| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
B-crystallin knockout mice demonstrate hyperproliferation and genomic instability
1
Departments of
* Ophthalmology and Visual Sciences,
Biochemistry and Molecular Biophysics,
Cell Biology and Physiology and
§ Genetics, Washington University School of Medicine, St. Louis, Missouri 63110, USA; and
¶ National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA
1Correspondence: Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8096, St. Louis, MO 63110, USA. E-mail: andley{at}vision.wustl.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
|---|
B-crystallin is a member of the small heat shock protein family and
can act as a molecular chaperone preventing the in vitro
aggregation of other proteins denatured by heat or other stress
conditions. Expression of B-crystallin increases in cells exposed to
stress and enhanced in tumors of neuroectodermal origin and in many
neurodegenerative diseases. In the present study, we examined the
properties of lens epithelial cells derived from mice in which the
B-crystallin gene had been knocked out. Primary rodent cells
immortalize spontaneously in tissue culture with a frequency of
10-5 to 10-6. Primary lens epithelial cells
derived from B-crystallin-/- mice produced
hyperproliferative clones at a frequency of 7.6 x
10-2, four orders of magnitude greater than predicted by
spontaneous immortalization (1)
. Hyperproliferative
B-crystallin-/- cells were shown to be truly immortal
since they have been passaged for more than 100 population doublings
without any diminution in growth potential. In striking contrast to the
wild-type cells, which were diploid, the
B-crystallin-/- cultures had a high proportion of
tetraploid and higher ploidy cells, indicating that the loss of
B-crystallin is associated with an increase in genomic instability.
Further evidence of genomic instability of
B-crystallin-/- cells was observed when primary
cultures were infected with Ad12-SV40 hybrid virus. In striking
contrast to wild-type cells, B-crystallin-/- cells
expressing SV40 T antigen exhibited a widespread cytocidal response 2
to 3 days after attaining confluence, indicating that SV40 T antigen
enhanced the intrinsic genomic instability of
B-crystallin-/- lens epithelial cells. These
observations suggest that the widely distributed molecular chaperone
B-crystallin may play an important nuclear role in maintaining
genomic integrity.—Andley, U. P., Song,, Z., Wawrousek, E. F., Brady, J. P., Bassnett, S., Fleming, T. P. Lens
epithelial cells derived from B-crystallin knockout mice demonstrate
hyperproliferation and genomic instability.
Key Words: molecular chaperone • nuclear • immortalization • ploidy
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
|---|
B-crystallin, a major protein of lens fiber cells,
is a stress-inducible chaperone that is constitutively expressed at low
levels in the lens epithelium and in numerous tissues, particularly
heart, kidney, skeletal muscle, lung, and brain (2
3
4
5)
.
However, the functions of B-crystallin in these tissues are unknown.
Several reports suggest a general cellular function of B-crystallin
linked with growth (4
5
6
7
8
9
10
11
12
13)
. Nearly 20% of B-crystallin
polypeptides isolated from the lens are phosphorylated in
vivo, which suggests its association with signal transduction
pathway(s) (4
, 11)
. Chinese hamster ovary cells induced to
express B-crystallin change its localization from the cytoplasm to
the nucleus during interphase (5)
, and B-crystallin
expression increases in mitotically arrested NIH 3T3 fibroblasts
(6)
. B-crystallin also accumulates in the developing
central nervous system (particularly in glial cells), in fibroblasts
transformed by the v-mos and Ha-ras oncogenes,
and in brain tumors of neuroectodermal origin (7
8
9
10
11
12
13
14)
.
Enhanced expression of B-crystallin is also observed in numerous
neurodegenerative diseases such as Alexander’s, Alzheimer’s,
Creutzfeldt-Jakob, multiple sclerosis, and Parkinson’s diseases
(11
12
13)
. B-crystallin-/- mice
develop muscle abnormalities but do not develop cataracts
(15)
. The major phenotype in these mice, lacking both
B-crystallin and small heat shock protein HSPB2 (encoded by an
adjacent gene that was also disrupted), is a progressive muscular
dystrophy that destroys a specific subset of skeletal muscles,
particularly those in the head and perivertebral regions. Since HSPB2
is not expressed in the lens (16)
, its deletion is not
expected to contribute to lens phenotypes. Lens epithelial cells
derived from mice lacking A-crystallin, a protein normally
associated with B-crystallin in the lens, have a diminished growth
potential (17)
. To study the physiological role of
B-crystallin, lens epithelial cells were cultured from
B-crystallin-/- mice. |
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
|---|
B-crystallin-/- lens epithelial cells were
used in this study. B-crystallin-/- mouse
lenses were obtained from mice with targeted disruption of the
B-crystallin gene and the adjacent HSPB2 gene (15)
. The
latter gene is not expressed in the lens and is not expected to give
lens phenotypes (15
, 16)
. To obtain primary lens
epithelial cells, capsule epithelial explants of wild-type (129SvJ) or
B-crystallin-/- mouse lenses (6–8 wk old)
were placed in 35 mm tissue culture plates containing Eagles minimum
essential medium-20% fetal bovine serum and cultured for 1–2 wk until
growth of cells was detectable (17)
. Primary cells were
passaged and plated on glass coverslips to facilitate microscopic
examination. Cultures from a pair of lenses were routinely placed
together in one tissue culture plate. To identify differences in growth
potential of fellow lenses from a given mouse, in some experiments each
of the two capsule epithelial explants from a mouse was cultured
separately. Once it was determined that the fellow lens epithelial
explants of a particular animal had the same proliferation behavior,
one epithelium of a pair of lenses was placed in fixative and the other
one was cultured. A-crystallin-/- cells
described previously (17)
were used as a control for some
experiments. Primary cultures of wild-type,
A-crystallin-/-, and
B-crystallin-/- cells were infected with
Ad12-SV40 hybrid virus as described previously (18)
.
Cell growth
Primary cultures of wild-type or
B-crystallin-/- lens epithelial cells in
passage 1 were subcultured into 24-well plates
(2x103 cells per well) for a period of 2 wk.
Attachment efficiency determined 2 h after plating was found to be
85%, and was the same for wild-type and
B-crystallin-/- cells. Cultures were fed
twice weekly. Cell numbers were measured on indicated days after
trypsinization and counting with a Coulter counter (17)
.
Hyperproliferative B-crystallin-/- cells
were plated at low densities to examine clonal growth
(17)
. To determine whether the hyperproliferative
B-crystallin-/- cells were immortal, they
were subcultured every week, the number of cells was counted, and the
population doubling level was determined as described previously
(18)
.
Cell cycle distribution
Wild-type or B-crystallin-/- cells
(106 cells) were washed with phosphate-buffered
saline (PBS) and cell pellets were labeled for 30 min on ice with
propidium iodide (50 µg/ml) in 0.1% sodium citrate containing 0.3%
Nonidet P-40 and 20 µg/ml ribonuclease A (pH 8.3). The percentage of
cells in each phase of the cell cycle was determined using a flow
cytometer (Becton-Dickinson FACScan, Rutherford, N.J.) and analyzed
using the Cell Quest software (17)
.
Immunofluorescence
Cells were fixed in 4% paraformaldehyde/PBS for 30 min at room
temperature, permeabilized in 0.1% triton X-100 for 30 min, and
blocked with 10% goat serum. To visualize the distribution of
B-crystallin, cells were incubated overnight at 4°C in a 1:50
dilution of a polyclonal antibody raised against bovine B-crystallin
(a kind gift from Dr. Joseph Horwitz). This primary antibody was used
because it gave a very low background in immunocytochemistry with the
B-crystallin-/- mouse lens slices. A
fluorescein-conjugated goat anti-rabbit immunoglobulin G (IgG) was used
as the secondary antibody. To visualize the distribution of
A-crystallin, cells were incubated overnight at 4°C in a 1:100
dilution of a monoclonal antibody against bovine A-crystallin (a
kind gift from Dr. Paul Fitzgerald). This primary antibody was used
because it has been shown to react with a high specificity with
A-crystallin in immunocytochemical detection (17)
and
gave no background with A-crystallin-/-
mouse lens slices. A lissamine-rhodamine-conjugated goat anti-mouse IgG
was used as the secondary antibody. To visualize the organization of
f-actin, fixed and permeabilized cells were incubated in a 1:50
dilution in PBS of a fluorescein phalloidin (Molecular Probes Inc.,
Eugene, Oreg.) methanolic stock solution (100 U/ml of methanol). Cells
were stained with fluorescein phalloidin for 20 min, washed 3 x 5
min in PBS, and viewed. Lens epithelial cells were viewed using a Zeiss
LSM 410 confocal microscope equipped with an Argon-Krypton laser.
Western blotting
Western immunoblotting was used to examine the expression of
A- and B-crystallin in primary mouse lens epithelial cultures
(17
, 19)
. The antibody used for immunoblot analysis of
A-crystallin was a monoclonal antibody to bovine A-crystallin (at
a dilution of 1:100), which has been shown to react with high
specificity with mouse A-crystallin (17)
. For
immunoblotting analysis of B-crystallin, a polyclonal antiserum
raised against the 21 amino acid carboxyl-terminal peptide of human
B-crystallin was used at a dilution of 1:1000. This antiserum showed
a high specificity for mouse B-crystallin. Immune complexes were
detected using 125I-protein A.
Cytogenetics
Karyotype analyses were done in the Cell Culture Laboratory of
Dr. Bharati Hukku, Children’s Hospital of Michigan (Detroit, Mich.)
using standard methods. Chromosome counts were determined in 51
metaphases, and 16 Giemsa-banded karyotypes were examined from each
culture (20
, 21)
.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
|---|
B-Crystallin was detected as a single protein band of 
20 kDa
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
immunoblot analysis in wild-type (129SvJ) mouse lens epithelial cells,
but was absent from B-crystallin-/-
epithelial cells derived from homozygous
B-crystallin-/- mouse lenses (Fig. 1A
). B-crystallin was readily detectable in the cytoplasm
of wild-type mouse lens epithelial cells by immunofluorescence but was
absent in the cultures derived from
B-crystallin-/- lenses (Fig. 1C
, E
). Very low background staining seen in the nuclei of the
B-crystallin-/- cells is most likely to be
due to the interaction of the rabbit antiserum used with nucleolar
antigen and not to B-crystallin. Since B-crystallin is normally
associated in the lens with A-crystallin in vivo, we also
examined the expression of A-crystallin in cultured lens epithelial
cells. A-crystallin expression can be detected in the cytoplasm of
wild-type and B-crystallin-/- cells. The
levels of A-crystallin were similar in wild-type and
B-crystallin-/- lens epithelial cells (Fig. 1B
, D
). Distribution of A-crystallin within the wild-type
and B-crystallin-/- cells was also similar,
indicating that knocking out B-crystallin does not affect the
expression of A-crystallin. The organization of the actin
cytoskeleton in wild-type and
B-crystallin-/- lens epithelial cells was
also examined. As shown in Fig. 1
, there were no significant
differences in the actin cytoskeleton of wild-type and
B-crystallin-/- cultured lens epithelial
cells.
|
Of 263 B-crystallin-/- explanted
capsule epithelia, 7.6% of the explants exhibited a high proliferative
ability as compared with wild-type cultures, producing a frequency of
hyperproliferation of B-crystallin-/- cells
of 7.6 x 10-2 (Table 1
). In contrast, the frequency of spontaneous immortalization of rodent
cells in culture is estimated to be 10-5 to
10-6 (1)
. The faster growing
colonies were observed only in the cells cultured
from the B-crystallin-/-
lenses, and 289 wild-type primary lens epithelial cultures produced no
hyperproliferative clones. Differences between proliferation of the
wild-type and B-crystallin-/- cells were
observed within 7 days of initiation of the culture. Examination of 173
A-crystallin-/- cultures also produced no
hyperproliferative cultures. These observations are consistent with the
fact that immortalization of mouse lens epithelial cells in culture is
a very uncommon event. No spontaneously immortalized mouse lens
epithelial cell lines are now available (22
23
24)
.
|
The hyperproliferative B-crystallin-/- cells
were shown to grow at a higher rate, with a doubling time of 1.5 days
as compared with 3 days for the wild-type cells. Ten days after
culture, the number of hyperproliferative
B-crystallin-/- cells was eightfold higher
than the wild-type or normal-growing
B-crystallin-/- cells (Fig. 2A
). The hyperproliferative cells can be considered to be
truly immortal, since they have already been passaged to >100
population doublings with no observable diminution in their
proliferative ability (Fig. 2B
). Expression of
A-crystallin in the hyperproliferative
B-crystallin-/- cells was observable at
levels similar to that in the normal-growing
B-crystallin-/- cells (Fig. 1F
, G
). Since growth rates can affect cell cycle distribution, we
examined propidium iodide-labeled cells by flow cytometry. As shown in
Fig. 2C
, the proportion of cells in the
G2/M phases was 9.6 ± 1.6% in wild-type
and 8.5 ± 2.0% in normal-growing
B-crystallin-/- cells, but increased to
27.7 ± 1.8% in the hyperproliferative
B-crystallin-/- cells.
|
Analysis of karyotypes of B-crystallin-/-
cells indicated the presence of cells with above normal chromosome
counts per metaphase (Fig. 3
). Of the 51 metaphases examined from a single hyperproliferative
B-crystallin-/- cell line, 60% were diploid
(2N), 28% were tetraploid, and 12% were of higher ploidy. Higher
ploidy metaphases were observed at an even greater level in the
B-crystallin-/- cells having normal growth
potential, with 23% diploid, 73% tetraploid, and 4% higher ploidy
metaphases. In contrast, wild-type cells were 91% diploid. These
observations suggest that B-crystallin has an important role in
maintaining genomic stability.
|
We next determined whether the hyperproliferative
B-crystallin-/- cells were capable of
growing on soft agar. A melanoma cell line, C8161, was used as positive
control and NIH 3T3 fibroblasts were used as a negative control.
Figure 4
shows that hyperproliferative
B-crystallin-/- cells (FGAB-1) did not grow
on soft agar in 3 wk, similar to NIH 3T3 fibroblasts (not shown). In
contrast, the C8161 cell line produced colonies in soft agar. This
observation suggests that the genomic instability introduced by
knocking out the expression of B-crystallin is in its early stages
of cellular evolution from normal to cancerous, and cells have not
acquired the ability to grow on soft agar (25)
. Consistent
with this observation, the hyperproliferative
B-crystallin-/- cells did not produce tumors
when injected subcutaneously into nude mice. In contrast, the C8161
cells produced well-developed tumors (data not shown).
|
To determine whether fellow lenses of a given animal produced
hyperproliferative clones, lens epithelial tissue from each capsule
epithelial explant of 39 B-crystallin-/-
mice was cultured in individual tissue culture plates, of which three
pairs of lens epithelial cultures produced hyperproliferative cells.
Moreover, whenever one culture from a given mouse produced
hyperproliferative cells, the epithelium from the contralateral lens
also produced hyperproliferative cells. This suggests that genomic
instability associated with loss of B-crystallin occurs early in
mouse development and at the level of individual animals, not
individual cells of a particular mouse. Furthermore, clonal cultures of
hyperproliferative primary cells plated at low density indicated that
each attached cell produced a hyperproliferative colony, suggesting
that most, if not all, lens epithelial cells of such an animal had
aberrant proliferative activity. To examine the morphology of the lens
producing hyperproliferative cells, tissue from one lens was placed in
culture and the fellow lens was fixed and sectioned. Examination of the
germinative region of the B-crystallin-/-
lenses giving rise to hyperproliferative lens epithelial cells
indicated a morphology that was not significantly altered from
age-matched wild-type or normal-growing
B-crystallin-/- lenses (Fig. 5
). The B-crystallin-/- mice did not get
cataracts and had no significant change in lens morphology during the
period examined from postnatal to 40 wk. Thus, it appears from the
observed ploidy changes and frequency of hyperproliferation of
B-crystallin-/- lens epithelial cells that
we have unmasked a phenotype of the
B-crystallin-/- lens that could not be
detected in vivo. This is consistent with reports that the
mitotic activity of the lens epithelium is enhanced by the removal of
fiber cells from the capsule epithelium (26)
. It also
suggests that epithelial–fiber interaction (27)
as well
as the presence of factors essential for normal growth and
differentiation masked the genetic instability of the
B-crystallin-/- cells in vivo.
|
Nontransformed B-crystallin-/- lens
epithelial cells (both normal-growing and hyperproliferative), like
wild-type and A-crystallin-/- cells, could
be maintained in confluent culture for extended periods (>60 days) and
remained viable. However, further evidence of enhanced genomic
instability of B-crystallin-/- lens
epithelial cells was obtained when primary cultures of wild-type and
B-crystallin-/- lens epithelial cells were
infected with the hybrid virus Ad12-SV40 to increase their ability to
proliferate. Although the wild-type,
B-crystallin-/-, and
A-crystallin-/- (used as a control) lens
epithelial cells produced cell lines that grew rapidly, only the lines
derived from wild-type and A-crystallin-/-
epithelial cells could be maintained in confluent cultures for periods
longer than 30 days without substantial cell death. In striking
contrast, B-crystallin-/- cells expressing
SV40 T antigen exhibited a widespread cytocidal response 2 to 3 days
after attaining confluence (Fig. 6
), indicating that SV40 T antigen-induced genetic instability results in
cell death of the B-crystallin-/- cells
only. To determine whether hyperproliferative primary cultures of
B-crystallin-/- lens epithelial cells also
displayed a cytocidal response in response to SV40 T antigen
expression, the hyperproliferative cells were infected with Ad12-SV40
hybrid virus. As observed with the normal-growing
B-crystallin-/- cells, expression of SV40 T
antigen also induced a cytocidal response in the hyperproliferative
B-crystallin-/- cells, although the cell
death occurred over a more protracted period of 10, instead of 3, days
after gaining confluence (data not shown).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
|---|
B-crystallin is a major lens protein expressed in numerous
non-lens tissues of vertebrates, consistent with its putative
nonrefractive cellular functions (3
, 11
12
13
14)
. It has been
shown to prevent the aggregation of denaturing proteins in
vitro, similar to other molecular chaperones, and its expression
increases under stress conditions (11
, 28
, 29)
. However,
the in vivo cellular functions of B-crystallin have not
been elucidated. B-crystallin normally is associated with
A-crystallin in the in vivo lens. During murine ocular
development, the transcription of B-crystallin precedes that of
A-crystallin (30)
. Recently, mice lacking the
A-crystallin gene were shown to develop cataracts (31)
and cultured lens epithelial cells derived from
A-crystallin-/- mice were shown to have a
decreased ability to proliferate (17)
. Unlike
A-crystallin-/- mice, the
B-crystallin-/- mice do not develop
cataracts (15)
. B-crystallin is particularly abundant
in muscle cells. B-crystallin-/- mice are
also deficient in the muscle-specific small heat shock protein HSPB2,
which is not expressed in the lens (16)
. [Using primers
specific to the HSPB2 gene and reverse transcriptase-polymerase chain
reaction, we did not detect any message in wild-type murine lens
epithelial cells (Z. Song and U. Andley, unpublished observations).]
These mice develop skeletal muscular dystrophy often characterized by
increased intracellular levels of desmin (15)
. Similarly,
patients who carry a missense mutation in B-crystallin (R120G)
develop desmin-related myopathy and cataracts (32)
.
The present work demonstrated that deletion of the murine
B-crystallin gene permits the emergence of lens epithelial cells
with an increase in polyploidy. However, a significant proportion
(23%) of normal-growing B-crystallin-/-
cells maintained normal ploidy. In contrast, 91% of the wild-type
cells were diploid. This suggests that the genomic instability of the
B-crystallin-/- cells is a property of the
culture population as a whole, perhaps due to a subtle defect in the
B-crystallin-/- cells. Note also that
hyperproliferation was never observed in the wild-type cultures, but
the B-crystallin-/- lens epithelial cells
demonstrated a wide variation in proliferative capacity. These results
suggest that the loss of B-crystallin may predispose cells to
undergo ploidy changes and hyperproliferation. During the cell cycle,
genomic changes such as spindle errors or spindle pole errors produce
aneuploidy or tetraploidy respectively in cells, whereas replication
errors produce chromosome aberrations (33)
. p53 monitors
genomic integrity at the G1 and
G2/M cell cycle checkpoints and cells lacking p53
show polyploidy or aneuploidy typical of many tumors
(33
34
35)
. The presence of tetraploid and higher ploidy
cells in B-crystallin-/- cultures suggests a
role of B-crystallin associated with cell cycle. Similarly, the
reported increase in expression of B-crystallin in mitotic
fibroblasts and its transient expression in the nucleus during
interphase of transfected CHO cells also suggest its involvement with
the cell cycle machinery (5
, 6)
. The phosphorylation of
A- and B-crystallin in vivo, their autokinase
activity, and the existence of enzymes in the lens, which can
dephosphorylate -crystallin, have previously suggested an
association of -crystallin with cellular signal transduction
pathways (36
37
38)
.
Maintenance of genomic integrity is important for cell survival, and
the tumor suppressor protein p53 is involved in this function
(39)
. Preservation of genomic stability encompasses many
factors, such as the maintenance of primary DNA sequence and the
preservation of chromosomal ploidy and structure (33)
.
Karyotypic alterations, including ploidy changes observed in the
B-crystallin-/- lens epithelial cells, are
common in cancer cells. It is well established that the expression of
oncogenes creates genomic instability (40
, 41)
. The
combination of p53 inactivation and oncogene expression markedly
accelerates the development of polyploidy, cell death, DNA
amplification, and tumor formation (33
34
35)
. Since genomic
instability is a hallmark of cancer cells, the overexpression of
B-crystallin during oncogene expression (9
, 10)
and in
tumors of neuroectodermal origin (7
, 8)
suggests that the
protein may accumulate as a stress response to maintain chromosomal
integrity during cellular evolution from a normal to a cancer cell.
This notion is consistent with the current finding that
B-crystallin-/- cells demonstrate genomic
instability.
The present work further supports a link between B-crystallin and
nuclear functions. When we introduced T antigen into the
B-crystallin-/- primary mouse lens
epithelial cells, it was found that unlike the nontransformed
B-crystallin-/- or wild-type lens epithelial
cells, SV40 T antigen-transformed cells could not survive confluence,
since >95% of the cells died by apoptosis a few days after gaining
confluence. This indicates that although the T antigen is normally a
growth enhancing protein, it triggers an apoptotic pathway in
postconfluent cells derived only from mice lacking B-crystallin.
Even though the mechanism by which cell death in the SV40 T-antigen
expressing B-crystallin-/- cell lines occurs
is not known, it is well established that oncogene expression creates
genomic instability (42
, 43)
. Together with other
oncoproteins or in the absence of tumor suppressor gene products,
oncogenes contribute to tumor formation by supporting accelerated
proliferation and deregulating cell cycle control and apoptosis
(44)
. The SV40 T antigen very likely enhances the genomic
instability inherent in B-crystallin-/- lens
epithelial cells. T antigen expression may also deregulate HSP25
expression, thus decreasing its level below a threshold necessary for
cell survival (45)
. The mechanism by which the loss of
B-crystallin enhances genomic instability needs further
investigation.
The hyperproliferation of B-crystallin-/-
lens epithelial cells that we observed in culture did not produce a
detectable in vivo lens phenotype, as shown by the lack of
significant differences in size and morphology of the lenses producing
hyperproliferative and normal-growing
B-crystallin-/- lens epithelial cells.
Previous studies suggest that the presence of contacts between lens
epithelial and fiber cells decreases the mitotic index of lens
epithelial cells in vivo (26
, 27)
. This may
explain the finding that in the
B-crystallin-/- lenses, genetic instability
or cataract formation did not occur in vivo. Aberrant
proliferation and apoptosis have been demonstrated in vivo
in the developing lenses of Rb-deficient mice (46)
and in
lenses of transgenic mice expressing polyoma large T antigen
(47)
, although these lenses showed no in vivo
lens tumor formation. Transgenic mice expressing viral oncoproteins E6
and E7 showed lens tumor formation (48)
. In the current
work, no tumors were noted in animals <40 wk old, but the formation of
tumors in older B-crystallin-/- mice could
not be evaluated (because the animals developed a severe skeletal
muscular dystrophy by 40 wk and were killed). Our studies indicate
that we have unmasked a subtle predisposition of
B-crystallin-/- lens epithelial cells to
undergo ploidy changes and hyperproliferation in culture. However, the
genomic instability we observed appears to be at early stages of
cellular changes that accompany evolution of normal to cancerous cells,
since the hyperproliferative B-crystallin-/-
cells did not grow on soft agar or produce tumors in nude mice. This
suggests that the number of genetic changes needed to cause
tumorigenicity has not occurred in the
B-crystallin-/- lenses.
The present studies indicate that culturing lens epithelial cells from
mice lacking B-crystallin resulted in effects that were extremely
different from those observed in cells derived from mice lacking
A-crystallin. The expression of A-crystallin is more lens
specific than that of B-crystallin, but is not induced by stress,
and A and B are complexed in 3:1 stoichiometry in lens fiber
cells (4
, 11)
. In contrast to the hyperproliferation and
ploidy changes observed in B-crystallin-/-
lens epithelial cells, the absence of A-crystallin decreased the
in vitro growth potential of cultured lens epithelial cells
(17)
, indicating that knocking out A or B-crystallin
affected the in vitro growth of lens epithelial cells in
opposite ways. This suggests that A and B-crystallin have
distinct and independent cellular functions in the lens. It will be
important in future work to determine whether A or B-crystallin
directly alter the regulation of the cell cycle in vivo.
Since secondary cataracts are primarily a growth problem, it would be
significant to understand whether the increase in lens epithelial cell
growth in secondary cataracts is associated with change in expression
of A or B-crystallins.
|
CONCLUSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
|---|
B-crystallin-/- mice suggest that
B-crystallin may be an important component of the cellular machinery
involved in maintaining genomic stability. By culturing lens epithelial
cells, a novel effect of knocking out a chaperone-like molecule was
unmasked. The unique phenomenon described here for the stress-inducible
chaperone B-crystallin may be particularly significant in growing
tissues, tumors, and neurodegenerative diseases. Genomic instability
has been associated with other dystrophic diseases such as myotonic
dystrophy (49
, 50)
, but it is not known whether it is
involved with the reported in vivo phenotype of the
B-crystallin-/- mice (15)
. It
is likely that maintaining genomic integrity is important in the lens,
since the anterior lens epithelial cells are held in
G0 phase of the cell cycle throughout life (even
though they are capable of mitosis when subjected to injury) and cell
division is restricted to the germinative zone (51
, 52)
.
The proliferation and ploidy changes of the
B-crystallin-/- cells provide a basis for
future work on B-crystallin interactions with cell cycle components,
including p53, the retinoblastoma protein Rb, cyclins, and
cyclin-dependent kinases.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Received for publication May 15, 2000.
Revision received June 15, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
|---|
B-crystallin gene is not restricted to the lens. Mol. Cell. Biol. 9,1083-1091B subunit of lens-specific protein -crystallin is present in other ocular and non-ocular tissues. Biochem. Biophys. Res. Commun. 158,319-325[Medline]
-crystallin. Proc. Natl. Acad. Sci. USA 89,10449-10453B-crystallin in Chinese hamster ovary cells suggests a nuclear role for this protein. Eur. J. Cell Biol. 78,143-150[Medline]
B-crystallin interacts with cytoplasmic intermediate filament bundles during mitosis. Exp. Cell Res. 253,649-662[Medline]
B-crystallin in astrocytic elements of neuroectodermal tumors. Cancer 68,2230-2240[Medline]
-crystallin/small heat shock protein/molecular chaperone gene in the lens and other tissues. Adv. Enzymol. Relat. Areas Mol. Biol. 69,155-201[Medline]
B-crystallin as candidate autoantigen in multiple sclerosis. Nature (London) 375,798-801[Medline]
B-crystallin in Parkinson’s disease. NeuroReport 10,2273-2276
B-crystallin gene knockout mice develop a severe fatal phenotype late in life. Invest. Ophthalmol. Vis. Sci. 39,S523
A-crystallin enhances lens epithelial cell growth and resistance to UVA stress. J. Biol. Chem. 273,31252-31261-crystallin/small heat-shock protein family. Mol. Biol. Evol. 10,103-126[Abstract]
A-crystallin gene induces cataract and cytoplasmic inclusion bodies containing the small heat shock protein B-crystallin. Proc. Natl. Acad. Sci. USA 94,884-889B-crystallin chaperone gene causes a desmin-related myopathy. Nature Genet 20,92-93[Medline]
-crystallin. Proc. Natl. Acad. Sci. USA 82,4712-4716This article has been cited by other articles:
|
|
 |
|
  M. Saint-Geniez, T. Kurihara, and P. A. D'Amore Role of Cell and Matrix-Bound VEGF Isoforms in Lens Development Invest. Ophthalmol. Vis. Sci., January 1, 2009; 50(1): 311 - 321. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-h. Xi, F. Bai, J. Gross, R. R. Townsend, A. S. Menko, and U. P. Andley Mechanism of Small Heat Shock Protein Function in Vivo: A KNOCK-IN MOUSE MODEL DEMONSTRATES THAT THE R49C MUTATION IN {alpha}A-CRYSTALLIN ENHANCES PROTEIN INSOLUBILITY AND CELL DEATH J. Biol. Chem., February 29, 2008; 283(9): 5801 - 5814. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-h. Xi, F. Bai, R. McGaha, and U. P. Andley Alpha-crystallin expression affects microtubule assembly and prevents their aggregation FASEB J, May 1, 2006; 20(7): 846 - 857. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Pappa, D. Brown, Y. Koutalos, J. DeGregori, C. White, and V. Vasiliou Human Aldehyde Dehydrogenase 3A1 Inhibits Proliferation and Promotes Survival of Human Corneal Epithelial Cells J. Biol. Chem., July 29, 2005; 280(30): 27998 - 28006. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Kumar, M. Kapoor, S. Sinha, and G. B. Reddy Insights into Hydrophobicity and the Chaperone-like Function of {alpha}A- and {alpha}B-crystallins: AN ISOTHERMAL TITRATION CALORIMETRIC STUDY J. Biol. Chem., June 10, 2005; 280(23): 21726 - 21730. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. K. Gangalum, M. J. Schibler, and S. P. Bhat Small Heat Shock Protein {alpha}B-Crystallin Is Part of Cell Cycle-dependent Golgi Reorganization J. Biol. Chem., October 15, 2004; 279(42): 43374 - 43377. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Der Perng, S. F. Wen, P. van den IJssel, A. R. Prescott, and R. A. Quinlan Desmin Aggregate Formation by R120G {alpha}B-Crystallin Is Caused by Altered Filament Interactions and Is Dependent upon Network Status in Cells Mol. Biol. Cell, May 1, 2004; 15(5): 2335 - 2346. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. A. Stronach, G. C. Sellar, C. Blenkiron, G. J. Rabiasz, K. J. Taylor, E. P. Miller, C. E. Massie, A. Al-Nafussi, J. F. Smyth, D. J. Porteous, et al. Identification of Clinically Relevant Genes on Chromosome 11 in a Functional Model of Ovarian Cancer Tumor Suppression Cancer Res., December 15, 2003; 63(24): 8648 - 8655. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Bai, J. H. Xi, E. F. Wawrousek, T. P. Fleming, and U. P. Andley Hyperproliferation and p53 Status of Lens Epithelial Cells Derived from {alpha}B-crystallin Knockout Mice J. Biol. Chem., September 19, 2003; 278(38): 36876 - 36886. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Xi, F. Bai, and U. P. Andley Reduced survival of lens epithelial cells in the {alpha}A-crystallin-knockout mouse J. Cell Sci., March 15, 2003; 116(6): 1073 - 1085. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-J. Sohn, M. Choi, J. Song, S. Chan, A. Medline, S. Gallinger, and Y.-I. Kim Msh2 deficiency enhances somatic Apc and p53 mutations in Apc+/-Msh2-/- mice Carcinogenesis, February 1, 2003; 24(2): 217 - 224. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. S. Mchaourab, E. K. Dodson, and H. A. Koteiche Mechanism of Chaperone Function in Small Heat Shock Proteins. TWO-MODE BINDING OF THE EXCITED STATES OF T4 LYSOZYME MUTANTS BY alpha A-CRYSTALLIN J. Biol. Chem., October 18, 2002; 277(43): 40557 - 40566. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. P. Andley, H. C. Patel, and J.-H. Xi The R116C Mutation in alpha A-crystallin Diminishes Its Protective Ability against Stress-induced Lens Epithelial Cell Apoptosis J. Biol. Chem., March 15, 2002; 277(12): 10178 - 10186. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-J. Sohn, S. A. Shah, S. Reid, M. Choi, J. Carrier, M. Comiskey, C. Terhorst, and Y.-I. Kim Molecular Genetics of Ulcerative Colitis-associated Colon Cancer in the Interleukin 2- and {beta}2-Microglobulin-deficient Mouse Cancer Res., September 1, 2001; 61(18): 6912 - 6917. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
6. C)
Flow cytometric analysis of propidium iodide-labeled wild-type,
normal-growing 
