Expression of the bcl-2 homologue bax in normal human skin, psoriasis vulgaris and non-melanoma skin cancers

ARTICLE

Apoptosis plays a major part in cell death during development and normal tissue turnover, and its modulation is also important in the genesis of tumours. Bcl-2 and its homologous proteins have emerged as one of the most important regulators of programmed cell death, playing a crucial role in the balance between cell survival and cell death.

Members of the bcl-2 family interact with each other by homo- and heterodimerization [1-4]. Although the precise mechanism of action of bcl-2 has yet to be fully clarified, one of the proposed functional models suggests that bcl-2 counteracts the apoptosis-inducing effect of bax (bcl-2 associated X-protein) [5, 6]. However, bcl-2/bax heterodimerization alone does not seem to be sufficient and bcl-2 phosphorylation is required for bcl-2 to exert its cell death suppressor activity [7]. Moreover, there is a recent evidence of the existence of heterodimerization-independent functions for both bax and bcl-2 proteins [8]. Bcl-2 and bcl-x_L, another cell-death suppressing factor, promote cell survival partly by blocking the release of cytochrome c from mitochondria, thus preventing activation of the caspase protease cascade and execution of programmed cell death [reviewed in 9]. Bcl-2 can also suppress cell death by inhibiting nuclear import of wild-type p53 following DNA damage [10].

Bax, a 21 kDa protein with approximately 21% sequence homology with bcl-2 [5, 11], has recently been reported to be present predominantly in the cytosol redistributing from its soluble to mitochondrial membrane-bound form in cells undergoing apoptosis [12, 13].

Bax has also been shown to mediate differentiation and apoptosis in cultured keratinocytes [14]. Besides skin, bax was found to be widely distributed in murine tissues, including gastrointestinal epithelia, hepatocytes, exocrine pancreas, respiratory tract, genitourinary tract, breast, several neuronal populations, cardiac muscle and vascular smooth muscle cells, as well as hematolymphoid cells, for example in thymic medulla and lymphocytes of lymph nodes [11]. Expression of bax was reduced in pre-neoplastic lesions and squamous cell carcinoma of the esophagus [15], and reduced bax expression was associated with poor response to chemotherapy and shorter survival in women with metastatic breast cancer [16].

In this study, we examined the presence and the distribution of the pro-apoptotic protein bax in normal human skin and several skin diseases and compared it with that of the apoptosis-suppressing bcl-2 oncoprotein.

Materials and methods

Immunohistochemical staining

Samples of normal human skin were obtained from 5 plastic surgery patients. Five biopsy specimens of psoriasis vulgaris, 5 keratoacanthomas, 15 squamous cell carcinomas and 17 basal cell carcinomas were selected from paraffin blocks from our department.

The paraffin sections were deparaffinized and rehydrated in xylene and graded ethanol series. To optimize the detection of bax, the sections on slides were boiled in 10 mM citrate buffer (pH 6) in a microwave oven for 5 minutes. After inactivation of endogenous peroxidase activity with 0.3% hydrogen peroxide in phosphate-buffered saline (PBS), the slides were preincubated with 1% bovine serum albumin/10% skim milk and then incubated with monoclonal mouse anti-human bax antibody (bax-alpha, clone 4F11, MBL, Nagoya, Japan) at a dilution of 1:200 at 4š C overnight. The reaction was developed with a standard technique using a 1:350 dilution of goat anti-mouse IgG (Immunotech, Marseilles, France) and streptavidin conjugated with horseradish peroxidase (Nichirei Inc., Tokyo, Japan). The visualization of the reaction with diaminobenzidine was followed by counterstaining with methylgreen. Negative controls were obtained by omitting the primary antibody.

Immunoblot analysis

Tissues from normal and psoriatic skin, squamous cell carcinoma and basal cell carcinoma were homogenized in extraction buffer (0.05 M Tris-HCl, pH 7.5, 2 mM phenylmethylsulphonyl fluoride, 1 mM ethylenediamine tetra-acetic acid, 10 µg/ml of pepstatin A, antipain, leupeptin, and chymostatin) supplemented with 1.5% sodium dodecyl sulphate (SDS), sonicated and centrifuged. After protein assay, tissue extracts were mixed with 1/3 volume of 4 x sample buffer (1 M sucrose, 8% SDS, 0.25 M Tris-HCl buffer, pH 6.8, 0.01% bromphenol blue), heated in the presence of 2-mercaptoethanol (5% of total volume) for 5 minutes at 95š C and stored at ­ 20š C. Aliquots containing 10 µg of total protein were size fractioned by SDS-polyacrylamide gel electrophoresis (12.5% gel) and transferred to a PVDF membrane (Immobilon™, Millipore Corp., Bedford, MA, USA). Blots were washed twice in Tris buffered saline (TBS), pH 7.5, preblocked with 5% skim milk/TBS at 4š C overnight, and then incubated with a 1:1,000 dilution of monoclonal mouse anti-human bax antibody for 1 hour at room temperature. Subsequently, they were incubated with peroxidase-conjugated anti-mouse immunoglobulin (DAKO A/S, Glostrup, Denmark) and the detection was performed using a BM chemiluminescence Western blot kit (Biochemica Boehringer Mannheim, Mannheim, Germany).

Results

Immunohistochemical staining

We observed positive bax staining in a cytosolic punctuate pattern in normal epidermis, with suprabasal keratinocytes being stained more strongly than the basal cell layer, which stained markedly weakly for bax (Fig. 1a). Keratinocytes of the follicular infundibulum and the lower follicle showed analogous bax protein immunoreactivity (Fig. 1b). Bax protein was found to be expressed in the sebaceous glands and also in the eccrine glands, with stronger staining in the outer layer of the coiled duct than in the pale cells of the eccrine glands (Fig. 1c). The apocrine glands, on the other hand, showed weak expression, mainly in the apical portions of the secretory cells (Fig. 1d).

Specimens of psoriasis vulgaris revealed positive bax staining, especially in the suprabasal layers (Fig. 2), that was similar to normal epidermis. Keratoacanthomas showed strong, predominantly diffuse staining for bax with the exception of the outermost layers of some tumour proliferations that were stained more weakly than the rest of the tumours (Fig. 3a). Squamous cell carcinomas showed only weak bax immunoreactivity (Fig. 3b), which was significantly weaker than that of the overlying epidermis, with the exception of well-differentiated tumour islands in two tumours that expressed immunostaining for bax comparable to that of normal suprabasal epidermis. Twelve basal cell carcinomas did not show bax immunostaining (Fig. 4) and the remaining 5 tumours showed only weak reactivity in tumour cells scattered within tumour nests and in palisading layers of some tumour formations. We could see numerous bax-positive, infiltrating lymphocytes in the specimens of psoriasis vulgaris, keratoacanthoma, squamous cell carcinoma and basal cell carcinoma (Figs. 2, 3 and 4), but only occasional positive lymphocytes in the mild inflammatory infiltrate in the specimen of normal hair follicle (Fig. 1b).

Immunoblot analysis

Western blot analysis (Fig. 5) confirmed the presence of 21 kDa bax protein in samples of normal skin, psoriasis vulgaris and squamous cell carcinoma. The level of bax protein expression in squamous cell carcinoma was apparently weaker than in normal and psoriatic skin. The expression of bax was not detected in basal cell carcinoma even on repeated immunoblot analysis.

Discussion

In this study, we have demonstrated that the pro-apoptotic protein bax is significantly expressed in normal human epidermis and its appendages. We found bax to be more intensely expressed in the suprabasal compartment in comparison with the basal cell layer, which stained markedly weakly for bax. Bcl-2, on the other hand, has been reported to be expressed in basal keratinocytes of the epidermis and the outer rooth sheath [17-19], but other authors found its expression to be confined mainly to melanocytes and also to Merkel cells, with the basal cell reactivity being variable, weak or even absent [20-24]. A recent in vitro study, however, demonstrated higher bcl-2 expression in keratinocytes than in melanocytes [25]. We observed slightly stronger immunoreactivity for bax in the suprabasal as compared to the basal epidermal compartment of psoriatic skin, in which reduced bcl-2 expression in the basal layer [26], and strong diffuse immunostaining for the anti-apoptotic bcl-x_L [24] and pro-apoptotic bak [27] have been reported. As certain pro-apoptotic and anti-apoptotic members of the bcl-2 family appear to be dysregulated in psoriasis vulgaris, further experiments are required to demonstrate the mechanisms involved in apoptosis of psoriatic keratinocytes.

Apoptosis has been reported to significantly influence the growth rate of tumours. Cell lines with high apoptotic rates in vitro tend to form slower growing tumours than those with low apoptotic rates [28]. Basal cell carcinoma, a slow growing tumour, has a doubling time of 9 days [29]. Continuous loss of tumour cells has been suggested [30] and a recent study clearly demonstrated that apoptotic cells outnumber mitotic cells in basal cell carcinoma [31]. The reason for this high apoptotic rate still remains enigmatic. Basal cell carcinoma overexpresses the anti-apoptotic bcl-2 protein [19, 32, 33], whereas there is minimal expression of the pro-apoptotic bax and bak, as we demonstrated in this and in our previous studies [27]. Moreover, almost no expression of the cell survival-promoting bcl-x_L and no expression of the apoptosis-inducing Fas has been detected [24]. A recent study has shown that bcl-2, when overexpressed at high levels in several solid tumour cell lines, results in paradoxical growth inhibition [34]. The pro-apoptotic bax and bak, on the other hand, have been reported to inhibit apoptosis in some circumstances [35, 36]. Both bcl-2 and bax may form cytotoxic channels in cells. Bcl-2/bax heterodimerization nullifies this channel activity and therefore promotes cell survival, suggesting that the ratio of bcl-2: bax is crucial and may determine whether apoptosis or cell survival will be promoted [37]. Hence, further research is needed to clarify interactions of both survival promoting and apoptosis triggering factors involved in the development and progression of basal cell carcinoma.

Our study revealed strong expression of bax in all keratoacanthomas examined, whereas squamous cell carcinomas stained only weakly for bax. Bcl-2 expression in keratoacanthoma is confined to the basal layer and the majority of squamous cell carcinomas do not express bcl-2 [33, 34, 38, 39]. It has been reported that wild-type but not mutant tumour suppressor p53 protein binds to the bax gene promoter region and thus stimulates the expression of bax [40, 41]. Since abnormal stabilization of p53 protein has been predominantly found in the outermost layers of keratoacanthomas, in contrast to a rather diffuse staining pattern in squamous cell carcinoma [42-44], our findings suggest that reduced expression of the pro-apoptotic bax protein in squamous cell carcinoma might be an important step in the development of this skin tumour, possibly related to dysfunctional p53, e.g. following UV radiation-induced p53 mutations.

CONCLUSION

Acknowledgement

We are grateful to Ms Yasue Yamamoto from the Department of Dermatology, Okayama University Medical School, for her excellent technical assistance.

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