ARTICLE
Heat shock proteins (HSP) of the HSP 70 family are called "chaperones"
because they protect the cell from environmental stress factors like acute
UV irradiation or hyperthermia. In contrast to HSP 72 [1], the role of
the small heat shock protein HSP 27 remains to be elucidated. Cellular
localization, structural organization and the state of phosphorylation
of HSP 27 is changed by environmental stress as heat, oxidative stress,
chemotherapeutics, heavy metals, cytokines and others [2, 3]. Cells transfected
with genes overexpressing HSP 27 showed increased resistance against hyperthermia
[4]. Moreover, HSP 27 is looked upon as a new marker of differentiation
and proliferation in keratinocytes [5-7]. Accordingly our group and others
found the protein in stratum spinosum and granulosum in normal human epidermis
[8, 9]. The biological role of this protein especially in carcinogenesis
has been controversially discussed. On the one hand, the absence of HSP
27 in the upper epidermal layers is a marker of malignancy, as squamous
cell carcinoma showed a loss of HSP 27 in oncogenically transformed cells
[8, 10]. On the other hand, HSP 27 was expressed proportionally to the
grade of aggressivity in human breast carcinoma and proportionally to
the resistance against chemotherapeutics [11-13].
Basal cell carcinoma (BCC) is the most frequent non melanoma skin cancer
in humans and is thought to be induced by chronic UV exposure [14]. In
order to detect potential differences in the biological behavior of BCC
in a high and low risk UV exposure group, we compared the age of patients
with BCC [15] and in a second step the immunohistochemical expression
of HSP 27, HSP 72 and bcl-2, a protein that protects against apoptosis
[16], in those two populations.
Materials and methods
Biopsies of primary (untreated) BCC, size from 1 to 3 cm in vivo,
were taken from German descendents in the rural population in the mountains
of Brazil, called Pommeranos (n = 15 BCC, n = 4 controls) and from Germans
of Baden-Württemberg (n = 14 BCC, n = 4 controls), both groups without
acute erythema. Both groups were compared for age distribution, size and
type of BCC with Wilcoxon's rank sum test and Fisher's exact test respectively.
All biopsies were derived from the facial area under local anesthesia
with mepivacain. The specimens were precooled in liquid nitrogen and snap
frozen or stored in buffered formalin for transport and then cryo-conserved
at minus 70° C or processed for paraffin sections.
The biopsies were classified according to Lever [17] in the H&E
staining. The cryostat and paraffin sections were labeled for HSP 27,
HSP 72 and bcl-2 (Table I)
with immunoperoxidase in a three step immunofluorescence [18], the paraffin
sections by the microwave method [19] and cryostat sections by APAAP staining
[18]. Second antibodies were used according to the staining procedures,
Vectastain Elite ABC-Peroxidase-kit (Camon, Germany), peroxidase-conjugated
rabbit anti-mouse IgM (DAKO, Denmark), APAAP dual system and biotin-conjugated
rabbit anti-mouse IgM (Amersham), streptavidin Texas Red (Amersham) for
the three step immunofluorescence. Controls for the labelling were performed
by replacing the primary antibody with phosphate-buffered saline or a
non relevant antibody. Chi-Squared and Fisher's exact test were used to
study for differences in immunohistochemical labelling.
Results
All fifteen Pommeranos were agricultural laborers while the fourteen
inhabitants from Baden-Württemberg in this study had indoor jobs.
Both patient groups showed skin type I-II according to the classification
of Fitzpatrick. The age range was similar in both groups with an average
age of 60,67 ± 15,11 years in Pommeranos and 67,88 ± 13,22 years
in Germans (Wilkoxon rank sum test, p > 0.05). BCC were classified
as solid (Pommeranos n = 7, Germans n = 8), six as keratotic (Pommeranos
n = 3, Germans n = 3), four as adenoid (Pommeranos n = 1, Germans n =
3) and four as fibrotic (Pommeranos n = 2, Germans n = 2). Size and type
of BCC were similar between both groups (Fisher's exact test, p > 0.05).
In normal human skin (controls) HSP 27 was increasingly expressed from
basal cell layers up to the stratum granulosum, spinosum and corneum,
in a patchy distribution. In keratotic BCC HSP 27 was expressed most prominently
in keratinocytes near to horn cysts (Fig.
1). In adenoid (Fig. 1),
solid (Fig. 2A) and fibrotic
(Fig. 3) BCC, HSP 27 could
be found in the tumour. In all histopathological types, HSP 27 was expressed
in the epidermis overlying the tumour. The antibody against HSP 72 is
recommended for use in cryostat sections, but proved to work in paraffin
sections, when pretreated with the microwave method. In normal skin, HSP
72 was in the basal cell layer and only in some suprabasal cells. In the
low cases a positive staining for HSP 72 was found (Fig.
2B), the number of labelled cells was lower than those stained
for HSP 27. Bcl-2 stained a few basal cells in normal skin. HSP 27 (Fig.
1, 2A, 3) and bcl-2 (not shown) were expressed in BCC through
all histopathological subtypes (Table
II). No statistical differences in labelling between Pommeranos
and inhabitants of Baden-Württemberg could be evaluated by immunohistochemistry
with these antiodies used (Chi-Square and Fisher's exact test, p >
0.05).
Discussion
Differences in age and body site distribution of the histological subtypes
of BCC are known. More than 50% of BCC occur in the face and neck [20].
In our study, no statistically significant differences in age between
the two groups could be found. Focusing on high and low UV exposure groups
we had decided to study BCC of all subtypes but all occuring in the facial
area.
HSP 27 is expressed according to differentiation and is found especially
in stratum spinosum and granulosum in normal skin or in hyperproliferating
epidermis in suprabasal cell layers in cryostat and paraffin sections
[5-9]. Therefore, in keratotic BCC, HSP 27 is increasingly expressed around
horn cysts, that are fully keratinized and represent "attempts at hair
shaft formation". In solid BCC, that are classified as "showing no differentiation"
[17], nevertheless, HSP 27 was labelled in central and peripheral cell
layers of the tumour and in the stroma. Adenoid BCC are positive for HSP
27 in the glandular-like structure forming cells surrounding the typical
lumina and interestingly, also fibrotic BCC show the protein in the elongated
strands of closely packed tumour cells. Our results are in contrast to
the study of Trautinger [8], who found HSP 27 only in BCC that show differentiation
(keratotic, adenoid) in paraffin sections with the same antibody used
in our study, but with a less sensitive method. We recommend staining
with the microwave method for HSP 27 in paraffin sections [9, 19].
Labelling for HSP 27 provides no help in the
differentiation between high and low UV exposure groups. The high rate
of BCC specimens positive for HSP 27 in both groups in our study, argues
for a role of HSP 27 not only in the differentiation of the normal epidermis,
but also in differentiation and proliferation of tumours. The role of
HSP 27 in carcinogenesis is controversial in different tissues. In humans,
in mamma carcinoma, in pituitary adenomas and in astrocytomas the expression
of HSP 27 stands respectively for tumour growth biological aggression
and tumor grading [11-13, 21, 22). In contrast, in Ehrlich aszites fluid,
high levels of HSP 27 reduce tumour growth [23]. Additionally, oncogenicity
in adenovirus transformed cells is also inversely correlated to HSP 27
expression [24]. As BCC is a low malignant tumour and HSP 27 is expressed
in all histopathological types of this tumour, the protein might reveal
protection against rapid aggressive malignant growth. Nevertheless, in
squamous cell carcinoma which is a UV induced epithelial tumour of the
epidermis with the potency to aggressive metastasizing, HSP 27 was negative
[10].
In contrast to HSP 27, HSP 72 was rarely expressed in BCC, which might
be explained by p53 that regulates the human HSP 70 promotor [25] and
induces apoptosis [26, 27]. In melanoma cells and colorectal cancer cells
in vivo HSP 72 enables tumoral cells to escape immunological surveillance
[28, 29]. In contrast to this, in primary human melanoma, HSP 72 was found
in 25% of patients, especially in patients who had significantly improved
survival rates [30]. Additionally, in human Ewing's sarcoma and osteosarcoma
cells, HSP 72 is expressed on the cell surface leading to an increased
susceptibility to lysis by CD3-natural killer cells [31]. The expression
of bcl-2, that antagonizes apoptosis, was found in benign naevi, BCC and
melanoma, but not in squamous cell carcinoma [24]. In this study, bcl-2
and HSP 27 were positive in BCC. It remains to be elucidated if there
is also modulation and interaction between p53, bcl-2 and HSP 27 in BCC.
The two populations studied, both skin type I or II [33], differ in
environmental factors such as extensive UV exposure in the rural agricultural
work in Pommeranos, descendants from Germans who imigrated 150 years ago,
and the indoor work in the inhabitants of Baden-Württemberg enrolled
in this study. However, in our samples no significant differences in the
age of patients with BCC and in the protein expression overall for HSP
27, HSP 72 and bcl-2 were found between the UV high risk group in Brazil
and the UV low risk group in Baden-Württemberg.
The immunohistochemical expression of the "stress triggered" HSP 27
and HSP 72 and of bcl-2 in BCC from two populations, different in their
environmental UV exposure, did not differ for the antibodies studied.
Bcl-2 positivity (in 93% of specimens) characterizes prolonged cell survival
in BCC, because bcl-2 blocks apoptosis. HSP 27 was expressed in all types
of BCC (> 79% of the specimens) and marks differentiation and proliferation
not only in normal human skin as previously known but also in BCC, implying
that HSP 27 and bcl-2 might characterize the progressive but slow-developing
malignant process in BCC.
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