Localization and quantification of intact, undamaged right-handed double-stranded B-DNA, and denatured single-stranded DNA in normal human epidermis and its effects on apoptosis and terminal differentiation (denucleation)

摘要

Quantification of two types of nucleic acids [double-stranded (ds-) and single-stranded (ss-) DNA] was performed to understand the distribution of DNA within the epidermal strata and to examine the effects of DNA structure on gene expression, viz., apoptosis and terminal differentiation. In addition, we examined the precise starting point of cell death within the epidermis (suprabasal layer); examined how DNA structure affects gene expression of melanocytes; and characterized the “transitional cells” located between the stratum granulosum and stratum corneum, viz., epidermal phase transition zone (EPTZ). Ultrasensitive anti-DNA antibody probes (ds-DNA, ss-DNA), the Feulgen reaction, histological stains (morphological characterization) and the terminal deoxyribonucleotidyl transferase (TUNEL) assay (apoptosis) were used to characterize cell death in normal human epidermis. This study characterized, for the first time, the deterioration of right-handed ds-B-DNA and the increase in denatured ss-DNA during epidermal maturation. For the first time, this approach also allowed for the quantitative and qualitative characterization of DNA content and structure in all epidermal strata, using anti-ds-B-DNA and anti-ss-DNA antibodies. In order to improve the retention and quality of DNA, a novel histotechnological processing procedure was used. The results indicate that the largest decline in DNA occurred within the stratum granulosum, followed by the EPTZ, and the stratum spinosum. Not all epidermal nuclei lost DNA, indicating two differentiating keratinocyte pathways, viz., apoptotic and non-apoptotic. Both pathways united in the stratum granulosum. These results suggest that keratinocyte terminal differentiation and apoptosis are distinct cellular events, cell death begins earlier than expected, and molecular epidermal events take place in a gradual and orderly manner within keratinocytes. During maturation, ds-B-DNA decreases as ss-DNA increases. Therefore, during differentiation of keratinocytes, both DNA content and DNA structure are altered.