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Altered Expression of bcl-2 Family Member Proteins
in Nonmelanoma Skin Cancer
Maryse Delehedde, Ph.D.
Song H. Cho, B.S.
Mona Sarkiss, M.D., Ph.D.
Shawn Brisbay, B.S.
Michael Davies, B.S.
Adel K. El-Naggar, M.D., Ph.D.
Timothy J. McDonnell, M.D., Ph.D.
Department of Molecular Pathology, The University
of Texas, M. D. Anderson Cancer Center, Houston,
BACKGROUND. Differentiation, proliferation, and cell death are coordinated tightly
within the epidermis. Alterations within keratinocytes that disrupt these processes
are believed to contribute to the development of nonmelanoma skin cancers
(NMSC). In the current study the authors examined the expression of selected
members of the bcl-2 gene family in the skin and in case-matched samples of
METHODS. Immunohistochemistry was performed on tissue sections using antibodies against bcl-2, bcl-x, bax, and bak. Case-matched frozen nonneoplastic skin
samples and tumor tissues were used for Western blot analysis.
RESULTS. In normal epidermis, bcl-2 oncoprotein is expressed in keratinocytes of
the basal layer but is down-regulated in suprabasal layers. The proapoptotic bax
protein is expressed at low levels in basal keratinocytes and is up-regulated in
suprabasal layers. The bcl-x and bak proteins both are expressed in the basal and
spinous strata but are down-regulated in the granular cell layer. Both bcl-2 and bax
were diffusely cytosolic whereas bcl-x and bak exhibited a distinct perinuclear
distribution. Squamous cell carcinomas (SCC) were negative for bcl-2 whereas
bcl-2 increased 5.5-fold in basal cell carcinomas (BCC). The distribution of bcl-x
and bax proteins within BCC and SCC overlapped and were associated with
squamous differentiation. Bax protein was increased twofold to threefold in NMSC.
An increase in bak protein also was observed in SCC. However, bak was diffusely
cytosolic within BCC in contrast to the perinuclear distribution in nonneoplastic
CONCLUSIONS. These findings suggest that altered expression of bcl-2 family members may play a role in the pathogenesis of NMSC. Cancer 1999;85:1514 –22.
© 1999 American Cancer Society.
KEYWORDS: bcl-2, skin cancer, apoptosis, differentiation.
Supported by Grant NCI P01 CA68233.
Dr. Maryse Delehedde is supported by a postdoctoral fellowship from the Fondation pour la Recherche Médicale.
Song Cho is supported by a Cancer Biology Training Grant NIH T32 CA60440.
Address for reprints: Timothy J. McDonnell, M.D.,
Ph.D., Department of Molecular Pathology, Box 89,
The University of Texas M. D. Anderson Cancer
Center, 1515 Holcombe Blvd., Houston, TX 77030.
Received May 11, 1998; revision received December 2, 1998; accepted December 2, 1998.
© 1999 American Cancer Society
balance between cell proliferation and cell death is necessary for
the normal development and maintenance of cellular homeostasis in the epidermis. Considerable evidence indicates that the development and progression of several types of cancer result from the
disruption of normal cell death and proliferation.1 In fact, molecular
alterations in genes known to regulate these two fundamental processes occur frequently in nonmelanoma skin cancer (NMSC). In this
context, activation of the ras gene family2,3 and inactivation of the p53
tumor suppressor gene4,5 are known to contribute to the pathogenesis
of NMSC.
We and others previously have reported that the antiapoptotic
bcl-2 protein is expressed at high levels in basal cell carcinomas
(BCCs) but is not expressed at detectable levels in squamous cell
carcinomas (SCCs) of the skin.6-10 The contribution of bcl-2 expression to multistep skin carcinogenesis recently was established in a
bcl-2 Family Member Proteins in Skin Cancer/Delehedde et al.
transgenic model that utilized the human keratin 1
promoter to target expression of a human bcl-2 transgene specifically to the epidermis.11 The HK1.bcl-2
transgenic mice had a reduced rate of apoptosis in
response to ultraviolet (UV) irradiation and were more
susceptible to tumor formation in response to UV and
chemical carcinogens.
Bcl-2 is now recognized to be a member of an
expanding multigene family of cell death regulators.12
Members of the bcl-2 gene family share significant
sequence homology and can be divided functionally
into two categories: the antiapoptotic members (bcl-2,
bcl-xL, mcl-1, and bcl-w) and the proapoptotic members (bax, bak, bad, bcl-xS, bid, and hrk). Current
evidence suggests that the differential expression of
antiapoptotic and proapoptotic bcl-2 family proteins
is an important determinant of the susceptibility of a
cell to undergo apoptosis.13 The potential contribution of these bcl-2 family members to the pathogenesis of NMSC essentially is unknown.
In this report, we examined the expression of selected members of the bcl-2 gene family in the normal
skin and in NMSC using immunohistochemical and
Western blot analysis.
Tumor Samples
Normal and corresponding NMSC specimens were
obtained from the pathology files of the Department
of Pathology at the University of Texas, M. D. Anderson Cancer Center in Houston. This material was
comprised of 17 cases of BCC, 4 cases of basosquamous carcinoma, and 14 cases of SCC varying from
well differentiated to poorly differentiated. Tissues
were excised prospectively after frozen-section evaluation to include tumor and adjacent normal skin in
certain blocks. When available, separate normal skin
and tumor samples corresponding to the tissue blocks
were snap frozen in liquid nitrogen and stored at -80
°C until used. The histopathologic diagnosis was established on routine sections stained with hematoxylin and eosin.
Immunohistochemical staining was performed on formalin fixed, paraffin embedded tissue samples. Serial
sections (5 mm) were deparaffinized in xylene and
rehydrated in descending concentrations of alcohols,
then placed in phosphate-buffered saline (PBS) for 5
minutes. Each specimen was evaluated by two pathologists (T.J.M. and A.K.E.N.).
bcl-2 Staining
After rehydration, sections were incubated with the
primary antibody, a mouse antihuman bcl-2 antibody
(Clone 124; Dako Co., Carpinteria, CA), at a dilution of
1:80 for 1 hour in a moist chamber. After washing in
Tris-buffered saline (TBS), the sections were incubated with rabbit antimouse antibody diluted 1:25 in
TBS for 30 minutes at room temperature. The slides
were rinsed in TBS and then incubated with alkaline
phosphatase antialkaline phosphatase (APAAP) at a
dilution of 1:50 for 20 minutes. To increase the intensity of the staining, the antimouse antibody and
APAAP steps were repeated for 3 cycles at 10 minutes
of incubation each. An alkaline phosphatase substrate,
Fuchsin (Dako Co.) was added and incubated for 10
minutes at room temperature in the dark and then
rinsed in water. Slides finally were counterstained with
Mayer hematoxylin (Sigma Chemical Co., St Louis,
MO) and mounted. Infiltrating lymphocytes served as
internal positive controls. Negative controls were
comprised of consecutive tissue sections of each case
in which the primary antibody was omitted.
bax, bcl-x, and bak Staining
To block nonspecific binding, the slides first were
covered in PBS containing 30% methanol and 0.3% of
hydrogen peroxide for 10 minutes. The anti-bax N-19,
anti-bcl-x S-18, and anti-bak N-20 antibodies were
obtained from Santa Cruz, CA. For bax and bcl-x staining, the sections then were incubated with 1% normal
goat serum in PBS for 20 minutes. After washing in
PBS, these slides were incubated for 1 hour at room
temperature with a 1:100 dilution of primary antibody
in PBS containing 1% normal goat serum. The slides
were rinsed with PBS and covered with a 1:100 dilution of peroxidase-conjugated goat antirabbit immunoglobulin (Ig) G antibody in PBS containing 1% goat
serum for 45 minutes at room temperature. Finally,
after an additional 30 minutes with Vectastain ABC
horseradish peroxidase (Vector Laboratories, Burlingame, CA), the staining was revealed with the chromogen diaminobenzidine and counterstained with
The same procedure was used for bak protein
staining except that the normal goat serum was replaced by 1% normal rabbit serum, the working dilution of the primary antibody was 1:50, and the secondary antibody was a rabbit antigoat IgG coupled
with horseradish peroxidase.
Proliferating Cell Nuclear Antigen Staining
Endogenous peroxidases first were quenched with
0.3% hydrogen peroxide in methanol for 10 minutes
CANCER April 1, 1999 / Volume 85 / Number 7
FIGURE 1. Distribution of bcl-2 family
members in normal epidermis. (A) In the
nonneoplastic epidermis, bcl-2 expression was confined strictly to basal keratinocytes. The suprabasal layers were
uniformly bcl-2 negative. (B) bcl-x staining was diffusely cytosolic within the
basal cells, but more intense, punctate,
and perinuclear in the keratinocytes of
the spinous layer. (C) The proapoptotic
bax protein was expressed at low levels
in basal keratinocytes and was up-regulated in the spinous and granular cell
layers. (D) bak staining was homogenous and mostly diffuse in the cytoplasm of the keratinocytes in the basal
layer, but exhibited a perinuclear and
punctate pattern in the differentiated
cells of the spinous layer (3 400).
and washed in PBS. Nonspecific binding was blocked
using 1% normal goat serum in PBS for 30 minutes.
The sections then were incubated with the monoclonal antiproliferating cell nuclear antigen (PCNA) antibody (PC 10; Dako Co.), diluted at 1:100 in PBS containing 0.5% Tween 20 and 0.5% bovine serum
albumin for 60 minutes.14 Negative controls were incubated with PBS without antibody. After washing in
PBS, the slides were incubated with a biotinylated
antimouse IgG secondary antibody diluted 1:100 with
1% goat serum in PBS for 30 minutes at room temperature. After an additional 30 minutes with Vectastain
ABC horseradish peroxidase (Vector Laboratories), the
substrate was added for the appropriate time period
(range, 5-15 minutes). This resulted in PCNA positive
cells being labeled brown. Slides were counterstained
with hematoxylin and mounted.
Gel Electrophoresis and Immunoblotting
Case-matched, frozen, nonneoplastic skin samples and
tumor tissues from ten patients were available for Western blot analysis. Tumor samples were comprised of at
least 75% neoplastic tissue as verified by frozen-section
evaluation at the time of acquisition. Aliquots of the total
crude protein extract were electrophoresed on 12.5%
sodium dodecyl sulfate-polyacrylamide gels. After transfer of the proteins onto nitrocellulose membranes, filters
were blocked overnight at 4 °C in blotting solution (PBS,
5% Carnation nonfat dry milk [Carnation,], and 0.05%
Tween 20). Incubation with the primary antibody (antihuman bcl-2 6C8, anti-bax N-19, anti-bcl-x S-18, and
anti-bak N-20 [all from Santa-Cruz, CA]) diluted at 1:500
was performed for 1 hour at room temperature in the
blotting solution. After 5 washes with 0.05% Tween 20 in
PBS, the filters were incubated with peroxidase-labeled
anti-IgG antibodies (goat antihamster, goat antirabbit,
goat antirabbit, and rabbit antigoat, respectively) diluted
1:1000 in the blotting solution. After several washes with
0.05% Tween 20 in PBS, immunoreactive proteins were
detected with enhanced chemiluminescence-Western
chemiluminescence detection on Hyperfilm (Amersham
Life Science, Arlington Heights, IL).
In the normal epidermis, bcl-2 expression was confined
to basal keratinocytes that exhibited cytoplasmic staining with perinuclear enhancement. The suprabasal keratinocytes uniformly were bcl-2 negative (Fig. 1A).
The distribution of bcl-x protein in non-neoplastic
epidermis varied from a high level of expression in
basal keratinocytes and that progressively was downregulated in the stratum spinosum and stratum granulosum (Fig. 1B). Bcl-x was diffusely cytosolic within
the basal keratinocytes, but distinctly punctate and
mostly perinuclear in keratinocytes of the spinous
layer. Keratinocytes of the granular cell layer showed
low or undetectable bcl-x immunoreactivity and the
cornified layer was bcl-x negative.
The localization of the proapoptotic bax protein in
the normal epidermis showed essentially no overlap
with the distribution of bcl-2 protein (Fig. 1C). Basal
keratinocytes rarely exhibited bax immunoreactivity.
The bax protein was diffusely cytosolic in keratino-
bcl-2 Family Member Proteins in Skin Cancer/Delehedde et al.
FIGURE 2. Distribution of bcl-2 family
members in basal cell carcinoma (BCC).
(A) bcl-2 protein was expressed strongly
in basal cell carcinomas Frequently, the
bcl-2 staining exhibited by the BCC was
increased compared with adjacent nonneoplastic basal keratinocytes. (B) bcl-x
and (C) bax staining appeared diffusely
cytosolic in BCC. (D) bak staining appeared diffusely cytosolic in marked
contrast to the punctate perinuclear pattern exhibited by the majority of nonneoplastic keratinocytes (3200).
cytes of the spinous and granular cell layers. Bax protein was undetectable in the cornified layer.
The distribution of the proapoptotic bak (bcl-2
homologous antagonist/killer) protein in the normal
epidermis is shown in Figure 1D. Bak protein was
present in keratinocytes from the basal layer to the
granular layer. Bak protein was homogenous and diffusely cytosolic in the keratinocytes of the basal layer,
but mostly was perinuclear in the differentiated cells
of the spinous layer. Keratinocytes of the granular cell
layer rarely exhibited bak immunoreactivity. Bak protein was not observed in the cornified layer.
Bcl-2 protein was expressed at high levels in all
BCCs examined in this study (Fig. 2A). The level of
bcl-2 protein present in the BCC frequently was more
intense than that observed in basal keratinocytes in
adjacent nonneoplastic epidermis. In contrast, neoplastic keratinocytes within BCCs expressed relatively
low levels of bcl-x protein. Bcl-x staining appeared
weak and diffusely cytosolic in the neoplastic cells
(Fig. 2B). Bax staining was heterogeneous in individual
cases of BCC and the subcellular staining pattern
mostly was cytosolic (Fig. 2C). Comparison of adjacent
tissue sections strongly suggests that expression of
bcl-x and Bax virtually is completely overlapping in
BCC. Bak staining within malignant keratinocytes in
BCC was diffusely cytosolic, which is in marked contrast to the punctate perinuclear pattern exhibited by
suprabasal epidermal keratinocytes (Fig. 2D).
SCC showed no immunohistochemically detectable bcl-2 protein (Fig. 3A). In these cases infiltrating
lymphocytes present in the dermis served as internal
positive controls for bcl-2 immunoreactivity. The dis-
tribution of bcl-x protein in SCC in general correlated
with areas exhibiting histologic evidence of squamous
differentiation (Fig. 3B). Moreover, in the poorly differentiated SCCs, we also observed enhanced immunoreactivity for bcl-x protein in individual tumor cells
that exhibited cytologic evidence of keratinization. bax
protein was expressed at relatively high levels in SCC
compared with nonneoplastic epidermis (Fig. 3C). The
distribution of bax protein within individual SCCs
overlapped with bcl-x in areas of squamous differentiation. Similar to bax, the distribution of bak protein
correlated with histologic evidence of squamous differentiation in SCC (Fig. 3D). In contrast to nonneoplastic epidermal keratinocytes, bak protein appeared
to be diffusely cytosolic, not perinuclear, in the majority of neoplastic cells of SCC and basosquamous
In those cases of basosquamous carcinoma exhibiting foci of both well differentiated SCC and BCC,
bcl-2 protein was limited to the basal cell component
of the tumor (Fig. 4A). Although the expression of bcl-x
protein also was found in the basaloid cells, it was
expressed more strongly in foci of squamous differentiation (Fig. 4B). As noted in BCC, the bax staining
pattern in the basal part of the tumor was heterogeneous. However, bax immunoreactivity was relatively
higher in the foci of squamous differentiation (Fig.
4C). The bak staining was increased in areas of squamous differentiation in basosquamous carcinoma
(Fig. 4D). In general, the staining intensity of bcl-x,
bax, and bak proteins was increased with the squamous differentiation in malignant keratinocytes of
both SCC and basosquamous carcinomas.
CANCER April 1, 1999 / Volume 85 / Number 7
FIGURE 3. Distribution of bcl-2 family
members in squamous cell carcinoma
(SCC). (A) All SCCs exhibited undetectable levels of bcl-2. Lymphocytes served
as internal positive controls. (B) The distribution of bcl-x protein correlated with
areas of squamous differentiation. bcl-x
protein was characteristically perinuclear and punctate in cells exhibiting
squamous differentiation. As shown in
panel C, bax was expressed uniformly in
SCC samples; the staining generally was
homogenous and diffusely cytosolic. (D)
The distribution of bak protein within
malignant keratinocytes was associated
with the squamous differentiation of the
tumors (3200).
Distribution of bcl-2 family
members in basosquamous carcinoma. (A)
In basosquamous carcinoma, bcl-2 protein
was detected within the neoplastic cells of
the basaloid component. (B) In contrast, the
bcl-x protein mainly was associated with
areas of squamous differentiation. Bcl-x
staining primarily was punctate and perinuclear in the neoplastic cells surrounding keratin pearls. (C) The level of bax protein appeared enhanced in areas of squamous
differentiation. (D) The distribution of the bak
protein was very diffuse in the cytoplasm of
the cells in basal part of the tumor, but
exhibited a characteristic pattern of perinuclear and punctate staining in areas of keratinization (3200).
The stromal components of the dermis and within
NMSCs exhibited negligible or undetectable levels of
the bcl-2 family proteins examined in this study with
the exception of infiltrating lymphocytes. The immunohistochemical observations for nonneoplastic epidermis and NMSC are summarized in Table 1.
Immunohistochemical evaluation of PCNA revealed that the majority of proliferating cells, as anticipated, were localized to the basal stratum in nonneoplastic epidermis (Fig. 5A). A loss of PCNA positivity
was associated with areas of keratinization and squamous differentiation in both SCC (Fig. 5B) and basosquamous carcinomas (Fig. 5C). In contrast, in BCC,
PCNA positive cells in general were scattered uniformly throughout the neoplasm (Fig. 5D). Tumor
cells exhibiting cytologic features consistent with apoptotic cell death (such as cell shrinkage, loss of junctional continuity, chromatin condensation, and formation of apoptotic bodies) generally comprised # 1%
of BCCs. Apoptotic cells present within SCC typically
were associated with areas exhibiting histologic evidence of squamous differentiation and keratinization
(data not shown).
Immunohistochemical analysis of the expression
of selected bcl-2 family member proteins in NMSC
was confirmed and extended using Western blot analysis. Total protein extracts were derived from casematched, adjacent nonneoplastic skin and NMSC
specimens (Figs. 6A and 6B). The level of bcl-2 protein
was increased approximately 5.5-fold in BCC when
bcl-2 Family Member Proteins in Skin Cancer/Delehedde et al.
Summary of Immunostaining Results for Nonneoplastic Epidermis
and NMSC
Nonneoplastic epidermis
Basal layer
Suprabasal layer
Basal cell carcinoma
(17 cases)
Squamous cell carcinoma
(14 cases)
Basosquamous carcinoma
Basal component
Squamous component
(4 cases)
NMSC: nonmelanoma skin cancer.
Staining intensity was normalized to infiltrating lymphocytes. Data are representative of all tissue
samples examined.
normalized to matched nonneoplastic epidermis (P ,
0.05). The level of bcl-2 protein in SCC was not demonstrably different from normal epidermis. Immunoblotting of nonneoplastic epidermis as well as
NMSC samples indicated that the bcl-x protein demonstrable by immunohistochemical techniques was
likely the antiapoptotic, 31-kilodalton (kD) bcl-xL form
of the protein because the proapoptotic, 19-kD bcl-xS
form of the bcl-x protein was undetectable in all nonneoplastic epidermis and NMSC samples examined.
However, levels of bcl-xS expression below the sensitivity of the immunoblot procedure cannot be excluded completely. The total amount of bcl-xL protein
in tumor samples was not significantly different from
the nonneoplastic epidermis. A 2-fold and a 3-fold
increase in the level of bax protein was observed in
BCC and SCC, respectively, compared with nonneoplastic epidermis (P , 0.05). A 2-fold increase in the
amount of bak protein was observed in SCC (P , 0.05).
However, no significant modulation of bak protein
was observed in BCC compared with nonneoplastic
The control of the individual processes of cell death,
cell division, and cell differentiation is inherently complex. Furthermore, the molecular regulation of each of
these processes must necessarily be integrated, temporally and cytoarchitecturally, within self-renewing
complex epithelia such as the epidermis. In this regard, an initially descriptive assessment of potentially
critical regulatory molecules within the epidermis may
provide insight into the mechanisms by which these
processes are regulated.
In this study, the expression and distribution of
specific apoptosis-regulating members of the bcl-2
gene family were assessed in the epidermis and in
NMSC using immunohistochemical and immunoblotting techniques. The keratinocytes of the basal layer of
the epidermis expressed high levels of the bcl-2 and
bcl-x proteins. Immunoblotting of nonneoplastic epidermis using anti-bcl-x antibodies demonstrated the
presence of the antiapoptotic, 31-kD bcl-x long form
of the protein. The proapoptotic, 19-kD bcl-x short
form of the protein either was not expressed or was
below the level of sensitivity of the immunoblot. Although to our knowledge antibodies that discriminate
bcl-xL from bcl-xS using immunohistochemistry currently are unavailable, our findings are consistent with
the interpretation that the predominant, if not exclusive, form of bcl-x expressed in nonneoplastic epidermis is bcl-xL.
To our knowledge immunohistochemical detection of bcl-x protein in basal keratinocytes has not
been demonstrated previously.15,16 The basis of these
discrepant findings potentially may be attributed to
tissue source (surgical specimens vs. autopsy), fixation
techniques (frozen-section, formalin, or Buin fixative),
or variations in the polyclonal anti-bcl-x antibodies
used in these studies. The inability to detect bcl-xS in
epidermal keratinocytes using Western blot analysis is
consistent with previous observations.16 In contrast to
the antiapoptotic bcl-2 and bcl-xL proteins, levels of
the proapoptotic bax and bak proteins are comparatively modest in basal keratinocytes. This may be related to the self-renewal capacity of these cells, which
would necessitate relative resistance to stress-induced
cell death. However, the expression and the cytoarchitectural distribution of the numerous other bcl-2 family members and other cell death regulatory proteins
within the epidermis largely is unknown.
Suprabasal keratinocytes preferentially expressed
the proapoptotic bax and bak proteins. This commitment to terminal differentiation is associated with the
immediate down-regulation of bcl-2 in suprabasal
keratinocytes and the loss of bcl-x expression in keratinocytes of the stratum granulosum. The relative
distribution of the proapoptotic and antiapoptotic
members of the bcl-2 family supports the contention
that terminal differentiation in the epidermis represents a specialized and tightly controlled physiologic
form of apoptosis.17-19
It was recently reported that bak protein is able to
interact preferentially with bcl-xL relative to bcl-2.20 It
is interesting to note that the distribution of bcl-x and
bak proteins within the epidermis is completely overlapping. A correlation between the expression of the
bcl-2-related proteins and keratinocyte differentiation
CANCER April 1, 1999 / Volume 85 / Number 7
FIGURE 5. Proliferative activity in nonneoplastic epidermis and nonmelanoma
skin cancer. (A) In nonneoplastic epidermis, proliferating cell nuclear antigen
(PCNA) was expressed predominantly in
basal keratinocytes as anticipated. (B) In
squamous cell carcinoma, PCNA positive
cells were not found in areas of keratinization (3200). (C) In basal cell carcinoma the PCNA staining was heterogeneous and a loss of PCNA positivity was
observed in the cells involved in the foci
of squamous differentiation (3200). (D)
In basosquamous carcinoma, only few
PCNA positive cells were found throughout the tumor (3400).
Immunoblot analysis of
bcl-2 family proteins in normal epidermis and in nonmelanoma skin cancer
(NMSC). (A) A representative Western
blot analysis of total protein extracts for
bcl-2 family proteins is presented. (B)
The results of scanning densitometry after normalization of expression to actin
loading and levels of expression in nonneoplastic epidermis show that the relative amount of bcl-x protein was not
substantially different from the nonneoplastic epidermis. In contrast, the relative amount of bax protein was modified
strongly in NMSC. Compared with bax
expression in normal tissue, a threefold
and twofold increase, respectively, in
the amount of the bax protein was observed in squamous cell carcinoma
(SCC) and in basosquamous carcinoma
(BCC). Moreover, a 5.5-fold increase in
the relative amount of bcl-2 protein was
observed in BCC. Finally, a twofold increase in the relative amount of bak
protein was observed in SCC but not
BCC. These findings confirm the relative
expression levels observed with immunohistochemical techniques.
bcl-2 Family Member Proteins in Skin Cancer/Delehedde et al.
in vitro recently has been reported.19 These authors
showed that the terminal differentiation of gingival
keratinocytes, induced by high calcium concentrations, is accompanied by a decrease in bcl-x and increase in bax. Together these findings indicate that
cell death regulatory proteins of the bcl-2 family are
expressed differentially within the epidermis and suggest that they may contribute to the normal cellular
growth, differentiation, and homeostasis.
It may be anticipated that altered expression of
these proteins could contribute to the pathogenesis of
NMSC. Our findings and those of others provide consistent evidence that BCCs express high levels of bcl-2
protein.6-9 In this report we demonstrated a fivefold
increase of bcl-2 protein in BCC compared with nonneoplastic epidermis. A corresponding increase in
bcl-2 protein was not observed in SCC. A comparatively modest, but significant, increase in the level of
bax protein was observed in SCC and BCC. It is interesting to note that an approximately twofold increase
in the level of bak protein was observed in SCC but not
BCC. The level of bcl-xL protein was not altered substantially in either SCC or BCC compared with nonneoplastic epidermis. The process of apoptosis is believed to be regulated by the ratio of antiapoptotic
proteins and proapoptotic proteins present in the
cell.13 This rheostat model may even apply to the role
bcl-2 family member proteins play in the process of
keratinocyte differentiation and their contribution to
skin carcinogenesis.
Direct evidence for the contribution of bcl-2 family proteins to multistep skin carcinogenesis recently
has been provided. Targeting of bcl-2 expression specifically to the epidermis was accomplished using a
human keratin 1 promoter construct.11 The bcl-2
transgenic protein was expressed transmurally in the
epidermis and resulted in focal areas of hyperplasia.
Keratinocytes from the bcl-2 transgenic mice were
resistant to apoptosis induction by UV irradiation and
chemical carcinogens. In addition, HK1.bcl-2 transgenic mice exhibited a shorter latency and higher frequency of tumor formation compared with control
littermates. A similar transgenic model used the human keratin 14 promoter to target expression of bcl-xL
specifically to the epidermis.21 The HK14.bcl-xL epidermis was resistant to cell death induction by etoposide and UV irradiation.
Moreover, the observed increase in the proapoptotic members of the bcl-2 family within NMSCs was
unexpected because recent evidence suggests that
these proteins may act as tumor suppressors. A reduction in the level of bax-a in breast carcinoma relative
to normal breast tissue has been observed.22 In metastatic breast carcinoma, reduced bax expression may
be predictive of a poor response to chemotherapy.23 In
addition frequent frame shift mutations of bax were
found in the mutator phenotype of colon adenocarcinomas, suggesting that bax inactivation may contribute to colorectal carcinogenesis.24 Direct evidence of
bax tumor suppressor activity has been provided from
experiments using genetically engineered strains of
mice.25 To our knowledge somatic mutations involving the bax gene product have not yet been reported in
NMSC nor have previously documented examples of
bax mutations been associated with an increase in the
steady-state levels of bax protein. Alternatively, the
elevated levels of death effector proteins observed in
SCC suggest that a cell death suppressor of the bcl-2
family other than bcl-2 or bcl-xL may be involved in
these neoplasms. Candidate proteins would include
MCL-1,26 A1,26 bcl-w,27 bfl-1,28 or an as yet unidentified antiapoptotic bcl-2 family member.
It is interesting to note that regions exhibiting
histologic evidence of squamous differentiation or keratinization in SCC and basosquamous carcinomas
showed immunohistochemical evidence of a reduction in bcl-2 protein and enhanced expression of
bcl-x, bax, and bak. bax protein generally was diffusely
cytosolic whereas bcl-x and bak showed a distinctly
punctate and perinuclear distribution. The significance of the subcellular distribution of these proteins
with respect to their ability to function in the regulation of cell death is an active area of investigation. In
this regard, recent observations suggest that the subcellular localization of bcl-2 may alter apoptosis sensitivity.29,30 In addition, the significance of the correlation between the modulation of these proteins with
loss of PCNA positivity, apoptosis induction, and acquisition of differentiated cytologic features remains
to be elucidated.
The results of the current study indicate that the
modulation of bcl-2 family member proteins is coordinated with differentiation in nonneoplastic epidermal keratinocytes and in NMSC. Ongoing studies will
determine whether variations in these proteins correlate with rates of apoptosis and clinical response to
nonsurgical therapeutic interventions. Studies also are
currently ongoing to evaluate directly whether inactivating mutations of death effector proteins contribute
to multistep skin carcinogenesis in vivo.
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