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).

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.

Novel DNA staining method and processing technique for the quantification of undamaged double-stranded DNA in epidermal tissue sections by PicoGreen probe staining and microspectrophotometry.

Histotechnological processing of DNA can cause damage to and loss of DNA and can change its structure. DNA probes have severe tissue-staining limitations. New DNA probes and improved histotechnology are needed to enhance the characterization of fixed tissue-bound DNA. Our team developed a novel DNA staining technique and histotechnological processing procedure that improves tissue-bound DNA retention and the qualification and quantification of intact double-stranded (ds)-B-DNA. We used the ultrasensitive PicoGreen ds-DNA probe for the histochemical characterization of ds-DNA. Fifteen fixatives were examined to determine which were best for preventing DNA denaturation and retaining original DNA content and structures. Our use of a microwave-vacuum oven reduced heating temperatures, shortened heating and processing times, and enhanced fixation. We achieved better qualitative and quantitative results by using superior tissue-acquisition techniques (e.g., reduced prefixation times) and improved histotechnology. We also compared our novel approach with archival tissues, delayed fixation, less sophisticated and conventional histological processing techniques, and by experimenting with preservation of tissue-bound ds-Z-DNA. Results demonstrate that our histotechnological procedure and nucleic acid staining method significantly improve the retention of intact, undamaged ds-DNA which, in turn, allows the investigator to more precisely quantify the content and structures of unaltered and undamaged tissue-bound ds-B-DNA.