Changes in the topological expression of markers of differentiation and apoptosis in defined stages of human cervical dysplasia and carcinoma

Abstract Objective We compared the capacity of cells in normal cervical epithelium, progressive stages of CIN, and invasive carcinoma to proliferate, differentiate, and undergo apoptosis. Methods We investigated 30 conizations showing regular squamous epithelium of the ectocervix, all stages of cervical preinvasive neoplastic lesions (CIN I to III), or invasive carcinoma. The expression of the cell proliferation and differentiation marker Ki67 and Mad-1, respectively, and of the apoptosis-related proteins bcl-2, active caspase-3, and DNase I was analyzed on paraffin sections by immunohistochemistry. The expression of DNase I or -like enzymes was also analyzed at the level of their gene transcripts by in situ hybridization. In addition, apoptotic events were identified by in situ end labeling of fragmented DNA (ISEL). Results Expression of Ki67 was restricted to suprabasal cells in normal cervical epithelium but increased with CIN severity and invasive carcinoma. ISEL demonstrated apoptosis in superficial layers of normal, CIN I, and CIN II epithelium, whereas in CIS (CIN III) and invasive carcinoma, ISEL-positive cells were additionally observed at varying epithelial locations. Bcl-2 immunostaining remained restricted to the basal layer of all preneoplastic and neoplastic stages. Active caspase-3 was present in the suprabasal layer and extended to all upper layers in normal epithelium and slightly decreased with increasing dysplasia. In invasive carcinoma it was restricted to few scattered cells. The differentiation marker Mad-1 extended from the spinous to the superficial layer in regular epithelium, but gradually shifted to more superficial layers with increasing CIN grade and invasive carcinoma. A similar topological change was observed for DNase I with increasing CIN grade. In CIS and invasive carcinoma, DNase I immunopositive cells were solely interspersed within neoplastic cells. In contrast, DNase I specific mRNA was present in all epithelial layers in CIN III and neoplasia, suggesting a translational block of the expression of DNase I or -like enzymes. Conclusions Our data indicate that the elevated proliferation observed with increasing CIN severity and carcinoma was not paralleled by a similar increase in cell elimination. Most of the dysplastic and neoplastic cervical epithelial cells appeared incapable of entering terminal differentiation and complete it by apoptosis, possibly due to their failure to express or activate apoptosis executing enzymes.