BAX protein is not expressed by basal cell carcinomas

K.ROSSEN, A.KARABULUT THORUP,* K.HOU-JENSEN† AND G.KRAG JACOBSENDepartment of Pathology, Gentofte University Hospital, Hellerup, *Department of Oral Pathology, School of Dentistry,University of Copenhagen, and †Department of Pathology, University Hospital, Rigshospitalet, Copenhagen, Denmark

Accepted for publication 21 April 1998

Summary BAX and related proteins encoded by the BCL2 gene family are involved in the regulation ofapoptosis. BAX is an apoptosis-promoting protein. The slow growth of basal cell carcinoma (BCC) hasso far been explained by a high apoptotic activity. We investigated immunohistochemically 27 BCCsfor expression of the apoptosis-promoting BAX protein. BCC did not express detectable amounts ofBAX immunohistochemically. The results indicate that apoptosis in BCC does not involve BAXprotein. We propose that the apoptotic pathway in BCC is regulated by either less common membersof the BCL2 gene family or bypasses the regulation of the BCL2 gene family.

Basal cell carcinomas (BCCs) are slow-growing tumourswith a high proliferative activity. The slow growth hasbeen explained by a concurrent high apoptotic activity.1

BAX is a proapoptotic member of the BCL2 gene familyof proteins. The BCL2 gene family represents a largefamily of homo- and heterodimerizing proteins, some ofwhich are apoptosis suppressing (e.g. BCL2 and BCL-xL), while others are apoptosis promoting (e.g. BAX,BAD and BAK).2,3

Overexpression of BCL2 is reported in BCC,4,5 and thequestion has been raised whether BCL2 is involved inthe regulation of apoptosis in BCC.1,5 In an attempt toanalyse further the importance of the BCL2 gene familyof proteins in the regulation of apoptosis in BCC, weinvestigated the immunohistochemical expression ofBAX in normal epidermis and different types of BCC.

Materials and methods

The study material comprised four samples of normalskin and 27 BCCs (eight superficial, seven nodular, sixinfiltrating and six morphea-like). The tissue was for-malin fixed and embedded in paraffin wax. The primaryantibody was a polyclonal rabbit antibody directedagainst human BAX protein (PC66; Oncogene ResearchProducts, Cambridge, U.K.). Deparaffinized and micro-wave-pretreated 3 mm sections were processed forimmunohistochemistry and incubated with primaryantibody diluted 1 : 25 in Tris-buffered saline, pH 7·6,with azide/bovine albumin overnight at 4 8C. Theimmunological reaction was visualized using a Dako

ChemMate detection kit (K 5003, peroxidase/AEC,rabbit/mouse; Dako, Glostrup, Denmark). A positivecontrol was achieved by running a biopsy fromnormal valvula ileocoelcalis (including ileal mucosa,colon mucosa and lymphatic tissue) concurrently. Asa negative control, the primary antibody was omitted.

Results

In positive cells, the BAX immunoreaction product waslocated in the cytoplasm, and a discrete perinuclearaccentuation was often seen. In epidermis and thefollicular infundibulum of normal skin, a weak tomoderate cytoplasmatic BAX immunoreactivity wasobserved in stratum malpighii (Fig. 1). The immuno-reactivity in stratum basalis was usually less pro-nounced than the expression in stratum malpighii.The lower portion of the hair follicle was negative. AllBCCs showed negative BAX immunoreaction, exceptthose of central areas with abortive terminal differentia-tion (Fig. 2).

Discussion

Apoptosis is a genetically determined, active processthat plays a part opposite that of mitosis in tissue sizeregulation. It is essential in multiple, biological andpathological processes including tumour progression.2

Apoptosis can be initiated by different signal-transdu-cing pathways that seem to converge on commoneffector mechanisms in which a major element is theactivation of caspases.2,3 The BCL2 gene family ofproteins have a central role in the regulation of

British Journal of Dermatology 1998; 139: 472–474.

472 q 1998 British Association of Dermatologists

Correspondence: K.Rossen, Department of Pathology, Bispebjerg Hos-pital, CHC, Bispebjerg Bakke 23, 2400 Copenhagen NV, Denmark.

apoptosis, as their level of action is located in the distalpart of the converging pathways.2 The BCL2 genefamily includes, among others, BCL2, BCL-xL, BAX,BAK and BAD.

BCCs are slow-growing tumours with a high prolif-erative activity, and the slow growth has been explainedby a concurrent high apoptotic activity.1

In the present study, we have shown that BCCs do notexpress detectable amounts of the apoptosis-promotingBAX protein immunohistochemically. Studies of theapoptosis-suppressing proteins BCL2 and BCL-xL showthat BCL2 is overexpressed in BCC4,5 and that BCL-xLexpression is minimal.4 The apoptosis-promoting pro-tein BAK is reported to be negative in BCC.6

The immunohistochemical profile of BAX, BAK andBCL2 expression in BCC is not in obvious agreementwith the interpretation of BCC as a neoplasm with highapoptotic activity. Three explanations for this can begiven: the apoptotic activity in BCC is regulated by lesscommon members of the BCL2 gene family, the apopto-tic activity in BCC is regulated by a BCL2 gene familyindependent pathway or the interpretation of BCC as aneoplasm with a high apoptotic activity is erroneous.

The BCL2 family of proteins is a still growing family.BAD, a new member of the BCL2 family, like BAX andBAK, is an apoptosis promoter that is opposed by BCL2and BCL-xL.7 Thus, BAD might regulate apoptosis inBCC.

BAX PROTEIN AND BASAL CELL CARCINOMA 473

q 1998 British Association of Dermatologists, British Journal of Dermatology, 139, 472–474

Figure 1. BAX immunoreactivity in normalepidermis. Note moderate cytoplasmicstaining in stratum malpighii and lesspronounced staining in basal keratinocytes(original magnification, × 400).

Figure 2. Basal cell carcinoma (BCC)without detectable BAX immunoreactivity(original magnification, × 400).

The family of caspases drive the effector process ofapoptosis.2,3 Recent data indicate that some of thesecaspases, named level 1 caspases, act upstream of theBCL2 gene family regulation, whereas other caspases,named level 2 caspases, act downstream of the BCL2gene family regulation.3 Thus, pathways activatinglevel 1 caspases might be responsible for the apoptoticactivity in BCC. Those pathways include the tumournecrosis factor and Fas signalling systems.2,3

The interpretation of BCC as a neoplasm with highapoptotic activity has been supported by electron micro-scopy1 and one end-labelling study.8 However, otherend-labelling studies point towards a low apoptoticactivity in BCC.4,9 When interpreting end-labellingresults, it must be taken into account that apoptosis isa highly dynamic process and that DNA fragmentationis probably a late event in this process.10 Therefore,caution is suggested in using end-labelling indices asdirect correlates of apoptotic activity. In the light of thisacknowledgment, we find that apoptosis is still the mostplausible explanation for the paradoxically slow growthrate of BCC and, in agreement with others, we considerBCC as a neoplasm with high apoptotic activity.1

On the assumption that BCC is a neoplasm with highapoptotic activity regulated by a BCL2 gene familyindependent pathway, the immunohistochemicalprofile of BAX, BAK and BCL2 expression can beexplained as an attempt to oppose this unidentifiedapoptotic factor.

In conclusion, it seems reasonable to assume thatapoptosis in BCC does not involve BAX and that theapoptotic activity in BCC is regulated by either lesscommon members of the BCL2 gene family or a BCL2gene family independent pathway.

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