Apoptotic DNA fragmentation can be revealed in situ: An ultrastructural approach

Abstract

A common pattern of apoptotic death is DNA cleavage, initially producing large fragments (50 kbp), followed by the production of nucleosomic/oligonucleosomic fragments. Nevertheless, apoptosis without DNA fragmentation, at least of the nucleosomic type, has been reported. To investigate the spatial relationship between DNA cleavage and chromatin condensation, we applied the TUNEL technique to the ultrastructural analysis of apoptotic cells. A modified method, utilizing a gold-conjugated antidigoxigenin antibody, was carried out on U937 versus Molt-4 cells, both exposed to UVB radiation or staurosporine treatment. Gold particle density in the different domains of apoptotic cells was evaluated by a four-way ANOVA test. Gold labelling was more strongly localised in condensed chromatin than in the diffuse chromatin. U937 cells, which evidenced in vitro oligonucleosomic fragmentation after both UVB and staurosporine treatments, revealed a significantly higher gold particle density, when compared with Molt-4, which did not show, on the other hand, oligonucleosomic cleavage even in the presence of < or = 50 kbp cleavage. Thus, a correlation between DNA fragment sizes and gold particle density appears. TUNEL applied to electron microscopy is an effective approach to study the relationship between apoptotic chromatin condensation and DNA cleavage. Both these events indeed appear in the apoptotic nucleus, but their reciprocal correlation is still greatly unknown. Microsc. Res. Tech. 2009. (c) 2009 Wiley-Liss, Inc.

Ultraviolet B (UVB) Irradiation-Induced Apoptosis in Various Cell Lineages in Vitro

“…The lack of endonuclease activation can be postulated in this apoptotic model, despite the existence of a metabolic pathway responsible for chromatin rearrangement and other apoptotic patterns [38]. In addition, after conventional agarose gel electrophoresis, a typical DNA ladder appeared in U937 cells, too (Figure 2A, lane 7) [44], as also described in another work [49]. …”

“…This review demonstrates that UVB radiation induces apoptosis in various cell lines using different approaches: Scanning (SEM) and Transmission (TEM) Electron Microscopy used to evidence morphological features, such as surface blebbing, cytoplasm shrinkage and chromatin condensation [38–41]; agarose gel electrophoresis and TUNEL reaction, the latter both at fluorescence and electron microscopy, used to investigate DNA [42–44]; and flow cytometry used to analyze cell cycle patterns and mitochondrial activity [45,46]. …”

“…Molt-4 cell response to UVB was investigated not only at the standard post-incubation time ( i.e. , 4 h), but also at 6, 8, 10 and 24 h and DNA cleavage never occurred [44]. The lack of endonuclease activation can be postulated in this apoptotic model, despite the existence of a metabolic pathway responsible for chromatin rearrangement and other apoptotic patterns [38].…”

C2C12 myoblast sensitivity to different apoptotic chemical triggers

“…It plays a key role in development, reproduction, aging, immune function and cell diseases. It is characterized by specific events, such as DNA fragmentation, chromatin margination and condensation, cell shrinkage, membrane blebbing and apoptotic bodies formation (Burattini et al, 2009).…”

Apoptotic ciliated cells on sputum smear

“…7,8 These changes are compatible with those observed in ultrastructural studies. 9,10 Other pathological factors may cause morphological alterations in airway ciliated cells such as viral injury, causing phenomena such as ''ciliocythopthoria'' 11,12 and aberrant ciliogenesis 13 ; smoking, with axonemal anomalies 14 and shortened cilia 15 ; and chronic bronchitis, with ciliary membrane abnormalities. 16 These alterations have been observed by means of light and electron microscopy both in cytology samples and biopsy specimens.…”