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J Biol Chem, Vol. 274, Issue 33, 23358-23367, August 13, 1999
From the Departments of Skin provides an attractive organ system for
exploring coordinated regulation of keratinocyte (KC) proliferation,
differentiation, senescence, and apoptosis. Our main objective was to
determine whether various types of cell cycle arrest confer resistance
to apoptosis. We postulated that KC cell cycle and cell death programs are tightly regulated to ensure epidermal homeostasis. In this report,
simultaneous expression of cyclin-dependent kinase inhibitors (p15,
p16, p21, and p27), a marker of early differentiation (keratin 1),
mediators of apoptosis (caspases 3 and 8), and NF- Normal human skin is covered by a multi-layered epidermis in which
keratinocytes (KCs)1 undergo
a continuous process of proliferation, differentiation and apoptosis
(1, 2). In this life-long self-renewing process, quiescent stem cells
are triggered to produce transiently amplifying cells, which then give
rise to early stages of differentiation followed by terminal
differentiation and death. This process is responsible for maintaining
the proper cutaneous thickness and barrier function of stratified
epithelium (2). Such dynamic tissue homeostasis involving cells in the
basal layer and throughout suprabasilar levels requires a delicate
balance of epidermal cells entering the proliferating pool
versus cells in various stages of differentiation,
senescence, and death (3). These cellular transitions take place within
a few cell layers each month over the span of several decades.
Although the relationship between proliferation, replicative senescence
and apoptosis has been extensively explored in dermal fibroblasts, less
is known regarding these complex processes in epidermis (4-9).
Furthermore, because dermal fibroblasts do not undergo terminal
differentiation like KCs, it is unclear whether differentiation-inducing treatments influence apoptotic-related events.
To fill this experimental void, various types of skin-derived KCs were
used to explore molecular mechanisms that regulate cell proliferation,
differentiation, senescence, and apoptosis in skin (10).
Our working hypothesis extends observations by previous investigators
who established there are multiple pathways leading to senescence and
protection from apoptosis in other organ systems (4-9), and that KCs
can also follow different biochemical pathways leading to cell cycle
arrest. Furthermore, we postulated that these pathways will have
distinctive characteristics as regards subsequent cell fate decisions
such as differentiation, senescence, and
susceptibility/resistance to apoptosis. The goal of this project was to begin to elucidate the phenotype and biochemical pathways regulating KC growth arrest, differentiation, and apoptosis. We postulated that there would be biochemical links between KC
replication, senescence, and apoptosis and designed experiments to
address how various types of KCs would respond to rapidly induced
growth arrest or spontaneous replicative senescence, followed by
exposure to high levels of ultraviolet radiation (UV light) that
acutely triggers apoptosis.
Primary KC cultures proliferate for several passages in a low calcium,
serum-free medium (11). Freshly isolated, proliferating, and relatively
undifferentiated KCs can become growth-arrested and subsequently follow
at least five different pathways. First, if growth supplements are
removed, KCs maintained in basal medium will become quiescent and
remain viable for at least several days, but retain the capacity to
re-enter the cell cycle (12). Another distinct method for inducing
reversible growth inhibition of KCs is exposure to anti-proliferative
agents such as transforming growth factor The purpose of this investigation was to delineate the response of KCs
to various stimuli that can influence all five potential pathways and
to examine the interactive behavior of both cell cycle regulatory
proteins that predominantly localize to the nucleus, with members of
the caspase family that are present in the cytoplasm and regulate
apoptosis (19). Because NF- Because the orderly process of KC proliferation, differentiation, and
apoptosis occurs with a high degree of fidelity in skin, we postulated
that the regulatory mechanisms involved in maintaining homeostasis
would reveal a remarkable degree of coordination among the pathways
that regulate the cell cycle (i.e. proliferation), and
several key molecular participants involved in caspase cascades (i.e. cell death). Interestingly, just as KC differentiation
is not required for cells to undergo apoptosis (19), we observed that early stages of differentiation were not required for
growth-arrested or senescent KCs to acquire an apoptotic-resistant
phenotype. Because the c-myc proto-oncogene is a central
regulator of cell proliferation, differentiation, and apoptosis,
expression of c-Myc protein levels were also examined (28, 29).
By comparing the behavior and response of normal KCs, senescent
KCs, and immortalized KCs to antiproliferative agents followed by UV
light, new insights were gained into the complex interactions of
molecular mediators that regulate KC proliferation, growth arrest,
differentiation, senescence, and apoptosis.
Cell Culture--
Primary KCs were isolated from freshly excised
neonatal foreskins as described previously (11) HaCaT cells, an
immortalized KC cell line, were obtained from Professor N. Fusenig
(Heidelberg, Germany) (26). Both normal KCs and HaCaT cells were
maintained in a low calcium (0.15 mM) KC growth medium
(KGM, Clonetics, San Diego, CA). Cells were treated with either KGM
alone or KGM containing various treatments for 8 and 48 h. In some
experiments, KGM was replaced with a basal medium (KBM) lacking growth
supplements. Treatments to reduce cell proliferation included addition
of recombinant IFN- Antibodies--
The anti-caspase-3/CPP32 antibody was used at
1:2,000 (c31720, Transduction Laboratories, Lexington, KY), and the
anti-caspase 8 and anti-poly(ADP)ribose polymerase antibodies were
purchased from CLONTECH (Palo Alto, CA). Antibody
against Bcl-x was obtained from Craig Thompson (University of Chicago)
and used as described previously (19). Antibodies against p15
(sc-612R), p16 (sc-468R), p21 (sc-397R), and p27 (sc-528R) were
purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), as were
antibodies against the p50 (sc-7178) and p65 (sc-109) subunits of
NF- Flow Cytometric Analysis--
Flow cytometry was performed on
single cell suspensions obtained using trypsin/EDTA as described
previously (19). Briefly, for cell cycle analysis propidium iodide
staining (50 µg/ml, Sigma) was performed following the
manufacturer's instructions. Also, terminal
deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling
(TUNEL) assay positive cells were detected following fixation and
staining as described previously (19).
UV Treatment and Suspension Culture Conditions to Induce
Apoptosis--
Apoptosis was induced by irradiating KCs with a
Panelite Unit (Ultralite Enterprises, Inc., Lawrenceville, GA) equipped
with four UVB bulbs (FS36T12/UVB-VH0) that have the majority of their output in the UVB range (65%), with minor output in the UVA and UVC
range (34 and 1%, respectively). KCs were irradiated with dish lids
removed using a dose of 25 mJ/cm2. The UV dose was
monitored with an International Light Inc. (Newburyport, MA) radiometer
fitted with a UVB detector. In selected experiments, cells were
pretreated with caspase inhibitors for 30 min prior to irradiation as
described previously (25). Another method for inducing apoptosis was to
place a single cell suspension of KCs in KGM medium containing 1.68%
methylcellulose (4000 centipoises, Sigma) for 48 h as described
previously (19).
Western Blot Analysis--
Nuclear cell lysate and whole cell
lysate were prepared to detect different proteins. In brief, for
nuclear lysates cells were washed with phosphate-buffered saline,
pelleted in buffer A (20 mM Hepes, pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM
dithiothreitol with 0.1% Nonidet P-40), incubated in ice for 15 min,
and microcentrifuged, and the supernatant was discarded. The pellet was
resuspended in buffer C (20 mM Hepes, 0.4 M
NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) for 15 min on ice. Cells were vortexed and microcentrifuged, and the
supernatant was saved and frozen at
30 µg of protein were loaded on 8-12.5% SDS-polyacrylamide gel,
transferred to Immobilon-p (polyvinylidene difluoride) membrane and
blocked in 5% nonfat powdered milk in TBST (50 mM Tris, pH 7.5, 150 mM NaC1, 0.01% Tween 20). The membrane was
incubated with the primary antibody in 2.5% powdered milk in TBST and
was washed extensively with TBST and then incubated with 1:1500 diluted anti-rabbit or mouse horseradish peroxidase (Amersham Pharmacia Biotech). Proteins were visualized with ECL reagents (Amersham Pharmacia Biotech) according to manufacturer's instruction. Loading of
proteins to verify equivalent distribution of proteins in each well was
confirmed by Ponceau S staining.
RNase Protection Assay--
Total cellular RNA was extracted
using Trizol Reagent (Life Technologies, Inc.). The RNase protection
assay was performed according to the supplier's instructions
(PharMingen, San Diego, CA). Briefly, human apoptosis template set
hAP0-5 was labeled with [ Electrophoretic Mobility Shift Assays and Supershift
Assays--
Electrophoretic mobility shift assays were performed as
described previously (24). In brief, 1 µg of poly(dI-dC) (Amersham Pharmacia Biotech) and 104 cpm of 32P-labeled
double-stranded oligonucleotide were incubated with 5 µg of nuclear
protein on ice for 30 min. The reaction mixture was separated on 4%
native polyacrylamide gel, dried, and autoradiographed. The NF- Analysis of p16 Gene in HaCaT Cells--
Examination of the p16
gene in HaCaT cells was performed initially by sequence analysis of
exons 1 and 2 using an automated DNA sequencer as described previously
(32). In addition, detection of p16 promoter methylation was performed
by Southern blot analysis as described previously (33). Briefly, 10 µg of total genomic DNA was isolated from HaCaT cells, normal KCs,
and a cell line known to have p16 DNA hypermethylation (SW13). DNA was
subjected to restriction endonuclease digestion with EcoRI
alone or EcoRI and SacII or EcoRI and
EagI (Life Technologies, Inc.). After running on 1% agarose
gel, DNA was transferred to nitrocellulose membrane. This membrane was
analyzed with a genomic DNA fragment (1.1-kilobase probe) containing
the promoter and exon 1 of human p16 gene as described previously
(33).
Differential UV-induced Apoptotic Response of Normal versus
Immortalized versus Senescent KCs--
When neonatal foreskin-derived
KCs are maintained at subconfluent density in a low calcium serum-free
medium, they proliferate and are highly sensitive to induction of
apoptosis by UV irradiation. A representative cell cycle profile for
proliferating normal KCs is presented in Fig.
1A in which less than 1% of
the cells have a sub-G0 DNA content and 5% are
TUNEL-positive. Typically, proliferating KCs have 45-55% of cells in
G1, 30-40% of cells in S, and 5-15% of cells in
G2M. 18 h after UV irradiation (25 mJ/cm2), subconfluent KCs undergo apoptosis with over 55%
of the KCs having sub-G0 DNA, and 39% becoming
TUNEL-positive (Fig. 1B). However, if KCs become confluent
and then exposed to UV (Fig. 1C), substantially less
apoptosis (17% sub G0 DNA, 22% TUNEL-positive) is
induced. If confluent KCs cultures have their growth supplements removed (i.e. washed and maintained in KBM for 24 h),
the resistance to apoptosis observed for the confluent cultures is
reduced, and over 70% of KCs have sub-G0 DNA and greater
than 40% of the cells are TUNEL-positive after UV exposure (Fig.
1D).
To further assess the consequences of growth-arresting treatments,
normal KCs at 50-60% confluence were treated for 24 h with 2 mM Ca2+, TPA, IFN-
Interestingly, in cultures of normal KCs that underwent spontaneous
replicative senescence (i.e. passages 3-5), the exposure to
UV did not induce apoptosis in these subconfluent cultured cells (Fig.
1I). Pretreatment of senescent KCs with the aforementioned growth-arresting agents did not alter this resistance to UV-induced apoptosis (data not shown).
To determine whether a similar phenotypic response would occur in
immortalized KCs, HaCaT cells were treated following the same protocol
as described above for early passage normal human KCs. HaCaT cells were
also growth-arrested by exposure to elevated Ca2+, TPA, or
IFN- Characterization of Caspase Cascade Involved in UV-induced
Apoptosis--
To determine the molecular mediators involved in the
UV-induced apoptosis, Western blot analysis was performed on whole cell extracts before and after UV exposure with a focus on the caspase 8, caspase 3, and poly(ADP)ribose polymerase. UV-induced apoptosis in KCs
is caspase-dependent, because it can be blocked by caspase inhibitors (25). Because caspase 3 is a primary executioner intermediate in this apoptotic pathway, the change in caspase 3 will be
highlighted (Fig. 3), although similar
changes were also observed for caspase 8 and poly(ADP)ribose polymerase
(data not shown). Prior to UV irradiation, proliferating normal KCs and
HaCaT cells had intact caspase 3, but after UV exposure both types of
cells undergoing apoptosis had proteolysis (i.e. activation) of caspase 3.
Normal KCs in either a confluent state or after pretreatment/wash with
Ca2+, TPA, IFN-
To determine whether HaCaT cells were capable of acquiring an apoptotic
resistant phenotype, several different caspase inhibitors such as
z-VAD, DEVD, and IETD, were utilized (25). Pretreatment of HaCaT cells
with these compounds prior to UV exposure decreased the apoptotic
response (data not shown), with concomitant prevention of the cleavage
of caspase 3 (Fig. 3). Overall, the degree of protection from
UV-B-induced apoptosis and extent of caspase 3 degradation using
these inhibitors was similar between KCs and HaCaT cells (data
not shown).
Early Differentiation Marker Expression by Normal and Immortalized
KCs--
Two differentiation-related proteins were measured before and
after the pulse/wash or continuous treatments including keratin-1 (present in suprabasilar KCs undergoing early differentiation) and
keratin-14 (detectable in basal layer KCs in vivo and
relatively undifferentiated cells in culture). Over 90% of cultured
normal KCs were keratin 14-positive (expect for small foci of
stratified clusters of KCs), and HaCaT cells were diffusely and
uniformly expressed keratin-14 (Fig. 4,
upper panel). In contrast, only rare focal clusters of
keratin-1-positive normal KCs maintained in KGM were present in
proliferating KCs (Fig. 4, middle panels). After exposure to
elevated calcium, approximately 20% of KCs became keratin-1-positive,
but no consistent or extensive induction of keratin-1 was seen after
exposure to TPA, IFN- Differential Expression of Cell Cycle Regulatory Proteins of Normal
versus Immortalized versus Senescent KCs--
Proliferating normal KCs
had low but detectable intranuclear levels of p21, p15, p27, and p16
(Fig. 5). Upon reaching
confluence-induced growth arrest, increased levels of all four
intranuclear proteins were observed (Fig. 5, left panel).
When KCs were growth-arrested with elevated Ca2+, TPA,
IFN-
KCs undergoing spontaneous replicative senescence were characterized by
marked elevation in p16 with a slight increase in p27 but without
changes in p15 or p21 levels (Fig. 5, middle panel). By
contrast to normal KCs, when proliferating HaCaT cells were examined,
only scant levels of intranuclear p21 was detected, and no p16 protein
was identifiable, whereas low levels of p15 and p27 were present (Fig.
5, right panel). Confluency did not produce complete growth
arrest, and only a slight increase in p27 levels was detected. Addition
of antiproliferative agents including elevated Ca2+, TPA,
IFN-
Given the complete absence of p16 in the HaCaT cells, the gene was
examined for mutation, but no mutations in either exon 1 or 2 were
identified. Next, the methylation status of the DNA was investigated,
and there was DNA hypermethylation in the promoter sequence and first
exon of p16 as portrayed in Fig. 6.
Constitutive and Inducible Levels of NF-
In contrast to these results by Western blot analysis, HaCaT cells had
relatively high constitutive intranuclear levels of p50 and p65 (Fig.
7, upper panel, right side), which was also confirmed by functional DNA binding (Fig. 7, lower panel,
right side) and supershift gel assays (data not shown).
Another difference noted was that after exposure to either TPA or
IFN-
Because there were significant differences in the constitutive and
inducible levels of NF-
The pattern of transcripts was very different in HaCaT cells.
Consistent with the constitutive levels of intranuclear NF- Expression of Cell Survival-related Proteins and c-Myc in Normal
and Immortalized KCs--
To explore expression beyond the
transcriptional level for various cell survival genes, Western blot
analysis was also performed. For normal KCs the relative levels of
TRAF-1 and TRAF-2 at the protein level were similar to the mRNA
levels for each of the different conditions portrayed in Fig. 8 (data
not shown). Similarly, for HaCaT cells, there was no TRAF-1 detected at
the protein level, and TRAF-2 levels were present constitutively with
no significant change following exposure to the antiproliferative
agents (data not shown). Because we had previously observed that in KCs
derived from psoriatic plaques there was overexpression of
Bcl-xL correlated with enhanced survival (37, 38), the
relative levels of Bcl-xL were examined. Western blot
analysis of Bcl-xL in normal KCs and HaCaT cells revealed
detectable Bcl-XL in both types of cells, but the
susceptibility of HaCaT cells to UV-induced apoptosis was not due to
any decrease or absence of Bcl-xL relative to the proliferating cells (Fig. 9).
Furthermore, the enhanced survival of normal KCs that had become
confluent or exposed to Ca2+, TPA, IFN-
Barely detectable levels of c-Myc are present in proliferating KCs,
that are slightly increased after exposure to IFN- Influence of Pharmacological Inhibition of NF- Given the constant thickness of the epidermis throughout life, we
postulated that there would be a tight link between the regulation of
cell proliferation (i.e. cell cycle programs) and cell death
(i.e. apoptosis). The results of these studies confirm our
hypothesis for KCs by providing evidence that regulation of proliferation is critical for determination of susceptibility to
apoptosis. We demonstrate, despite multiple experimental conditions that impact the cell cycle such as spontaneous replicative senescence, confluency, or exposure to several different antiproliferative agents,
that all of these growth-arresting pathways lead to apoptosis resistance in normal KCs. By contrast, the immortalized KCs
(i.e. HaCaT cells) that have abnormal antiproliferative
responses remain susceptible to apoptosis. Although detailed molecular
exploration for each of these responses is beyond the scope of this
study, initial focus on the role for NF- Two sets of results point to an important role for NF- There are several other notable observations in this report that link
regulators of cell cycle with resistance to apoptosis. When normal
human foreskin-derived KCs are proliferating, they express low nuclear
levels of p15, p16, p21, and p27 as well as p50 and p65 subunits of
NF- There were also several differences observed in the pattern of cell
cycle regulatory proteins present in the nucleus of normal KCs cultured
under various conditions. Confluency-induced growth arrest produced the
most striking enhancement of all four CDKIs measured with accompanying
resistance to apoptosis. This result may be of particular relevance
to in vivo conditions because KCs in skin are tightly
aggregated with close cell-cell contact as simulated by the confluency
experiments. The elevation in extracellular calcium ion concentration,
while inducing growth arrest, only partially induced early markers of
differentiation (i.e. keratin-1) in a relatively small
percentage of KCs, compared with a marked increase in p21. Recently,
p21 was found to inhibit differentiation of murine KCs (40). A previous
investigation noted that TPA or elevated calcium ion levels could
up-regulate p21 in KCs (41), but the current work indicates that
induction of p21 can also be triggered by several other
growth-arresting agents including IFN- The immortalized HaCaT cells had a substantially different profile of
CDKIs compared with the normal KCs. HaCaT cells had barely detectable
nuclear levels of p15 or p21, and there was complete absence of p16.
The only consistently detectable CDKI that was constitutively expressed
was p27, which was further enhanced after exposure to the
antiproliferative agents: TPA and TGF- In this report, the first evidence documenting the silencing of the p16
gene in HaCaT cells is presented. Although there is evidence for p16
alterations in several skin-derived cancers and cell lines (48, 49),
the HaCaT cells have not been previously shown to harbor such an
alteration in which the DNA encoding sequence within exons 1 and 2 lacks a mutation, but the p16 promoter region is hypermethylated.
The most dramatic phenotypic difference between normal KCs and HaCaT
cells was revealed by their respective responses to UV light-mediated
induction of apoptosis. Previous reports using fibroblasts and myocytes
documented that CDKIs play a role in protecting cells from apoptosis
(4-9, 50), and our results using KCs also clearly demonstrate a
correlation between growth arrest, induction of CDKIs, and resistance
to apoptosis. Indirect evidence to support a link between CDKIs and
apoptosis was revealed using the HaCaT cells in that these cells fail
to induce either p16 or p21 and remain highly susceptible to apoptotic
stimuli. To explore the molecular mechanism of this anti-apoptotic
phenotype, the expression of NF- However, a role for NF- It is conceivable that the exposure of neonatal KCs to these
growth-arresting agents converts these cells to a phenotypic state that
resembles premature senescence (57). We postulate that on one hand
HaCaT cells have become immortalized by several genetic mutations that
disrupt cell cycle regulation and NF- In conclusion, these results highlight potential cross-talk between
pathways that regulate cell proliferation and cell survival (58-62).
Given the qualitative and quantitative differences in CDKI profiles
induced by various antiproliferative treatments, it appears there are
multiple pathways leading to G1 arrest in KCs. Clearly
susceptibility to apoptosis is also linked to the cell cycle activity
of KCs, and the anti-apoptotic mechanism in KCs requires properly
regulated NF- We thank Dr. N. E. Fusenig (Divison of
Carcinogenesis and Differentiation, German Cancer Center, Heidelberg,
Germany) for providing HaCaT cells.
*
This work was supported by NCI, National Institutes of
Health Grant RO-1CA49133 (to M. O. D.)The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
2
V. Chaturvedi and B. J. Nickoloff,
unpublished observations.
The abbreviations used are:
KC, keratinocyte;
CDKI, cylin-dependent kinase inhibitor;
TGF, transforming growth
factor;
IFN, interferon;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
TUNEL, terminal
deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid.
Apoptosis in Proliferating, Senescent, and Immortalized
Keratinocytes*
,,,
Pathology,
§ Radiation Oncology, and ¶ Medicine, Loyola University
Medical Center, Maywood, Illinois 60153
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B were analyzed in
three types of KCs. By comparing the response of proliferating, senescent, and immortalized KCs (HaCaT cells) to antiproliferative agents followed by UV exposure, we observed: 1) Normal KCs follow different pathways to abrupt cell cycle arrest; 2) KCs undergoing spontaneous replicative senescence or confluency predominantly express
p16; 3) Abruptly induced growth arrest, confluency, and senescence
pathways are associated with resistance to apoptosis; 4) The
death-defying phenotype of KCs does not require early differentiation; 5) NF-B is one regulator of resistance to apoptosis; and 6) HaCaT cells have undetectable p16 protein (hypermethylation of the
promoter), dysfunctional NF-B, and diminished capacity to respond to
antiproliferative treatments, and they remain highly sensitive to
apoptosis with cleavage of caspases 3 and 8. These data indicate that
KCs (but not HaCaT cells) undergoing abruptly induced cell cycle arrest or senescence become resistant to apoptosis requiring properly regulated activation of NF-B but not early differentiation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-) (13). These
initial two growth-arresting pathways can be reversed if quiescent
cells are subsequently stimulated to re-enter the cell cycle and
proliferate by addition of competence and progression factors following
withdrawal of TGF- (14). A third pathway involves growth arrest such
that no further proliferation is possible, and growth arrest does not
induce early markers of diffentiation (i.e. keratin 1), such
as after exposure to phorbol ester and/or interferon 
(IFN-) (15,
16). A fourth pathway involves irreversible growth arrest and early
differentiation, which occurs when extracellular calcium ion
concentration is increased (17). A fifth pathway for KCs is to undergo
replicative senescence, in which case they remain viable and
metabolically active but not capable of any further replicative
expansion (18).
B is a key transcription regulator in KCs
and plays a critically important role in both regulation of the cell
cycle as well as influencing cell death pathways, particular focus was
directed at this transcription factor (20-22). In addition to the
aforementioned cell cycle arresting agents, UV light was also used
because it can induce apoptosis in human skin and is regarded as an
important etiological factor in development of skin cancer (23-25).
The death defying behavior of normal skin-derived KCs was compared with
senescent KCs and immortalized HaCaT cells (26, 27).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(103 units/ml, Genentech Inc., San
Francisco, CA), 12-O-tetradecanoylphorbol-13-acetate (TPA, 5 nmol/liter; Sigma) or transforming growth factor- (10 ng/ml; Upstate
Biotechnology, Inc., Lake Placid, NY) or increasing the extracellular
calcium ion concentration to 2.0 mM. Counting of cells was
performed manually using a calibrated slide chamber hemacytometer. The
caspase inhibitor Z-VAD-FMK and IEDT were purchased from Enzyme System
Products (Livermore, CA), and DEVD was purchased from Calbiochem (La
Jolla, CA). In some experiments KCs were pre-exposed for 2 h to
the proteasome inhibitor MG132 (0.1-1 µM, BIOMOL
Research Laboratories, Inc., Plymouth Meeting, PA) to inhibit NF-B
activation as described previously (30). SW13 cells were obtained from American Type Culture Collection.
B, the antibody against c-Myc (clone 9E10), and the antibody
against heat shock protein 27 (sc-1048).
80 °C. For the whole cell
lysate, KCs were washed with phosphate-buffered saline and were
incubated in ice for 15 min in CHAPS buffer (31). Cells were
microcentrifuged, and supernatants were saved and frozen at 80 °C.
Protein concentration of each sample was determined by Lowry assay.
-32P]uridine triphosphate.
RNA (10 µg) and 8 × 105 cpm of labeled probes were
used for hybridization, and after RNase treatment, the protected probes
were resolved on a 5% sequencing.
B
oligonucleotide had the following sequence: 5'-AGT TGA GGG GAC TTT CCC
AGG C-3'. Competition analysis was performed by adding excess unlabeled
oligonucleotides. For supershift experiments, 4 µg of rabbit
polyclonal antibodies against p50, p65, RelB, and c-Rel (Santa Cruz)
subunits of NF-B were incubated with 5 µg of nuclear proteins for
30 min on ice, prior to the addition of 32P-labeled probe.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Fig. 1.
Phase contrast microscopic appearance and
flow cytometry profiles of proliferating human KCs pretreated with
various growth-arresting agents before (A) and after
(B-H) exposure to UV light as well as senescent KCs
(I). Insets depict extent of apoptosis
by either TUNEL positivity or by propidium iodide staining (% sub-G0 DNA content).
, or TGF- and then
washed and maintained in KGM for an additional 24 h. In this
scenario, there is irreversible growth arrest for all of the treatments
except TGF-. KC cultures exposed to elevated Ca2+, TPA,
IFN-, or TGF- had a reduction in proliferation assessed by manual
cell counting of viable cells revealing substantial reductions compared
with untreated cells in KGM of 79 ± 8, 92 ± 8, 93 ± 6, and 83 ± 6%, respectively. After the pulse/wash treatments, the dishes were then exposed to UV light (25 mJ/cm2), and
the KCs were examined as before. KCs pulsed/washed with Ca2+ (Fig. 1E) were highly resistant to
UV-induced apoptosis (10% sub G0; 14% TUNEL-positive), as
were KCs pulsed/washed after exposure to TPA (Fig. 1F) or
IFN- (Fig. 1G). However, TGF--treated KCs were not as
consistently protected as revealed by 45% sub-G0 DNA content and over 32% TUNEL-positive cells (Fig. 1H).
Resistance to apoptosis for these treatments was not unique to UV
light treatment, because similar results were observed when different
KC cultures treated as above with identical pulse/wash protocols were
trypsinized followed by 48 h of suspension in methylcellulose
(data not shown). When the results using 24 h pulse/wash were
compared with 48 h of continuous exposure similar levels of
protection from UV-induced apoptosis were observed, with the exception
that continuous growth arrest produced by 48 h of treatment with
TGF- enhanced the resistance to apoptosis (29% sub
G0; 23% TUNEL-positive) compared with the pulse/wash protocol.
as determined by manual cell counting, although with an overall
diminished antiproliferative response. Compared with untreated HaCaT
cells, cultures (n = 3) exposed for 72 h to
elevated Ca2+, TPA, IFN-, or TGF- had reductions in
cell proliferation of 38 ± 5, 31 ± 85, 68 ± 8, and
54 ± 11%, respectively. In marked contrast to growth-arrested
normal KCs, none of the treatments (i.e. either pulse/wash
or continuous) reduced the extent of apoptosis present in HaCaT cells
after UV exposure (Fig. 2). To better
view the HaCaT cultures, only cell cycle DNA profiles are presented (although similar trends in TUNEL assays were also identified; data not
shown). Briefly, subconfluent HaCat cells had only approximately 3% of
the cells undergoing spontaneous apoptosis (sub-G0 DNA: Fig. 2A), whereas after UV exposure subconfluent (Fig.
2B) or confluent cultures (Fig. 2C) or cells
placed in KBM (Fig. 2D) had markedly increased numbers of
cells with sub-G0 DNA (48-58%). Pulse/wash treatments
using Ca2+, TPA, IFN-, or TGF- prior to UV exposure
provided no protective effects (Fig. 2, E-H, respectively).
Also, 48 h of continuous exposure to these antiproliferative
treatments did not change the ability of UV light to induce apoptosis
in the HaCaT cells (data not shown).
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Fig. 2.
Phase contrast microscopic appearance and
flow cytometry profiles of immortalized human HaCaT cells pretreated
with various antiproliferative agents before (A) and after
(B-H) exposure to UV light. Similar to Fig. 1,
propidium iodide staining revealed percentage of apoptotic cells
reflected by sub-G0 DNA content (insets).
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Fig. 3.
Western blot detection of caspase 3 before
and after UV light in normal KCs (left panel) and
HaCaT cells (right panel). Note that apoptosis
was characterized by cleavage of caspase 3. KCs prior to UV exposure
have detectable caspase 3 (first lane), but after UV light
(second lane) no intact caspase 3 was present, and the
indicated antiproliferative treatments rendered antiproliferative KCs
less susceptible to UV-induced caspase 3 cleavage. In contrast to KCs,
HaCaT cells had all caspase 3 cleaved after UV exposure; even with
pretreatment with antiproliferative agents. Only the inhibitors (DEVD,
ZVAD, and IETD) blocked caspase 3 cleavage in HaCaT cells.
, or TGF- had less evidence of caspase
3 cleavage upon subsequent UV exposure, which correlated with
diminished induction of apoptosis. HaCaT cells treated in a similar
fashion had no or barely detectable intact caspase 3 levels, consistent with the induction of apoptosis under these conditions induced by UV.
, or TGF- (Fig. 4, middle
panels). Furthermore, normal KCs undergoing spontaneous replicative senescence were devoid of detectable keratin-1 expression except for only rare, scattered cells (data not shown). HaCaT cells
either before or after exposure to the treatments failed to express any
keratin-1 except for rare isolated cells (Fig. 4, lower
panels). Thus, the induction of early differentiation markers such
as keratin-1 was not a prerequisite for the anti-apoptotic phenotype of normal KCs exposed to either elevated calcium, TPA, IFN-, or TGF- or senescent KCs.
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Fig. 4.
Expression of keratin 14 (undifferentiated NN
KCs in upper panels) or keratin 1 (early
differentiation marker) in normal human KCs (middle
panels, NN cells) or immortalized HaCaT cells (lower
panels) before and after exposure to elevated calcium ion
concentration, TPA, IFN- , or
TGF-. Note diffuse cytoplasmic expression
of keratin-14 in both proliferating normal (NN) KCs (except
for central spontaneously differentiating enlarged KC) and all HaCaT
cells. While raising extracellular calcium ion levels triggered
clustered KCs to enhance keratin-1 expression, the other
antiproliferative agents had minimal to no effect on either KC or HaCaT
keratin 1 expression.
, or TGF-, there was a G1 arrest (data not shown) that was maintained after UV irradiation (Fig. 1). The growth arrest
mediated by elevated Ca2+ or exposure to IFN- involved
elevated intranuclear levels of p21 and p27 without changes in p15 or
p16 levels (Fig. 5, left panels). TPA-mediated growth arrest
was primarily associated with induction of p21, whereas TGF- induced
predominantly p15 and p27. There was no significant or consistent
change in the cytoplasmic levels of heat shock protein 27 expression
under any of these conditions involving either KCs or HaCaT cells.
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Fig. 5.
Composite profile of nuclear cell cycle
regulatory proteins before and 8 h after exposure to indicated
anti-proliferative agents involving normal KCs (left
side), normal KCs undergoing spontaneous growth
arrest-replicative senescence (middle), and HaCaT
cells (right side). Heat shock protein 27 levels
in the cytoplasm were also analyzed from cells grown under identical
conditions. Note the relatively low constitutive levels of the CDKIs
and the differential expression depending on the antiproliferative
agent and type of cell analyzed.
, or TGF- did not produce enhanced nuclear levels of p21 or
detection of p16. Some of these antiproliferative treatments did induce
higher levels of either p15 or p27.
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Fig. 6.
Genomic Southern blot to analyze the p16
promoter region reveals that HaCaT cells, but not normal KCs, p16 is
hypermethylated. The left three lanes reveal HaCaT
cells, the middle three lanes are normal KCs, and
right three lanes are SW13 cells. For each cell type, the
DNA was probed after digestion with restriction enzymes (first
lane, EcoRI alone), after EcoRI plus
SacII digestion (second lane), or after
EcoRI plus EagI (third lane). Note
that although DNA digestion had no effect on the HaCaT cells or SW13
cells indicating p16 promotor methylation, characteristic hybridization
fragments (700 and 919 base pairs (bp)) were detected for
KCs.
B in Normal KCs and
HaCaT Cells--
Because transcriptional activation by NF-B can
induce several cell survival genes (21, 22), the levels of the two
subunits (i.e. p50 and p65), as well as DNA binding capacity
were examined in this system. By Western analysis, proliferating KCs
had relatively low but consistently detectable intranuclear p50 and p65
components of NF-B (Fig. 7,
upper panel, left side). After exposure to TPA or
IFN-, there was rapid induction (within 30-60 min) and enhancement of both intranuclear levels. The functional activity of the nuclear p50/p65 was confirmed by DNA binding assays (Fig. 7, lower
panel, left side) and supershift gel analysis in which
p50 and p56 subunits were identified but not RelB or c-Rel (data not
shown). However, addition of 2 mM calcium did not produce
detectable increase in either p50 or p65 by Western blot or gel shift
assays (data not shown).
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Fig. 7.
Constitutive and inducible nuclear levels of
NF- B subunits in KCs and HaCaT cells.
Upper panels, Western blot analysis for p50 and p65 subunits
after exposure to IFN- (103 units/ml) for indicated time
points (30 min to 8 h). Lower panels, kinetic analysis
of DNA binding by p50 and p65 subunits before and after IFN- or TPA
treatment. Note that although there is induction of levels for both
p50/p65 heterodimers as well as p50 homodimers in KCs after either
IFN- or TPA exposure, the high constitutive levels of p50 and p65
subunits do not change in HaCaT cells using the same treatment
protocol.
, unlike normal KCs in which this treatment triggered nuclear
translocation of p50/p65, no change in the relative levels of either of
these subunits was observed in HaCaT cells by Western blot analysis
(Fig. 7, upper panel, right side). The lack of
further enhancement for p50 and p65 levels in stimulated HaCaT cells
was confirmed by DNA binding (Fig. 7, lower panel,
right side) and supershift gel assays (data not shown).
B in normal KCs and HaCaT cells, we sought to
determine whether there would be differences in the expression of
anti-apoptotic transcripts triggered via NF-B activation (34-36).
Because HaCaT cells did not resist apoptosis and did not activate
NF-B, our hypothesis was that HaCaT cells would differ from normal
KCs by not up-regulating apoptotic-resistant transcripts, as one
mechanism to explain the response to UV light. To explore the
transcriptional patterns of normal KCs and HaCaT cells with respect to
cell survival pathways, RNase protection assays were performed. Fig.
8 reveals a panel of mRNA transcripts
for numerous apoptosis-regulating genes. In proliferating normal
KCs, several anti-apoptotic transcripts were present including XIAP,
TRAF-2, and cIAP-1 mRNAs (34-36). However, after normal KCs are
exposed to elevated calcium ion levels, TPA, IFN-, or TGF-,
several of these transcripts become more abundant including TRAF-1 and cIAP-2 mRNAs. By scanning laser densitometry, normal KCs had a 18-fold induction of TRAF-1 mRNA by TPA, a 1.8-fold increase by Ca2+, a 1.7-fold increase by IFN-, and a 2.0-fold
increase byTGF-. There was a 3.1-fold increase in cIAP-2 mRNA by
Ca2+, as well as a 23.7-fold increase by TPA, a 2.6-fold
increase by IFN-, and a 1.8-fold increase by TGF-. The
constitutive presence of L32 and glycereldehyde-3-phosphate
dehydrodgenase served as loading controls. The other abbreviations
represent human testosterone-repressed prostate message-2 (TRPM2), CRAF
for TRAF3, and CART for TRAF4 as described previously (36).
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Fig. 8.
RNase protection assays for normal KCs
(left panel) and HaCaT cells (right
panel) before and 6 h after exposure to indicated
antiproliferative agents including elevated Ca2+ ion, TPA,
IFN- , or TGF-.
The specific mRNA transcripts detected are labeled with
arrows on the left side of each panel. Note the
differences in the constitutive and inducible level of transcripts
encoding several anti-apoptotic protein for KCs, but the lack of any
changes in transcriptional levels in the HaCaT cells. The nomenclature
is from the suppliers' kit. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
B, many
of the transcripts were also constitutively present such as XIAP,
TRAF-2, cIAP-1, and cIAP-2 mRNAs. However, even after exposure to
elevated calcium ion concentrations or TPA, IFN-, or TGF-, no
induction of any of the cell survival gene products in this assay was
identified. The lack of such induction correlated with the lack of
induction of NF-B and the highly sensitive phenotype of both
proliferating and growth-arrested HaCaT cells to UVB-induced apoptosis.
, or TGF-
could not be correlated to the relative levels of Bcl-xL
(Fig. 10).
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Fig. 9.
Bcl-xL levels (upper
panels) and c-Myc levels (lower panels) in
normal human KCs and HaCaT cells before and 48 h after exposure to
the indicated antiproliferative agents. Note that except for
slight induction of Bcl-XL by IFN- in KCs, minimal
enhancement in BCL-XL levels, was observed for the other
conditions in either KCs or HaCaT cells. c-Myc protein levels were
either undetectable or barely detectable in normal KCs before and after
IFN- exposure, but c-Myc was constitutively more prominent in HaCaT
cells and further enhanced by IFN- treatment.
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Fig. 10.
Influence of pharmacological inhibition of
NF- B function on UV-induced apoptosis in KCs
using the proteasome blocker MG132. Panel A reflects the
Western blot analysis of levels of p50 and p65 subunits of NF-B in
nuclear extracts of KCs before and after IFN- exposure (6 h) in the
absence and presence of MG132 (1 µM). Note the
preincubation for 2 h with MG132 prior to addition of IFN-
reduced total nuclear levels of p50 and p65. Panel B
portrays the phase microscopic appearance of KCs before (upper
panels) and after UV exposure (lower panels) in KCs
treated either with medium alone (untreated; first set of panels),
MG132 alone (1 µM; second set of panels), IFN- alone
(103 units/ml; third set of panels), or MG132 (1 µM-2 h) followed by IFN- (24 h). Note the minimal
effects of MG132 by itself on the KCs, but the ability of pretreatment
with MG132 to abrogate the anti-apoptotic phenotype of IFN- treated
KCs after UV exposure. Panel C represents the percentage of
KCs undergoing apoptosis as assessed by propidium iodide staining and
flow cytometry with sub-G0 DNA quantitation. The
bars on the left represent the results in the
absence of UV light exposure for IFN- (103 units/ml)
and/or MG132 (either 0.1 µM or 1 µM) in
which KCs were treated for 48 h. The bars on the
right represent data from two to three experiments that were
normalized so that the percentage of UV-induced apoptosis of untreated
proliferating KCs was 100%, and then the effects of pretreatment with
IFN- (103 U/ml; 24 h) or MG132 (either 0.1 µM or 1 µM; 2 h) followed by IFN-
(24 h) and subsequent UV irradiation were examined. Note that as seen
in panel B, pretreatment of KCs for 2 h with MG132 (1 µM) partially negated the anti-apoptotic response of KCs
to IFN-. Error bars indicate standard deviation.
(Fig. 9). By
contrast, HaCaT cells have constitutively high levels of c-Myc, which
were only minimally changed in response to IFN- (Fig. 9).
B Activation on
UV-induced Apoptosis--
To determine whether the death-defying
phenotype of IFN- treated KCs was related to NF-B activation, KCs
were pretreated with the proteasome inhibitor (MG132), and the
subsequent phenotype of the KCs was examined before and after IFN-
treatment followed by UV irradiation. Fig. 10A reveals that
at 1 µM (but not 0.1 µM), MG132
preincubated with KCs for 2 h blocked induction of NF-B activation by IFN-. IFN- itself, MG132 at either 0.1 or 1 µM, and the combination of IFN- plus 1 µM MG132 did not induce significant apoptosis (<5%)
over a 48-h incubation period (Fig. 10, B and C). Next, MG132 was added 2 h before IFN- (103
units/ml), and after 24 h the KCs were irradiated with UV light. By blocking IFN--induced NF-B activation with 1 µM
MG132, the anti-apoptotic phenotype of the KCs was reduced. Thus,
although IFN- pretreatment could reduce the extent of apoptosis
(sub-G0 DNA) by 24 ± 8%, preincubation with MG132 (1 µM) prior to IFN-, led to an increase in the extent of
apoptosis in these treated KCs to 52 ± 8% when subsequently
analyzed 24 h hours after exposure to UV light.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B in regulating KC apoptosis was pursued.
B activation
in regulating KC apoptosis. First, although normal KCs responded to
several antiproliferative agents such as IFN- or TPA by activating
NF-B (and several key anti-apoptotic transcripts), no such
induction was observed in HaCaT cells, which were unable to acquire a
resistance to apoptosis compared with normal KCs. Secondly, when this
activation of NF-B was blocked by MG132, the resistance to apoptosis
by normal KCs was reduced. As discussed later, it is clear that there
might be other pathways beyond that regulated by NF-B, and we are
currently investigating several other mechanistic leads to more fully
explain the basis for resistance to apoptosis induced by other conditions.
B, and these replicating cells are highly susceptible to
apoptosis, with rapid activation of the caspase cascade involving
caspases 8 and 3 after UV light exposure. When neonatal KCs, which have
tremendous replicative potential in vivo, are
growth-arrested in vitro by exposure to anti-proliferative agents such as elevated calcium ion levels, IFN-, TPA or TGF- or
by allowing the cells to become confluent or following spontaneous replicative senescence, the normal KCs acquire a resistance to apoptosis. In contrast, immortalized HaCaT cells responded quite differently to the antiproliferative agents with less complete growth
arrest and at the same time remained highly susceptible to UV-induced
apoptosis. Besides the well documented inactivation of both p53 alleles
in HaCaTs (39), this apoptosis-related investigation uncovered several
other previously unknown abnormalities in HaCaT cells, such as DNA
hypermethylation involving the p16 promoter region of the gene
resulting in no detectable protein, constitutively elevated levels of
p50/p65 subunits of NF-B, a failure to undergo further elevation of
NF-B levels, lack of induction of several transcripts associated
with cell survival proteins, constitutively elevated c-Myc, and no
resistance to apoptosis under the conditions examined.
as well as TGF-. Moreover,
because the other antiproliferative treatments also strongly induced
p21, it is possible that the failure of TPA, IFN-, or TGF- to
enhance keratin-1 expression was related in part to the induction of
p21. Our results are in agreement with an earlier report that
up-regulation of these CDKIs was not sufficient by itself to induce
differentiation of KCs (42). The reversible growth arrest induced by
TGF- led to enhanced levels of p15 (43), whereas spontaneous
replicative senescence was associated most prominently with an increase
in p16 (18). These differences in induction of specific CDKIs in
response to various antiproliferative agents point to multiple pathways
by which normal KCs undergo growth arrest.
(44). Given the relatively
high constitutive levels of c-Myc in HaCaT cells, we postulate that
more significant growth arrest was not induced by these
antiproliferative agents in HaCaT cells, relative to normal KCs with
lower levels of c-Myc, because of the previous reported ability of
c-Myc to abrogate growth arrest mediated by p27 (28, 29). Given the
complex role c-Myc plays in regulating apoptosis (45-47), more work is
required to determine the significance of c-Myc overexpression in KCs.
B and several cell survival genes
and proteins were studied. In many cell types including KCs, NF-B
transcriptional activity mediates enhancement of cell survival gene
products (20, 21). Cell survival genes of interest in this study
included Bcl-xL because transgenic mice overexpressing
Bcl-xL yield epidermal KCs with resistance to apoptosis
(51), as well as IAP genes (34, 35), and TRAF1/TRAF2 (36). As regards
NF-B, dramatic differences were noted between normal KCs and HaCaT
cells. Using several different assays, it was clearly established that
HaCaT cells have a dysregulated NF-B pathway in which there is
extremely high constitutive intranuclear levels of p50 and p65.
Although such abnormalities have been seen in other cell lines, this is the first report documenting such a dysregulated state in HaCaT cells
(52, 53). RNase protection assays confirmed the electrophoretic mobility shift assay results, in that HaCaT cells did not respond to
either TPA or IFN-, as did normal KCs, by enhanced NF-B nuclear translocation and up-regulation of several cell survival mRNA transcripts and proteins. We postulate that proliferating normal KCs
were susceptible to apoptosis in part because they did not produce
either TRAF-1 or c-IAP-1. It was recently demonstrated that expression
of TRAF-1 with c-IAP-1 substantially increased the anti-apoptotic
response in a different human cell system (36). The precise mechanism
by which the regulatory events described herein mediate the resistance
to apoptosis remains largely unknown. Because elevated Ca2+
ion levels do not trigger NF-B activation, it is possible that the
Ca2+ mediated anti-apoptotic mechanism is significantly
different from the mechanism involving exposure to either IFN- or
TPA, which do activate NF-B. Furthermore, the exact contribution of the various NF-B-inducible gene products such as XIAP, c-IAP-1, c-IAP-2, TRAF-1, and TRAF-2 are currently under investigation.
B in the anti-apoptotic phenotype of IFN-
treated KCs was demonstrated using a pharmacological approach with the
proteasome inhibitor MG132. By inhibiting the activation of NF-B,
IFN- treated KCs become more susceptible to UV-induced apoptosis in
agreement with an earlier report focusing on IL-1 and KC survival (30).
Possible mechanisms involving cell cycle regulators such as p21 and the
caspase cascade as seen in other cell types (54, 55) were found not to
be operative in this system involving
KCs.2 There was also no
modulation of heat shock protein 27 to suggest a role for this protein
in the anti-apoptotic phenotype of KCs (56).
B activation in a way that also
prevents them from undergoing replicative senescence. This resistance
to induction of senescence by the HaCaT cells is accompanied by an
inability of these cells to acquire an anti-apoptotic phenotype. The
resistance to apoptosis in this system could not be attributed solely
to levels of Bcl-XL because HaCaT cells contained similar
levels of Bcl-XL compared with normal KCs but remained
highly sensitive to UV-induced apoptosis.
B activity. Once a KC becomes growth-arrested, it
may or may not begin to express early differentiation markers, but
resistance to apoptosis does not require induction of
differentiation. Future studies are required to better understand the
molecular complexities associated with regulation of KC
proliferation, differentiation, senescence, and apoptosis.
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Pathology, Skin Cancer Research Laboratories, Loyola University Medical Center, Cardinal Bernardin Cancer Center, 2160 S. First Ave., Maywood,
IL 60153. Tel.: 708-327-3241; Fax: 708-327-3239; E-mail: Bnickol@luc.edu.
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