The characterization of DNase II and DNase I activity was undertaken to discriminate their different roles in physiological nuclear degradation during lens fiber cell differentiation. The activity of both nucleases determined in a new assay allows to discriminate DNase II from DNase I in the same extract. In fibers, both types of nuclease activities are found and appear higher than in epithelial cells. Specific polyclonal antibodies directed against these two nucleases reveal by Western blot analysis the presence of various DNase isoforms. DNase II like-nuclease, present in fibers, is represented by three major bands (60, 23, and 18 kDa), which are not detected, at least for two of them (60 and 23 kDa), in epithelial cells. DNase I like-nuclease pattern in fiber cells shows a single 32-kDa band, while several bands can be detected in epithelial cells. Immunocytochemistry studies show both nucleases present in lens cell sections. DNase II is, as usual, in cytoplasm of epithelial cells, but it appears strikingly concentrated in the nuclei of fibers. DNase I is always concentrated in nuclei of epithelial and fiber cells. DNA degradation observed in agarose gels shows that DNase II-activating medium cleaves the DNA from fiber cells more efficiently than DNase I-activating buffer. In addition, DNase II antibody is able to prevent this degradation. These results suggest a specific involvement of DNase II in nuclear degradation during lens cell differentiation.Apoptosis or programmed cell death occurs in many physiological and pathological situations where selection of cells is required (1-4). In 1980, a landmark study (5) revealed that glucocorticoids induced extensive DNA degradation in rat thymocytes in vitro at the onset of cell death. DNA cleavage occurred in a very specific pattern producing fragments of DNA that were multiples of 180 -200 base pairs. This indicated that the chromatin was cleaved at the linker DNA between nucleosomic cores. The characteristic ladder was first shown by Hewish and Burgoyne (6) To date, three different endonucleases have been involved in DNA fragmentation leading to nucleosomal appearance. Some authors, such as Peitsch et al. (8), claimed that the well characterized pancreatic deoxyribonuclease (DNase I) was constitutively expressed in cells of tissues potentially primed for apoptosis. This 30-kDa nuclease, active at neutral pH, could be responsible for DNA cleavage into oligonucleosomes during cell death. On the other hand, Barry and Eastman (9), studying apoptosis in Chinese hamster ovary cells, were unable to detect a Ca 2ϩ -Mg 2ϩ -dependent endonuclease. Instead, they identified another endonuclease, which was cation-independent, with optimal activity at pH 5. This enzyme was proposed to be DNase II (9, 10). Finally, Hughes and Cidlowski (11) showed a lower molecular weight nuclease, an 18-kDa peptide (termed NUC 18), which was activated by Ca 2ϩ and Mg 2ϩ and related to cyclophilin (12). This peptide appears as a novel enzyme whose activity correlates with apoptosis in thymocy...

The most widely recognized biochemical change associated with the majority of apoptotic systems is the degradation of genomic DNA. Among the enzymes that may participate in this cleavage, the acidic cationindependent DNase II is a likely candidate since it is activated in many apoptotic cells. To better understand its role, we purified and sequenced a DNase II extracted from porcine spleen. Protein sequencing of random peptides demonstrated that this enzyme is derived from a ubiquitous serpin, the leukocyte elastase inhibitor (LEI), by an acidic-dependent posttranslational modification or by digestion with elastase. We call this novel enzyme L-DNase II. In vitro experiments with purified recombinant LEI show that the native form has no effect on purified nuclei whereas its posttranslationally activated form induces pycnosis and DNA degradation. Antibodies directed against L-DNase II showed, in different cell lines, an increased expression and a nuclear translocation of this enzyme during apoptosis. Since the appearance of the endonuclease activity results in a loss of the anti-protease properties of LEI, the transformation from LEI to L-DNase II may act as a switch of protease and nuclease pathways, each of which is activated during apoptosis.Apoptosis, or programmed cell death, is a mechanism of cell clearance in many physiological processes such as embryogenesis, metamorphosis, and tumor regression (2). Although the signals inducing apoptosis are very different, nuclear condensation, membrane blebbing, and formation of apoptotic bodies are morphological features common to all apoptotic cells. By far the most widely recognized biochemical change is the degradation of genomic DNA. The identity of the enzymes responsible for this cleavage is the subject of considerable debate. Several endonucleases have been proposed to be responsible for DNA fragmentation. Ca 2ϩ -plus Mg 2ϩ -dependent endonucleases in thymocytes, such as NUC 18/cyclophylin A (16), DNase I (21), DNase ␥ (29), and a new 97-kDa DNase (18), are examples. Mg 2ϩ -dependent, Ca 2ϩ -independent endonucleases have been implicated in human myeloid cell line apoptosis (7,8). Barry and Eastman (1) implicated DNase II, a cation-independent acidic endonuclease, as the enzyme that degrades DNA in apoptosis associated with intracellular acidification. We have shown in our laboratory the involvement of DNase II in nuclear degradation in terminally differentiating lens fiber cells (32).To date, our knowledge of the molecular structure of DNase II is very limited. The enzymatic properties of DNase II from different tissues and animals were found to be very similar, but its physical and chemical properties showed high variability. For instance, the molecular mass of mammalian DNase II ranges between 26 and 45 kDa. The reasons for this variability remain unknown (15).To better understand the biology of DNase II, the knowledge of its protein sequence seemed to be a mandatory step. In this study, we showed that this ubiquitous L-DNase II arises from leukocyte elastase...

Lens cells demonstrate a terminal differentiation process with loss of their organelles including nuclei. Chromatin disappearance is characterised by the same changes as most apoptotic cells, i.e. condensation of chromatin and cleavage into high molecular weight fragments and oligonucleosomes. The endo-deoxyribonucleases (bicationic (Ca2+, Mg2+), mono-cationic (Ca2+ or Mg2+) and acidic non-cationic dependent nucleases) are present in lens fibre cells. Our results suggest that the acidic non-cationic nuclease (DNase II) plays a major role in chromatin cleavage. This nuclease, known to be lysosomal, is found in lens fibre nuclei and only an antibody directed against DNase II inhibits the acidic DNA cleavage of lens fibre nuclei. In addition, there must be another DNase implicated in the process which is not DNase I but appears to be a Ca2+, Mg2+ dependent molecule. Regulation of these DNase activities may be accomplished by the effect of post-translational modifications, acidic pH, mitochondrial release molecules, growth factors or oncogenes. Finally, fibre cells lose organelles without cytoplasmic elimination. The survival of these differentiated cells might be due to the action of survival factors such as FGF 1.