Elastase is not required for L-DNase II activation during apoptosis in developing chicken neural retina.

During retinal development, the neuronal death is carried out by the mechanism of apoptosis. Among the different endonucleases activated, L-DNase II seems to be responsible for most of DNA degradation in this tissue. L-DNase II derives from LEI (Leukocyte Elastase Inhibitor) by a post-translational modification carried out by elastase in apoptosis induced in vitro. In this study, we investigated whether elastase could be implicated in apoptosis occurring during retinal development. Although elastase and LEI/elastase complex are colocalized in retinal sections, the LEI/elastase complex, detected by Western blot, does not change at all stages of development. However, at pH 4 retinal extracts show an enhanced activation of the L-DNase II. These results suggest that an acid protease, such as a cathepsin, may be implicated in neuronal retinal apoptosis.

Involvement of L-DNase II in nuclear degeneration during chick retina development.

During the development of the neural retina, 50% of the neurons die physiologically by apoptosis. In the chick embryo, the apoptotic wave starts at E8 and ends at E18, with a peak at E11. The onset of apoptosis is accompanied by the activation of several degradative enzymes. Among these, the activation of the endonucleases leads to the degradation of the genomic DNA of the cell which is thought to be the final event in apoptosis. Here, we have investigated the endonucleases activated during apoptosis associated with retinal development. We have found that Ca2+-Mg2+-dependent endonucleases, as well as acid endonucleases are activated. The results obtained in vitro using purified nuclei from chicken retina indicate that the endonuclease activity resulting from the activation of L-DNase II, an acid DNase is responsible for most of the DNA degradation observed in these cells.

Differential involvement of DNases in HeLa cell apoptosis induced by etoposide and long term-culture.

We have applied to human HeLa cells two different stimuli of apoptosis: the antitumoral drug etoposide, and a more 'physiological' death condition, obtained by growing cells in the same medium for long time periods, for up to 10 days. Analysis of different parameters demonstrated that in both experimental systems the same apoptotic features are visible. However, the DNA degradation pattern appeared to be different, suggesting the involvement of different DNases. In this view, we have analyzed the activity and expression of Ca2+-Mg2+-dependent and acid DNases. We have observed that DNase I is not modulated during apoptosis. In contrast, the acid L-DNase II (derived from Leukocyte Elastase Inhibitor by post-translational modification), recently identified in our laboratory, is mainly active in the apoptotic pathway induced by long term-culture. Furthermore, we have provided evidence that while caspase 3 is activated by both inducers, caspase 1 is essential only for the etoposide-induced apoptosis.

L-DNase II, a molecule that links proteases and endonucleases in apoptosis, derives from the ubiquitous serpin leukocyte elastase inhibitor.

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 cation-independent 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.

Analysis of nuclear degradation during lens cell differentiation.

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.

On the use of Zn2+ to discriminate endonucleases activated during apoptosis.

One approach to discriminate among specific DNases in apoptosis is to use inhibitors specific for each endonuclease. Zn2+ is known to inhibit Ca(2+)- and Mg(2+)-dependent endonuclease enzymatic activities during apoptosis. Acidic DNases were thought to be insensitive to Zn2+. In this paper, we analyse the effects of Zn2+ on activity of DNase II, either purified or in nuclei from lens fiber cells. These cells follow a physiological nuclear degeneration with DNase II accumulation in their nuclei. We show that Zn2+ is able to inhibit also this acidic endonuclease at a concentration of 1-6 mM. At a higher concentration of Zn2+, DNA is extensively degraded during the assay, masking the inhibition of the enzyme. This DNA degradation in the presence of Zn2+ has led to an overestimation of the activity of DNase II in studies of apoptosis. Hence, Zn2+ cannot be used to specifically identify one endonuclease among the different DNases involved in nuclear degradation during programmed cell death.

Involvement of DNase II in nuclear degeneration during lens cell differentiation.

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.

Characterisation of eye-lens DNases: long term persistence of activity in post apoptotic lens fibre cells.

Fibre cells in the ocular lens exhibit a constitutive apoptotic process of nuclear degradation that includes chromatin breakage, generating a ladder pattern of DNA fragments. This process is intrinsic to the normal terminal differentiation program. Despite the loss of nucleus and cytoplasmic organelles, the terminal differentiated fibre cells remain in the lens during the whole life span of the individual. The lens cells thus provide a unique system in which to determine the presence and fate of endonucleases once the chromatin has been cleaved. We report here on the presence of DNase activity in nucleated and anucleated lens cells. Using a nuclease gel assay and double-stranded DNA as substrate, we found active 30 and 60 kDa DNases. The enzymatic activities were Ca(2+), Mg(2+) dependent, and active at neutral pH. The relative amount of these forms changed during development and aging of the lens fibre cells. Both forms were inhibited by Zn(2+), aurintricarboxylic acid, and G-actin. The proteins were also separated by SDS-PAGE, renatured after removing SDS and incubated in the presence of native DNA adsorbed to a membrane. Therefore it was possible to demonstrate, by means of a nick translation reaction, that the enzymes produced single strand cuts. Based on these findings we propose that these chick lens nucleases are probably related to DNase I.

DNA strand breakage during physiological apoptosis of the embryonic chick lens: free 3' OH end single strand breaks do not accumulate even in the presence of a cation-independent deoxyribonuclease.

Epithelial cells from the lens equator differentiate into elongated fiber cells. In the final steps of differentiation, the chromatin appears quite condensed and chromatin breakdown into nucleosomes occurs. DNA breaks due to an endodeoxyribonuclease activity corresponding to at least two polypeptides of 30 and 40 kDa have been identified. To identify the nature and the developmental appearance of initial breaks, nick translation reaction was followed both biochemically and in situ in fiber and epithelial cells from chick embryonic lenses. There is no accumulation of single-strand breaks (SSB) with 3'OH ends in lens fiber cells during embryonic development. Such damage can be increased in these cells by treatment with DNAase I indicating the absence of an inhibitor of the nick translation reaction in fiber cells. However, there are indications of the presence of DNA breaks with blocked termini when the phosphatase activity of nuclease P1 is used. The presence of breaks is also indicated by the large amounts of (ADP-ribose)n found in lens fibers particularly at 11 days of embryonic development (E11) as ADP-ribosyl transferase binds to and is activated by DNA strand breaks. Incubation of lens cells in vitro, which causes nucleosomal fragmentation only in fiber cells, produces SSB with 3'OH ends in both epithelia and fibers. Incubation for short periods, observed in experiments in situ, induces SSB first in the central fiber nuclei, which are late in differentiation. This may indicate that these SSB play a physiological role. Long incubations produce larger numbers of SSB in epithelia than fibers. The SSB in the fibers may have been converted into double-strand breaks (D SB), seen as nucleosomal fragments, and therefore no longer act as substrates for nick translation. The nuclease activity responsible for SSB production is independent of divalent cations and could be implicated in lens terminal differentiation.

DNAase activities in embryonic chicken lens: in epithelial cells or in differentiating fibers where chromatin is progressively cleaved.

Lens is an organ composed of a layer of epithelial cells and a mass of fibers. During terminal differentiation, epithelial cells from the equatorial region elongate into fibers, nuclei change shape, the chromatin appears much condensed in the last step of differentiation and the DNA breaks down into nucleosomes. The pattern of DNAase activities has been recorded at different chick embryonic stages (11 and 18 days) using polyacrylamide gel electrophoresis with DNA substrate in the gel matrix. Two DNAases (30 and 40 kDa) have been observed in lens epithelia and fibers at both stages. However, the activities of both of the enzymes are augmented in fiber cells. The 30 kDa DNAase requires and Ca2+ and Mg2+ (5-15 mM) to hydrolyze the DNA substrate while the 40 kDa-activity is inhibited by added divalent cations (5-15 mM). The 30 kDa protein is inhibited by Na+ and is probably an endonuclease. Both nuclease activities probably are involved in the cleavage of fiber chromatin into nucleosomes during lens terminal differentiation, but variables such as chromatin configuration, unmasked DNA sequences, presence of cations, and pH gradients probably determine the extent of involvement of each DNAase.

Lens fiber differentiation correlated with activation of two different DNAases in lens embryonic cells.

In order to identify the different DNAases present in the lens differentiating tissue, we have used an assay which reveals their activity directly on DNA-containing gels after SDS polyacrylamide gel electrophoresis. DNAase renaturation from nuclear embryonic lens extracts does not occur after separation in 0.1% SDS polyacrylamide gel electrophoresis in contrast to that observed with purified micrococcal nuclease. When the SDS concentration in the running buffer and separating gel is decreased to 0.075%, renaturation of lens DNAase and enzyme activities are observed. Isoelectrofocusing was carried out in a polyacrylamide gel which was overlaid with an agarose gel containing DNA, permitting the visualization of the pI of DNAase activity. The presence of several DNAase isoenzymes was demonstrated in 11-day embryonic lenses. In epithelial lens nuclei, high molecular weight (MW) isoenzymes with basic pI were predominant. In post-mitotic fiber lens nuclei, two lower MW isoenzymes with acidic pI were detected as well as high MW activity with a basic pI.

Increased sensitivity of various genes to endogenous DNase activity in terminal differentiating chick lens fibers.

In the lens, epithelial cells from the equatorial zone differentiate into postmitotic elongated fibers. One aspect of this differentiation is nuclear shape transformation and DNA degradation. This process is controlled by DNase activity which in fiber nuclei increases with development. DNase activity is also present in the epithelial cell nuclei which appears to be non-functional but could be activated in vitro by exogenous addition of Ca2+. We have analyzed the possible selective action of endogenous DNase on 3 genes involved in lens terminal differentiation, namely delta-crystallin, beta-tubulin and vimentin, and on 1 gene not thought to participate in this process, ovalbumin. We have compared restriction DNA patterns of these genes in nuclei isolated from 11-day-old chick embryos and incubated in Ca2+-free medium or in fresh epithelial and fiber lens tissue at 11 and 18 days of development. During incubation in vitro of 11-day fiber nuclei, there is a net increase in the sensitivity of the delta-crystallin, beta-tubulin, ovalbumin and vimentin chromatin to the endogenous DNase. The vimentin gene appears to be more stable than the beta-tubulin and delta-crystallin genes indicating a degree of specificity of the endogenous DNase activity. In the epithelial nuclei, the lens-specific genes appear to be more stable but paradoxically there is a net degradation of the ovalbumin gene. In freshly isolated tissues the 4 genes were detected in epithelial and fiber cells at 11 and 18 days. Furthermore, in the mature fibers in which the nuclei were degenerating, the latter genes were still not completely digested.

Effect of X-irradiation and vitamin C on DNA degradation and endogenous DNase in embryonic chick lens cells.

The lens is an organ in which epithelial cells become elongated fibers. During this process, nuclei are transformed and the DNA is degraded. In previous studies, we described an autodigestion of the chromatin in isolated fiber nuclei but not in epithelial nuclei, but the level of DNAase activity was found to be identical in both epithelial and fiber nuclei of lenses at 11 days of development. In this study, we have investigated the possibility that x-irradiation might stimulate the nuclear endogenous activity responsible for chromatin breakdown or epithelial cells to a level comparable to that observed in fiber cells. We have observed that x-irradiation does not increase the nuclear epithelial DNAase activity. Conversely, vitamin C, suspected to prevent cataract formation by protecting DNA against free radical formation, has a damaging effect on the DNA of the lens of chick embryo in vitro.

Chromatin condensation and terminal differentiation process in embryonic chicken lens in vivo and in vitro.

During embryonic chick lens differentiation, the epithelial cells become transformed into elongated fibres. Concomitantly, the fibre nuclei undergo degeneration and high molecular weight (HMW) DNA breaks down due to nuclear endodeoxyribonuclease activity. An electronmicroscopic study of lens epithelial and fibre nuclei was made at different stages of chick embryonic development, both in vivo and in vitro. The in vitro conditions are conducive to the expression of endogenous endodeoxyribonuclease activity in fibres. In both conditions we observed condensation of chromatin. The organization of some nuclear material into distinct linear arrays followed by streaming of nuclear material into the cytoplasm is recorded only in vitro. Such a condition may lead to acceleration of the process of aging in lens fibres.

Nuclear endogenous Ca2+-dependent endodeoxyribonuclease in differentiating chick embryonic lens fibers.

During terminal differentiation of lens epithelial cells into fiber cells, nuclei become pycnotic and DNA degradation occurs. We investigated the putative role in this process of an endogenous DNAase. After incubation of isolated nuclei of both cell types at 37 degrees C, DNAase activity was revealed by DNA size analysis on 0.3-1% neutral and alkaline agarose, one- and two-dimensional gels. This DNAase activity is more prominent in lens fiber nuclei than in epithelial nuclei at all the embryonic stages probably because of a preexisting higher concentration of divalent cations in the former. This activity is calcium or magnesium dependent in both types of nuclei.

DNA repeat size in chick embryonic lens epithelium, lens fiber, brain and liver cell nuclei.

The DNA repeat size is determined by micrococcal nuclease digestion kinetics and subsequent electrophoresis of the products among various chick embryonic tissues. The repeat size is found to be not significantly different from 193 to 197 bp, for brain and liver at 11 days and for lens epithelium and fiber at different embryonic stages. However, the pattern of micrococcal digestion seems to reveal an overall chromatin modification as a function of development in the lens fibers.

Nuclear ADP-ribosylation in the chick lens during embryonic development.

Nuclear ADP-ribosyltransferase is present in cells from the chick lens throughout embryonic development. The activity does not decrease when the cells become post-mitotic and commence terminal differentiation but declines slowly in both epithelia and fibre cells. At all stages studied the enzyme retains its ability to be activated by DNA strand breaks induced either by X-irradiation or by the action of an endogenous endonuclease. There is no correlation between the enzyme activity or the levels of its substrate NAD+ and the changes in DNA repair capacity which have been observed during the development of the lens.

Changes in the DNA breakage and crystallin synthesis of embryonic chicken lenses cultured in a tryptophan-deficient medium.

In vitro experiments were performed in order to understand the biochemistry of tryptophan-deficient cataract. Eleven-day-old embryonic chick lenses were cultured in vitro for 3 hr, one and three days in a tryptophan-deficient medium. DNA breakage was followed on sucrose gradient and water-soluble protein synthesis was analysed by SDS-PAGE coupled with fluorography. A medium lacking tryptophan delays the DNA degradation and decreases the synthesis of all soluble proteins including the crystallins.