(Received for publication, June 22, 1995; and in revised form, September 20, 1995 )
From the
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, 2, 3, 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) when they demonstrated the
Ca- and Mg
-dependent autodigestion
of DNA in isolated liver cell nuclei. To explain this process, a
Ca
- and Mg
-specific endonuclease
was postulated by Wyllie et al.(5, 7) .
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-Mg
-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
and Mg
and related to cyclophilin(12) .
This peptide appears as a novel enzyme whose activity correlates with
apoptosis in thymocytes. It seemed quite obvious from the above that
there was conflicting evidence on which class of enzyme was responsible
for the nucleosomal ladder formation.
The lens was found to be a very interesting model for the study of DNases. This avascular organ localized in the posterior segment of the eye is composed of a layer of epithelial cells overlying a concentric array of elongated cells, the fibers. The whole organ is surrounded by the lens capsule. In the equatorial region of the lens, the epithelial cells sequentially differentiate into fiber cells. One of the most striking features of lens cell differentiation is the disappearance of lens cell nuclei followed by the same degradation phenomena, classically described in apoptotic cell nuclei. The chromatin appears quite condensed in the last step of differentiation(13) . The DNA was cleaved between nucleosomes(14, 15) , and one variant of histone H1 (H1-2) was missing in the embryonic fiber cells(16) , probably contributing to the action of endonuclease on linker DNA. In this tissue, however, the degradation of nuclei was not present in scattered cells, as was usual in apoptotic tissues, but occurred in a well synchronized population of cells. Moreover, microdissection allows the complete separation of the nucleated, undifferentiated epithelial cells, attached to the capsule, from the underlying fiber mass. Thus, we could compare cells of the same origin but different in metabolism and cellular state of differentiation. In addition, this was a physiological system, and no laboratory manipulation (such as irradiation or addition of drugs) was needed to induce the nuclear breakdown.
In the lens, DNase activity was detectable in fiber nuclei, resulting in double-stranded breaks, as judged by the appearance of the classical oligonucleosomal ladder(15) . However, there was no accumulation of single strand breaks with free 3`-OH ends (17) as expected if DNase I was responsible for such breaks. This led to the hypothesis of the presence of another DNase responsible for nucleosomal degradation of DNA in these cells.
In the present paper, we have discriminated DNase I and DNase II activity by an original assay in lens cells. These enzymes were immunocharacterized by Western blot and immunochemistry using a commercial anti-DNase I and a new polyclonal antibody against DNase II. DNase II showed some molecular forms (60 and 23 kDa) detected only in fiber cells. DNase II was immunolocalized in the nuclei of these cells while it remained cytoplasmic in epithelial cells. Moreover, only DNase II antibody was able to inhibit the in vitro DNA degradation in fiber cells. These results allow us to propose the participation of DNase II in lens cell differentiation.
To distinguish between DNase I and DNase II in the same
sample, their activities were measured at different pH and cationic
conditions. Fig. 1shows the DNase I activity in cationic buffer
at neutral pH (dark bars) and in acidic, non-cationic medium (white bars). Note that no enzymatic activity was observed in
Tris-EDTA buffer, but a regular decrease of non-digested DNA was
measured when DNase I was incubated in a neutral buffer containing
Ca and Mg
. Fig. 2shows the
DNase II activity in both buffers used above for DNase I. In this case,
the DNase II digested the DNA when incubated in an acidic buffer, but
it was inactive when incubated in a neutral, Ca
and
Mg
-containing buffer. We used this technique to
measure both kinds of nucleases in lens cells from 18-day-old chick
embryos. Fig. 3shows the DNase activities in the epithelial and
fiber extracts presented in ng of DNA digested per min either per mg of
protein (A) or per 10
cells (B). Note
that in both epithelial and fiber cells the DNase II-like activity
appeared greater than the DNase I-like activity. In contrast, the DNase
I/DNase II ratio was the same for both populations of cells, DNase II
representing approximately 60% of the total DNase activity.
Figure 1:
Measurement of DNase I activity. 1
µg of H-labeled DNA was incubated with increasing
concentrations of DNase I in a neutral
Ca
-Mg
-containing medium (dark
bars) or in an acidic medium (white bars). The
non-digested DNA was then precipitated and
counted.
Figure 2:
Measurement of DNase II activity. 1 µg
of H-labeled DNA was incubated with increasing
concentrations of DNase II in a neutral
Ca
-Mg
-containing medium (dark
bars) or in an acidic medium (white bars). The
non-digested DNA was then precipitated and
counted.
Figure 3:
DNase I and DNase II activities in lens
fiber and epithelial cells. The results are expressed in ng of DNA
digested per min, per mg of protein (A) and in ng of DNA
digested per min, per 10 cells (B). 1 µg of
H-labeled DNA was incubated in the presence of epithelial
or fiber cells extracts in a neutral
Ca
-Mg
-containing medium (dark
bars) or in an acidic medium (white
bars).
Figure 4:
Western blot analysis of DNase II in lens
epithelial and fiber cells. Proteins from lens epithelial (100 µg)
and fiber (500 µg) cells were separated using 12% (A) and
15% (B) acrylamide slab gels, transferred to Immobilon P, and
detected using an anti-DNase II antibody (A) or preimmune
serum (B). Lanes 1 and 5, DNase I; lanes
2 and 6, DNase II; lanes 3 and 7,
epithelial cells; lanes 4 and 8, fiber cells. The arrow heads indicate the two bands of 100 and 18 kDa labeled
in epithelial cells (lane 3) and the three major bands of 60,
23, and 18 kDa labeled in fiber cells (lane 4). Open
diamond indicates the crystallin
mass.
Similar experiments were performed using a commercial antibody against DNase I (Fig. 5). The DNase I antibody recognized specifically DNase I, a protein of about 32 kDa (lane 1), and did not react with DNase II (lane 2). In epithelial cells, three major bands of 18, 32, and 60 kDa were labeled (lane 3). In fiber cells, only a single band of 32 kDa was seen (lane 4). Note that the high amount of proteins loaded on the gel increased the background.
Figure 5:
Western blot analysis of DNase I in lens
epithelial and fiber cells. Proteins from lens epithelial (100 µg)
and fiber (500 µg) cells were separated using 12% acrylamide slab
gel, transferred to Immobilon P, and developed using an anti-DNase I
antibody. Lane 1, DNase I; lane 2, DNase II; lane
3, epithelial cells; lane 4, fiber cells. The arrow
heads indicate the three major bands of 60, 32, and 18 kDa labeled
in epithelial cells (lane 3) and the band of 32 kDa labeled in
fiber cells (lane 4). The open diamond indicates the
crystallin mass.
Figure 6:
Effect of anti-DNase I and anti-DNase II
antibodies on DNases activities. 1 unit of DNase I (panel A)
or 4 units of DNase II (panel B) were preincubated in neutral
Ca-Mg
-containing medium (dark
bars) or in an acidic medium (white bars) in the presence
of increasing concentrations of DNase I antibody (A) or DNase
II antibody (B). After 30 min at 37 °C, 1 µg of
H-labeled DNA was added to start the reaction. The
non-digested DNA was precipitated and counted. No cross-reaction was
recorded (data not shown).
To investigate the
involvement of DNases I and II in DNA degradation, we prepared lens
epithelial nuclei (Fig. 7A) and lens fiber nuclei (Fig. 7B). The incubation of fiber nuclei in a neutral
buffer containing 10 mM Ca and
Mg
generated a smear (lane 5), which was not
inhibited by DNase I antibody (lanes 6 and 7). The
same results were obtained with epithelial nuclei (lanes 1 and 2). Lanes 8, 9, and 10 have been
loaded with fiber nuclei incubated in Tris-EDTA, pH 5.75. Under these
acidic conditions, in the control, the DNA cleavage was seen along the
tract (lane 8), and cleavage inhibition was noted in the
presence of increasing amounts of DNase II antibody (lanes 9 and 10). In lane 10, there was no DNA smear, and
the DNA of high molecular weight appeared protected by the antibody
against DNase II. In epithelial nuclei, the DNA degradation obtained
under acidic conditions was not modified by the DNase II antibody (lanes 3 and 4). In addition, the DNase II antibody
was not able to inhibit the smear generated when both epithelial and
fiber nuclei were incubated in a neutral, cationic buffer (data not
shown).
Figure 7:
Effect of the anti-DNase I and anti-DNase
II antibodies on DNA cleavage. 10 nuclei from lens
epithelial cells (A) or fiber cells (B) were
incubated in both neutral cationic and acidic medium in the absence or
in the presence of an anti-DNase I or DNase II antibody. Arrow
heads show the position of the sample wells. Incubation in
neutral, cationic medium: control, lanes 1 and 5.
DNase I antibody added, lane 6 (2 µl) and lanes 2 and 7 (5 µl). Incubation in acidic medium: control, lanes 3 and 8. DNase II antibody added, lane 9 (2 µl) and lanes 4 and 10 (5
µl).
Figure 8: Section of an 18-day-old chick embryo lens stained with 1% toluidine blue. Ep, epithelial cells; AP, annular pad; OF, outer fibers; IF, inner fibers. The black bar represents 300 µm.
Figure 9: Immunolocalization of DNase I and DNase II in outer fiber cells. Transversal sections of lens from 18-day-old chick embryos were incubated with polyclonal antibody against DNase I (b), DNase II (d), or with preimmune serum (f). a, c, and e show the same sections stained with the nuclear stain, DAPI. White bar represents 30 µm.
A similar study is shown in Fig. 10for the central epithelial cells. The nuclear staining DAPI (Fig. 10a) shows that the DNase I antibody (Fig. 10b) labeled the nuclei of epithelial cells. In contrast, the DNase II antibody labeled the cytoplasm of epithelial cells (Fig. 10d), and no significant labeling of the epithelial cell nuclei was observed (Fig. 10, c and d, white arrows). Note that a strong labeling of the lens capsule was also observed.
Figure 10: Immunolocalization of DNase I and DNase II in lens epithelial cells. Transversal sections of lens from 18-day-old chick embryos were incubated with polyclonal antibodies against DNase I (b) or DNase II (d). a and c show the same sections stained with the nuclear stain, DAPI. White arrows indicate two epithelial cell nuclei. White bar represents 30 µm (a and b) or 20 µm (c and d).
To further study DNases, immunological methods remain a very powerful tool. An antibody against DNase I is commercially available. DNase II antibodies with low titre have been obtained by two laboratories(9, 30) . Thus, we prepared a polyclonal antibody against DNase II having a high titre, which does not cross-react with DNase I and is able to inhibit DNase II activity in vitro.
An increase of DNase activities in fiber cells is in agreement with the involvement of these enzymes in nuclear degeneration. It is interesting to note that there is no change in the DNase I/DNase II ratio between epithelial and fiber cells. This means that, in terms of activity, we have no elements to involve preferentially one or the other nuclease in the differentiation process of lens fibers.
Several forms of DNase II-like molecules have also been found. Two forms of 100 and 18 kDa are present in epithelial cells, and three major bands of 60, 23, and 18 kDa are detected in fiber cells. Two of these (60 and 23 kDa) are found only in fiber cells and may be incriminated in lens cell differentiation process.
When considering the immunohistochemistry data, DNase I is found mainly in the nuclei of both epithelial and fiber cells, an already described subcellular localization for DNase I(36) . DNase II, known as a lysosomal enzyme(37, 38, 39) , is observed in the cytoplasm of epithelial cells and also in the lens capsule. Strikingly, DNase II is highly concentrated in all fiber cell nuclei. This enzyme has already been seen in cell nuclei(40, 41) , but its presence there has been related to DNA replication(42) . This nuclear localization is seen only in fiber cells cleaving their DNA and may reflect a specific function of DNase II in DNA degradation.