression of Bcl-2 (Morales-
fluid (Nassif et al., 2004). Antibody of these two diseases, along with seb- Ducret et al., 1995), and Bcl-xL is over-
blocking of the Fas–Fas-L interaction orrheic and actinic keratosis, verru- expressed in SCC (Wrone-Smith et al.,
appears protective in this disease and cae, and lichen simplex chronicus, is 1999). A relatively higher apoptotic
represents the rationale for treatment expression of Survivin (Bowen et al., rate in basal-cell carcinoma (Staunton
with intravenous Ig, a therapy that has 2004). Unlike other known IAPs and and Gaffney, 1995), however, may
been used with remarkable success most apoptotic inhibitors, Survivin account for the slow growth of clini-
in adults (Prins et al., 2003) and chil- is generally not expressed in normal cal basal-cell carcinoma lesions. The
dren (Tristani-Firouzi et al., 2002). The skin. Interestingly, we found in psoria- presence of Survivin in pre-malignant
potential involvement of additional sis and lichen simplex chronicus that lesions (Bowen et al., 2004) suggests
molecules such as TNF, which is also Survivin expression is localized to the that its expression represents an early
found in lesional blister fluid (Nassif et upper third of the epidermis, whereas step in KC transformation. In SCC,
al., 2004), may explain why efficacy of keratoses, basal-cell carcinoma, and expression of Bcl-2 (Hantschmann
Ig therapy does not correlate directly squamous-cell carcinoma (SCC) reveal and Kurzl, 2000), Bcl-xL (Matsumoto
with concentration of Fas/Fas-L-block- staining in all epidermal layers (Bowen et al., 2001), and Survivin (Lo Muzio
ing antibodies (Prins et al., 2003).
et al., 2004).
et al., 2001) is associated with meta-
In graft-versus-host disease, a com-
In psoriasis, there is decreased stasis or poor prognosis. Many stud-
mon complication of allogeneic bone spontaneous KC apoptosis in lesional ies have shown that targeting these
marrow transplantation, damage to the skin (Laporte et al., 2000), which cor- molecules in KC cell lines results in
skin and other tissues is largely medi- relates with decreased levels of cas- apoptosis (Grossman et al., 1999).
ated by activated cytotoxic T lympho- pase-14 (Lippens et al., 2000). KCs in TNF-related apoptosis-inducing ligand
cytes. The term ‘satellite-cell necrosis,’ psoriatic plaques exhibit a phenotype (TRAIL) (Stander and Schwarz, 2005),
referring to degenerating KCs associ- reminiscent of that of senescent KCs, its receptor (Bachmann et al., 2001),
ated with adjacent lymphocytes, is characterized by resistance to apop- and Fas-L (Bachmann et al., 2001) are
a misnomer, as the KCs are apoptotic tosis compared with normal KCs and reduced in actinic keratosis and SCC.
rather than necrotic. These lympho- lack of p53 activation (Qin et al., 2002; Progression of SCC is also associated
cytes express Fas-L and induce KC Wrone-Smith et al., 1997). Multiple with constitutive activation of KC sur-
apoptosis through direct cell contact factors likely contribute to increased vival signaling pathways. In malignant
and secretion of cytokines, primarily resistance of KCs to apoptosis in this KCs, signaling through EGFR activates
TNF-α. Animal models have revealed disease. First, it is possible that psoriatic the transcription factor Stat3 (Quadros
that both Fas-L and TNF-α are impor- KCs are resistant toTNF signaling, given et al., 2004), which drives expression
tant in the pathogenesis of graft-ver- that TNF-α is paradoxically elevated in of multiple antiapoptotic molecules,
sus-host disease. When donor T cells psoriatic skin. For unknown reasons, including Bcl-xL and Mcl-1 (Buettner
are derived from mice deficient in Fas- maintenance of disease is dependent et al., 2002). Activation of the EGFR–
L, there is reduced mortality and skin on TNF-α, and this represents the basis Stat3 axis is associated with SCC pro-
disease (Baker et al., 1996). Although for multiple novel biologic therapies gression (Bowman et al., 2000; Rubin
antibodies againstTNF-α similarly con- targeting this cytokine. Other cytokines Grandis et al., 1998), and activation
fer protection, targeting of both Fas-L involved in psoriasis include IL-15, a of the Akt/NF-κB pathway is com-
and TNF-α is required for complete potent inhibitor of KC apoptosis in vitro monly seen in SCC (Nakayama et al.,
abrogation of graft-versus-host disease that is upregulated in psoriatic skin 2001). The role of some of these regu-
(Hattori et al., 1998). TNF-α appears to (Ruckert et al., 2000) and has proven lators and pathways in promoting skin
be more important than Fas-L, howev- to be a viable target in animal models cancer development in experimental
er, in mediating beneficial graft-versus- (Villadsen et al., 2003). In addition to models is discussed below.
www.jidonline.org 251

D Raj et al.
Epidermal apoptosis
a Normal apoptosis sensitivity
Apoptosis as a Cancer Defense
Mechanism
UVB
UVB
UVB
Apoptosis in the skin acts as a defense
against cancer at two levels. First,
apoptotic elimination of KCs in the
upper epidermis results in removal of
cells that may have acquired pre-malig-
nant genetic alterations. Second, apop-
tosis may be directly induced in KCs
exposed to carcinogenic stimuli, usual-
ly as part of the DNA damage response.
This mechanism would be particularly
important in eliminating KC stem cells
that are long-lived and not destined for
apoptotic turnover.
p53 mutation
Apoptosis
Mutant clone
Papilloma
SCC
b
Increased apoptotic resistance
UVB
UVB
UVB
p53 mutation
Apoptosis
Mutant clone
Papilloma
SCC
Role of p53. As noted above, KC apop-
totic responses in vivo are generally
dependent on p53, which is activated
and regulated in cells by multiple fac-
tors (Vogelstein and Kinzler, 1992).
Levels of p53 are potentially sup-
pressed in normal KCs by a number
of regulators, including PTEN (which
inhibits Akt), products of the p38 MAPK
pathway, and the Retinoblastoma pro-
tein; p53 is activated by ATM-medi-
ated phosphorylation, and p19ARF
protects it from degradation by inhibit-
ing MDM2. p53 delays cell cycle pro-
Figure 5. Schematic model depicting multiple roles of apoptosis in the various stages of UVB-induced
skin carcinogenesis. Under normal conditions (a), UVB causes a p53 mutation (black cell) that confers
apoptosis resistance. However, continued UVB exposure also induces apoptosis in neighboring
keratinocytes (white cells, thick arrow), facilitating expansion of the p53-mutant clone. Ultimately, large
clones develop into papillomas (thick arrow), and some papillomas that acquire additional mutations
will convert to SCC. Under conditions of increased apoptosis resistance (b), as seen in the skin of
K14-Survivin mice, creation of initial p53-mutant cells is enhanced (increased number of black cells,
thick arrow) but fewer neighboring keratinocytes undergo apoptosis; this results in the formation of
smaller but more numerous p53-mutant clones. This reduced clonal expansion leads to formation of
fewer papillomas, but the increased apoptosis resistance selects for mutations that facilitate malignant
conversion to squamous-cell carcinoma (SCC) (thick arrow).
gression (in G1 by inducing the Cdk2/ of UVB exposure. Further impairment of p53 can be detected in KCs from clini-
cyclin E inhibitor p21Cip1, and in G2 p53 and other genes through additional cally normal sun-exposed skin with a
through the Cdc25 inhibitor 14-3-3σ), UVB-induced mutations may then lead frequency of up to 1% (Nakazawa et
presumably until DNA damage can be to even greater resistance to apoptosis al., 1994), and clonal patches of p53-
repaired (Harris and Levine, 2005), or and acquisition of pre-malignant chang- mutated KCs are larger and more fre-
promotes apoptosis through transcrip- es. Consistent with this scenario, p53- quent in sun-exposed skin (Jonason et
tional and non-transcriptional mecha- deficient mice are highly susceptible to al., 1996). In mouse models, the same
nisms. Transcription of proapoptotic UVB-induced SCC (Li et al., 1998).
regulators of the mitochondrial path-
p53 mutations are present in clones and
subsequent SCC (Kramata et al., 2005).
ways (Bax, Noxa, Puma, Apaf-1) and Clonal model of squamous-cell carci- Moreover, development of these p53-
death receptor pathways (Fas, death noma. Given the proapoptotic func- mutated clones correlates with tumor
receptors 4 and 5) may be activated tion of p53 in UVB-treated KCs, it is not induction in a mouse model (Rebel et
by p53, and that of antiapoptotic mol- surprising that the p53 gene is disabled al., 2001). These findings substantiate
ecules (Bcl-2, Bcl-xL, Survivin) may by mutation in most SCCs (Brash et al., a clonal basis for SCC and suggest that
be repressed (Yu and Zhang, 2005). In 1991). Commonly, these mutations p53 mutation is an early event in UVB-
addition, p53 can translocate to the are C→T and CC→TT transitions, also induced skin carcinogenesis (Figure 5a).
mitochondria and interfere with Bcl-2 known as 'UVB signature' mutations
family proteins, resulting in cytochrome (Kress et al., 1992). Most SCCs exhibit defined role for p53-mediated apop-
c release (Chipuk et al., 2004). loss of heterozygosity with respect to tosis in suppressing the development
This clonal hypothesis suggests a
Exposure of KCs to UVB does not p53 and have point mutations on the and progression of SCC. At the earli-
always result in apoptosis but rath- remaining allele (Basset-Seguin et al., est stage, apoptosis would be predicted
er may cause mutations in the p53 1994). With respect to SCC precursors, to act selectively on normal but not on
gene that lead to apoptosis resistance. p53 mutations are found in most actinic p53-mutant cells, promoting forma-
Compromise of p53 function through keratoses (Ziegler et al., 1994). Different tion of a p53+/– clone. During continu-
UVB-induced
mutation
conceiv- p53 mutations occur in separate kerato- ous UVB exposure, there is subsequent
ably undermines the apoptosis-based ses, but all cells within a single precur- clonal expansion in which mutant cells
defense mechanism, allowing p53- sor lesion tend to have the same muta- extend beyond the borders of their origi-
mutant KCs to survive repeated cycles tion (Ziegler et al., 1994). Mutations in nal stem-cell compartment (Zhang et al.,
252 Journal of Investigative Dermatology (2006), Volume 126

D Raj et al.
Epidermal apoptosis
2001). Large clones either regress or ulti- engineered to modulate KC apoptosis, may play a more complex role in con-
mately form a papilloma. Transition from however, demonstrate unexpected phe- trolling skin tumorigenesis than previ-
papilloma to SCC (malignant conversion) notypes. Although p53-deficient mice ously expected.
involves new mutations (Yuspa, 1998), are more susceptible to UVB-induced
and thus apoptosis would be expected to tumor formation (Li et al., 1998), para- Apoptosis promotes early tumorigenic
exert a negative or protective influence doxical findings have emerged from events. It is curious that agents required
at this stage as well (Figure 5a).
studies in mice with reduced (but not for experimental induction of skin
absent) p53. Chemical carcinogenesis cancer — namely 9,10-dimethyl-1,
studies with mice on a p53+/– back- 2-benzanthracene and UVB — are also
Role of Apoptosis in Animal Models of
Skin Cancer
ground
demonstrated
increased potent apoptotic stimuli in KCs (Allen
Antitumorigenic effects of apoptosis. resistance to tumor formation com- et al., 2003). This may merely reflect
A number of animal models support pared with that in wild-type animals the need to induce mutations in p53
the notion that KC apoptosis suppress- (Greenhalgh et al., 1996; Kemp et al., and other genes but, alternatively, may
es tumor formation. It is worth noting, 1993), although the rate of malignant indicate a requirement for apoptosis in
however, that in most cases neither conversion was enhanced (Kemp et al., some early steps of cutaneous carcino-
forced expression of apoptotic resis- 1993). These results, contrasting with genesis. Perturbations of KC apoptotic
tance factors nor deletion of proapop- those in K14-MDM2 mice (Ganguli et balance might then potentially result
totic molecules is purely oncogenic al., 2000), may reflect multiple mecha- in multiple (and unpredictable) effects
(that is, sufficient to cause skin tumors). nisms of p53 regulation and targets of on carcinogenesis. Zhang et al. (2001)
In these models, a mutagenic stimulus MDM2. Consistent with these findings, examined p53-mutant clones in multi-
such as UVB or a chemical carcinogen Chaturvedi et al. (2005) reported that stage UVB carcinogenesis and found
like 9,10-dimethyl-1,2-benzanthracene RNA interference-mediated reduction that, when UVB exposure was discon-
is also needed. For example, whereas of p53 levels in KCs enhances rather tinued, clones ceased expanding and
p53-deficient animals spontaneously than inhibits UVB-induced apoptosis.
regressed; this suggested that UVB was
develop lymphoma and sarcoma, topi- Although NF-κB is a survival sig- driving clonal expansion by a physio-
cal application of 9,10-dimethyl-1,2- nal in KCs, as is noted above, and is logic mechanism other than creation of
benzanthracene or chronic UVB is activated in many tumor types, NF- additional mutations. It seemed plau-
required for development of cutaneous κB blockade in the skin, surprisingly, sible that this dependence of clonal
SCC. As is noted above, these animals promotes KC transformation and SCC expansion on sustained UVB exposure
are more prone to UVB-induced SCC formation (Dajee et al., 2003). Mice might reflect a requirement for apop-
(Li et al., 1998). Similarly, K14-MDM2 transgenic
for
IκB
demonstrate tosis in adjacent KCs, providing space
mice are more susceptible to chemical increased KC apoptosis but also sponta- to accommodate the expanding clone
carcinogenesis (Ganguli et al., 2000). neously develop SCC (van Hogerlinden (Figure 5a). Indeed, formation of iso-
Mice transgenic for Bcl-xL also dem- et al., 1999). Similarly, increased KC lated ‘imprisoned’ clones, character-
onstrate enhancement of chemical- apoptosis in mice deficient in Cox-1 is ized by increased cell number without
induced tumors as well as an increased not associated with reduction in UVB- increased area, was observed follow-
rate of malignant conversion (Pena et induced tumors (Pentland et al., 2004). ing withdrawal of UVB during clonal
al., 1998). Genetic alterations in prima- In addition, several mouse models with expansion (Zhang et al., 2001).
ry KC survival pathways also predispose increased KC apoptosis resistance are
To test this possibility, we examined
to cutaneous tumorigenesis. For exam- less susceptible to skin carcinogenesis. p53-mutant clones in apoptosis-defec-
ple, the K5-PTEN-flox/flox mice with For example, K14-Bcl-2 (Rossiter et tive K14-Survivin mice at multiple steps
constitutive Akt activation demonstrate al., 2001) and K14-Survivin (Allen et during UVB carcinogenesis (Zhang et
accelerated tumor onset with chemical al., 2003; Zhang et al., 2005) mice are al., 2005). We found that, under condi-
treatment (Suzuki et al., 2003). resistant to sunburn-cell formation but tions of reduced apoptosis, formation of
Treatment with an EGFR inhibitor yield reduced numbers of chemical- small p53-mutant clones was enhanced,
reduces UVB-induced skin tumorigen- and UVB-induced papillomas. These consistent with a role for apoptosis in
esis (El-Abaseri et al., 2005). Similarly, mice have normal skin, whereas others suppressing new mutations by remov-
mice deficient in Stat3 are resistant to with increased susceptibility to tumori- ing mutant cells. Formation of medium
chemical carcinogenesis (Chan et al., genesis have hyperkeratosis (Ganguli et and large clones, on the other hand, was
2004). The HK1.bcl-2 mice demonstrate al., 2000; Rodriguez-Villanueva et al., impaired, suggesting that inhibition of
increased susceptibility to chemical car- 1998) — although some mice with such apoptosis impairs clonal expansion; and
cinogenesis (Rodriguez-Villanueva et susceptibility, for example, Bcl-xL trans- tumor yield was reduced. Papillomas that
al., 1998). Taken together, these studies genic mice, do not have skin hyper- formed under these conditions, however,
support a role for apoptosis resistance keratosis (Pena et al., 1998). Thus, the demonstrated an increased tendency
in the development of SCC.
lack of correlation between apoptosis for malignant conversion. These results
resistance and susceptibility to tumor support a revised model, with apoptosis
Paradoxical role of apoptosis in car- formation and malignant conversion restraining stages that involve new muta-
cinogenesis. Other mouse models in these studies suggests that apoptosis tions — initiation and malignant conver-
www.jidonline.org 253

D Raj et al.
Epidermal apoptosis
CO NFLICT O F INTEREST
The authors state no conflict of interest.
Bowen AR, Hanks AN, Allen SM, Alexander
A, Diedrich MJ, Grossman (2003)
sion — but promoting clonal expansion
of existing mutant cells and develop-
ment of papillomas (Figure 5b). These
opposing roles of apoptosis in restraining
and promoting early steps in skin car-
cinogenesis may explain the divergent
phenotypes observed in the animal
models of dysregulated apoptosis des-
cribed above.
D
Apoptosis regulators and responses in human
melanocytic and keratinocytic cells. J Invest
Dermatol 120:48–55
ACKNOWLEDGMENTS
Supported by NIH grants KO8 AR48618 and
RO3 AR048953 and by the Huntsman Cancer
Foundation. We apologize to those colleagues
whose work we neglected to cite, primarily
because of limitations on space and the number
of references allowed.
Bowen AR, Hanks AN, Murphy KJ, Florell SR,
Grossman D (2004) Proliferation, apoptosis,
and survivin expression in keratinocytic
neoplasms
and
hyperplasias.
Am
J
Dermatopathol 26:177–181
Bowman T, Garcia R, Turkson J, Jove R (2000)
STATs in oncogenesis. Oncogene 19:2474–
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