1
Marion Bessin Liver Research Center, Albert Einstein College of Medicine,
Bronx, New York, NY 10461, USA
2
Cancer Research Center, Albert Einstein College of Medicine, Bronx, New York,
NY 10461, USA
3
General Clinical Research Center, Albert Einstein College of Medicine, Bronx,
New York, NY 10461, USA
4
Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx,
New York, NY 10461, USA
5
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York,
NY 10461, USA
*
Author for correspondence (e-mail:
sanjvgupta{at}pol.net
)
Accepted May 3, 2001
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SUMMARY |
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Key words: Growth factor, Hepatocyte, Liver, Ploidy, Regeneration
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INTRODUCTION |
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Hepatic polyploidy accompanies late fetal development and postnatal
maturation (Sigal et al.,
1999) and its onset in the
adult liver is well recognized (Brodsky and Uryvaeva,
1977
; Carriere,
1969
; Alison and Wright,
1985
). Moreover, weaning and
commencement of feeding (Dallman et al.,
1974
; Barbason and Houbrechts,
1974
), compensatory liver
hypertrophy following partial hepatectomy (Bucher et al., 1963; Brodsky and
Uryvaeva, 1977
; Sigal et al.,
1999
), toxin and drug-induced
liver disease (Bohm and Noltemeyer,
1981
; Madra et al.,
1995
; Kato et al.,
1996
; Aardema et al.,
1998
), as well as
administration of specific growth factors and hormones (Printseva et al.,
1989
; Cruise et al.,
1989
; Torres et al.,
1999
) may induce hepatic
polyploidy. Although liver growth control has long been studied, whether the
replication potential of polyploid hepatocytes is altered remains unresolved,
in part, owing to difficulties in distinguishing between cellular DNA
synthesis and generation of daughter cells (Simpson and Finckh,
1963
, Solopaev and Bobyleva,
1981
). More recently,
transplanted cells were shown to integrate into the liver parenchyma and then
to repopulate the host liver (Gupta et al.,
1995
). Regulated proliferation
in transplanted cells, with no proliferation within the normal liver, and
extensive proliferation in animals where endogenous host hepatocytes were lost
selectively, permitted establishment of clonogenic type assays in intact
animals (Rhim et al., 1994
;
Overturf et al., 1997
; Mignon
et al., 1998
; Laconi et al.,
1998
; Gupta et al., 1999a;
Guha et al., 1999b).
Here we provide evidence from studies utilizing a variety of systems, including a genetically defined cell transplantation system in F344 rats, to demonstrate the proliferation capacity of polyploid rat hepatocytes. We found evidence of oxidative DNA injury in polyploid hepatocytes isolated from rats subjected to two-thirds partial hepatectomy (PH). Moreover, we were able to recapitulate polyploidy in cultured primary rat hepatocytes following mitogenic stimulation in the setting of oxidative DNA injury.
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MATERIALS AND METHODS |
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Two-thirds PH was performed under ether anesthesia between 8 and 10 AM
according to Higgins and Anderson (Higgins and Anderson,
1931), without restrictring
food or water intake. Several DPPIV-deficient rats were treated with 30 mg/kg
retrorsine (Sigma Chemical Co., St. Louis, MO) intraperitoneally at 6 and 8
weeks of age as described previously (Laconi et al.,
1998
). Four weeks after the
final retrorsine dose, animals were subjected to two-thirds PH followed by
injection of 5x106 F344 rat hepatocytes into the spleen as
described previously (Gupta et al.,
1995
; Gupta et al., 1999a).
Intrasplenic injection deposits hepatocytes into liver sinusoids and cells
integrate subsequently in the liver parenchyma (Gupta et al.,
1997
; Gupta et al.,
1999b
).
Hepatocyte isolation and culture
The liver was perfused in situ with collagenase to isolate hepatocytes, as
described previously (Gupta et al.,
1997). Hepatocytes were plated
on collagen-coated dishes at 4x104 cells/cm2 in
RPMI 1640 medium containing penicillin, streptomycin, amphotericin B and 10%
fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA). The cell
viability was estimated by exclusion of Trypan Blue dye. The culture medium
was changed 3 hours after cell attachment. Cells were cultured for up to 5
days and harvested by trypsinization and centrifugation under 500
g for 5 minutes at 4°C. Cells were washed twice with cold
phosphate-buffered saline (PBS) pH 7.4.
Radiation of isolated hepatocytes
A Gamma Cell-40 Irradiator (Atomic Energy of Canada, Ottawa, Canada) with
Cesium-137 as the source was used. Radiation dose was administered at 84
cGy/minute and cells were radiated to a total of 10-30 Gy. For transplantation
studies, hepatocytes were suspended in RPMI 1640 medium at
2x106 cells/ml, radiated to 30 Gy immediately after cell
isolation and transplanted into animals within 2 hours of isolation.
DNA synthesis rates
After specified periods, [3H]thymidine (3 µCi, 70 Ci/mmole,
ICN Radiochemicals, CA) was added to cultured hepatocytes for 1 hour. Cells
were washed twice with cold PBS and DNA was extracted as described (Gupta et
al., 1992). 3H
activity was measured by liquid scintillation counting in an aliquot, and DNA
microquantitation by fluorimetry. All experiments were in triplicate at least.
To induce DNA synthesis, 20 ng/ml transforming growth factor
(TGF
), 0.1 µM norepinephrine and 25 µM vasopressin (Sigma) were
added to the culture medium in some experiments.
Flow cytometric analysis of cell ploidy
Changes in cell profiles were analyzed in isolated nuclei as described
previously (Sigal et al.,
1999). Cells were harvested by
trypsinization, washed with PBS once and resuspended in citrate buffer (250 mM
sucrose, 40 mM trisodium citrate.2H2O and 5% DMSO). Cells stored at
-80°C were rapidly thawed to 37°C and lyzed in 100 µl citrate
buffer containing 900 µl trypsin, 3.4 mM trisodium citrate.2H2O,
0.1% NP-40 and 1.5 mM spermine tetrahydrochloride, pH 7.6, at room temperature
for 10 minutes with occasional mixing. The nuclei were stained with 0.04%
propidium iodide on ice. Flow cytometry was carried out with the FACSTAR plus
machine (Becton Dickinson, San Jose, CA). Typically, approximately 10,000
events were collected. Data were analyzed with Lysis II software. A laser
scanning cytometer (Compucyte, Cambridge, MA) was used to document cell ploidy
in parallel. Each condition was examined at least in triplicate and
experiments were reproduced on more than five occasions.
Total glutathione content
Unless specified, all chemicals below were from Sigma. Harvested cells were
stored at -80° in 6% salicylic acid. Cells were thawed to 4°C and
disrupted by ultrasonication, as described previously (Anderson,
1985). Cell debris were
eliminated by pelleting at 10,000 g for 10 minutes at 4°C.
To 100 µl supernatant, 800 µl 0.3 mM nicotinamide adenine dinucleotide
phosphate, reduced form (NADPH), 100 µl of 6 mM 5.5'-dithiobis
(2-nitrobenzoic acid) and 0.5 U glutathione reductase were added. Changes in
absorbance at 412 nm were measured for 2 minutes. Glutathione standards were
prepared in a linear range with 100 µM stock solution. Protein content was
assayed in aliquots using the Bradford assay. All experiments were done in
triplicate.
Catalase activity
Frozen cells were thawed on ice, ultrasonicated and then centrifuged at
10,000 g for 10 minutes at 4°C according to the methood of
Luck (Luck, 1965). To 10-40
µl supernatant, 3 ml H2O2-phosphate buffer was added
in a silica cuvette. The time (T) for change in optical density at 240 nm from
0.45 to 0.40 was measured at room temperature. The catalase activity was
derived from the formula 17/T=units/assay mixture. Protein content was
determined in aliquots using the Bradford assay. Data were expressed as
catalase units/µg protein using triplicate conditions.
Lipid peroxidation
Harvested cells were suspended in 100 µl deionized water and
peroxidation was measured using a calorimetric kit, according to the
manufacturer's instructions (Calbiochem-Novabiochem Corp., San Diego, CA). All
conditions were in triplicate.
Localization of senescence-associated ß-galactosidase (SABG)
activity
Cultured cells were fixed in 0.5% glutaraldehyde in PBS (pH 7.4) at room
temperature for 10 minutes. Cells were washed and incubated at 37°C with
5-bromo-4-chloro-3-indolyl ß-galactoside (X-gal) in either PBS or citric
acid/sodium phosphate buffer, pH 6.0, according to the method of Dimri et al.
(Dimri et al., 1995) for 18
hours.
Tissue analysis
Samples from the median liver lobe were frozen in methylbutane at -70°C
and 5 µm thick cryostat sections were prepared.
To demonstrate oxidative DNA injury, sections were fixed in 70% ethanol and stained with anti-8-oxo-2' dG (Trevigen, Gaithersburg, MD, Catalog No. 4355-MC-100) was according to the manufacturer's instructions. Briefly, sections were digested with RNase (100 µg/ml) for 1 hour at 37°C. DNA was denatured by immersion in 4 M HCl for 7 minutes and neutralized in 50 mM Tris-base. After blocking with 10% fetal bovine serum, tissues were incubated with antibody diluted 1:300, at 37°C for 16 hours. Antibody binding was localized with a biotinylated multilink secondary antibody (Biogenex, San Ramon, CA). Endogenous peroxidase was quenched with 3% H2O2 in methanol for 30 minutes. The Vectastain ABC system (Vector Laboratories, Burlingame, CA) was used for visualization of antibody binding and color was developed with the enhanced diaminobenzidine substrate (Dako Corporation, Carpinteria, CA).
Transplanted cells were identified in DPPIV-deficient F344 rats by
detecting DPPIV activity histochemically as described previously (Gupta et
al., 1995). Tissue sections
were fixed in chloroform-acetone (1:1 vol/vol) at 4°C. Liver from normal
F344 rats was included as a positive control and liver from DPPIV-deficient
rats, which were not subjected to cell transplantation, served as a negative
control.
For morphometric analysis, multiple photomicrographs of all conditions were
obtained. The number of transplanted cell foci in 44-56 consecutive
high-magnification fields was determined. Individual transplanted cells in
each of these foci were identified as described previously (Sokhi et al.,
2000). To establish the number
of transplanted cells composing each cell focus, 43-66 cell foci were scored
in each animal and the data tabulated for comparison.
Statistical analysis
The data were analyzed with the SigmaStat software (Jandel Scientific, San
Rafael, CA), and expressed as mean±s.d. The significance of differences
was analyzed by the Student's t-test, 2 test,
Mann-Whitney rank correlation test for non-parametrically distributed data,
Kruskall-Wallis one way analysis of variance (ANOVA), and Dunn's test to
isolate groups differing from others. P values <0.05 were
considered significant.
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RESULTS |
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Analysis of oxidative injury
Depletion of cellular glutathione content and catalase activity and
increase in lipid peroxidation were used to assess the onset of oxidative
injury. Hepatocytes were isolated from three normal rats and three rats on
which two-thirds PH had been performed 5 days previously. The total cellular
glutathione content, as well as catalase activity, declined significantly in
hepatocytes from animals subjected to PH
(Table 1). Also, hepatocytes
from animals subjected to PH exhibited greater lipid peroxidation. Our lipid
peroxidation assay demonstrated malonaldehyde and 4-hydroxyalkenal
accumulation in cells. The findings were in agreement with induction of
oxidative stress in hepatocytes after PH.
|
Immunostaining with an antibody that recognizes 8-hydroxyguanine adducts established that PH caused oxidative DNA injury. The portion of the liver that was removed served as an internal control for each animal (n=3). Liver samples were obtained from animals at 5 days after PH for the analysis. In control livers, only occasional hepatocytes showed 8-hydroxyguanine adducts (Fig. 1). Additional biliary and arteriolar cells also showed immunostaining. In contrast, 5 days after PH, 8-hydroxyguanine adducts became apparent in most hepatocytes. The nuclei of these cells with oxidative DNA injury often had a `stippled' appearance, possibly indicating extensive adduct formation. On occasion, cells with evidence for oxidative DNA adduct formation were found to be undergoing apoptosis. Morphometric analysis showed that in control livers, 8±6 nuclei per high-magnification field (x400) had evidence for oxidative DNA adduct formation. After PH, this increased several-fold, with 90±20 nuclei per high-magnification field showing oxidative DNA damage, P<0.001, t-test.
|
To further establish the consequences of oxidant stress in hepatocytes, we exposed cells to ionizing radiation, which induces release of free radicals. Hepatocytes were irradiated after attachment to culture dishes and they were then cultured for 1, 3 and 5 days. After radiation, hepatocytes from normal control rats showed dose-dependent decreases in glutathione and catalase activity, along with increased lipid peroxidation, which was in agreement with oxidative injury (Fig. 2). Hepatocytes from livers after PH showed a similar pattern of oxidative injury, albeit with greater changes in glutathione, catalase and lipid peroxidation after radiation. At 5 days after 30 Gy radiation, glutathione content in hepatocytes from livers after PH was only 50% of that in control hepatocytes, 19±0.6 nmol/µg protein versus 10±0.3 nmol/µg protein, P<0.005, t-test. Catalase activity decreased in control hepatocytes after radiation. This decrease was more pronounced in hepatocytes from livers after PH, where catalase activity following radiation became undetectable by the third day of culture. Finally, when lipid peroxidation was examined in cells cultured for 5 days after radiation, hepatocytes from PH livers showed 2-fold greater lipid peroxidation compared with cells from the normal liver, P<0.05. These findings indicated that hepatocytes exposed to oxidative injury after PH were more susceptible to radiation-induced oxidative injury.
|
Does oxidative injury increase susceptibility for hepatic
polyploidy?
If oxidative injury were responsible for cellular DNA damage, induction of
polyploidy would be affected. Nuclear DNA was analysed with flow cytometry
after cell culture for 5 days. Hepatocytes were studied on four occasions from
unperturbed rats and rats subjected to PH.
Culture for 5 days altered the initial ploidy classes of hepatocytes. In cells from the unperturbed liver, the diploid fraction decreased from 29±1% to 25±0.6% after 5 days, P<0.001, t-test, the tetraploid cell fraction was unchanged at 67±3% and 68±2%, p=n.s., and the octaploid cell fraction increased from 1±0.5% to 6±0.5%, P<0.001. Ploidy of cells from the normal liver began to resemble initial ploidy distributions of cells from liver 5 days after PH, where the diploid cell fraction constituted 19±2%, the tetraploid cell fraction 75±3% and the octaploid cell fraction 6±1%. However, after 5 days of culture, hepatocytes from PH livers showed more diploid cells, 46±2%, P<0.001, t-test, and fewer tetraploid cells, 48±3%, P<0.001, t-test, while the fraction of the polyploid cells remained unchanged, 5±0.3%. Such diploid cell enrichment could have arisen from transition of cells to the next ploidy class with depletion of the most highly polyploid cells from the culture. Alternatively, diploid cells could have proliferated selectively.
The response of cultured cells to radiation provided further information. For these assays, cells were attached to tissue culture plates for 24 hours, exposed to either 10 Gy or 30 Gy radiation and then cultured for an additional 4 days. Changes in cell ploidy are summarized in Table 2. In hepatocytes from unperturbed livers, diploid cell fractions decreased modestly after radiation, albeit significantly, without much change in the tetraploid or the octaploid cell fractions. In contrast, after radiation, hepatocytes from PH animals showed a two-fold increase in the proportion of octaploid cells to approx. 10%. SABG activity was used to demonstrate whether oxidative injury induced senescence-type changes in cells from PH livers. The fraction of cells with pancytoplasmic SABG staining was analyzed in at least 1000 cells in 10 random microscopic fields for each cell type. Radiation increased SABG expression by 2.6±0.5-fold (2.7±0.3% versus 7.2±1.5%, P<0.001, t-test). These findings suggested that oxidative injury caused cell ploidy to increase with activation of senescence-type changes.
|
Hepatic polyploidy and cell proliferation
To demonstrate whether polyploidy perturbed the ability of cells to
proliferate, we used an in vivo assay in DPPIV-deficient F344 rats. Treatment
of rats with retrorsine and two-thirds PH prior to cell transplantation
induces extensive proliferation in transplanted cells (Laconi et al.,
1998).
Groups of four to six DPPIV-deficient rats were established as follows. Group I received hepatocytes from unperturbed F344 rats; Group II received hepatocytes from F344 rats 5 days after two-thirds PH; Group III received hepatocytes radiated to 30 Gy from unperturbed F344 rats; and Group IV received hepatocytes irradiated to 30 Gy from F344 rats 5 days after two-thirds PH. Two rats in each group were sacrificed at 2 days after cell transplantation to determine whether cells had engrafted in the liver. All rats were sacrificed 10 days after cell transplantation to analyze cell proliferation.
Two days after cell transplantation, similar numbers of transplanted cells were observed in the liver of all DPPIV-deficient recipients. Transplanted cells were distributed in periportal areas at this time with 1-3 cells per portal area, which indicated that engraftment of the cells was unperturbed (Fig. 3). Ten days after cell transplantation, Group I animals showed large foci of transplanted cells, with 53±30 cells per focus. In contrast, Group II recipients showed far smaller foci of transplanted cells, with 18±11 cells per focus, representing a 2.9±1.7-fold decrease in cell numbers, P<0.001, Mann Whitney rank sum correlation. Group III recipients showed smaller foci of transplanted cells, similar to Group II recipients, with 22±13 cells per focus, which indicated a decline in the number of transplanted cells by 2.4±1.4-fold, P<0.001. The size of transplanted cell foci was the smallest in Group IV animals, which received hepatocytes from PH liver and radiation, with only 4±3 transplanted cells per focus, which was 13.3±7.5-fold less than Group I cell recipients, P<0.001. Additional data from these studies are shown in Table 3.
|
|
Recapitulation of cellular polyploidy following oxidative injury
If oxidative injury were causally involved in polyploid induction, this
change should be reproducible in suitable systems. To demonstrate this, we
studied primary hepatocytes from unperturbed rats. We reasoned that as
polyploid cells contain greater amounts of DNA, advancement of ploidy would
require induction of cellular DNA synthesis by adding growth factors, such as
TGF, to the culture medium. It was hoped that this would mimic some
aspects of PH-induced hepatic DNA synthesis. Also, in some experiments, we
added norepinephrine (NE) and vasopressin (VP), which are released after PH
and modulate growth factor responses in cultured hepatocytes. To induce
oxidative injury, cultured cells were exposed to 30 Gy radiation. Assays
involving cells cultured for 48 hours, to verify the bioactivity of
TGF
, showed 3- to 5-fold greater DNA synthesis rates in stimulated
cells, P<0.001, t-test. NE and VP increased
TGF
-induced DNA synthesis further by between 1.4 and 1.6 fold,
P<0.01, t-test. As expected, radiation of cells before
adding TGF
abrogated hepatocellular DNA synthesis. The final
experimental protocol involved addition of TGF
to hepatocytes within 2
hours of culture, radiation 24 hours after the start of culture and analysis
of cells after 4 more days in culture with TGF
.
The findings from one of two experiments providing similar results are
shown in Fig. 4. Exposure to
TGF by itself increased enrichment of octaploid cells to approx. 10%,
P<0.001,
2 test. After cell culture with
TGF
and either NE or VP, cellular ploidy advanced further, with
octaploid hepatocytes constituting up to 15%, P<0.001. Moreover,
radiation of cells cultured with TGF
alone or with the addition of
either NE or VP advanced cell ploidy even more with decline in diploid cell
fractions and rise in octaploid cell fractions. The advancement in cell ploidy
was verified to be associated with the appearance of megalonuclei in our
cultured cells.
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DISCUSSION |
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The findings provide direct evidence for impaired postmitotic activity in
polyploid cells, which has implications in liver growth control (Gupta,
2000). Moreover, our findings
should have wider biological implications because polyploidy is an ubiquitous
process in tissues and organs, particularly after various types of
hypertrophic stimuli, e.g., hypertension (myocardial cells), lactation
(mammary glands), or pregnancy (endometrial cells), microbial infection
(lymphocytes), oncogenesis (multiple tumors and cell lines) etc. (Brodsky and
Uryvaeva, 1977
; Gupta,
2000
). In other situations,
reactive oxygen species are generated after exposure to xenobiotics, metals,
such as iron and copper, mineral dusts and chemotherapeutic drugs. The
hyroxyl-free radicle reacts aggressively with other molecules leading to
8-hydroxy adducts of guanine (Toraason et al.,
1999
). Consequential DNA
repair results in 8-hydroxy-2'-deoxyguanosine adducts, as shown above.
Further evidence for the role of oxidative injury in polyploidy is provided by
studies showing that in transgenic mice overexpressing copper-zinc-superoxide
dismutase and glutathione peroxidase, which are antioxidants, PH-induced
hepatic polyploidization is decreased (Nakatani et al.,
1997
). Similarly, treatment
with aminoguanidine, which attenuates oxidative stress, decreased polyploidy
(Diez-Fernandez et al.,
1998
).
If one considers models of cell lineage-dependent organ development, where
replacement of epithelial cells helps maintain structure-proliferation units
(Slack, 2000), extreme
polyploidy would be expected to be associated with cell senescence and
eventual cell loss. Sigal et al. (Sigal et al.,
1999
) showed that SABG
expression, p21 expression and apoptosis were induced by PH in remnant
hepatocytes, which are associated with terminal cell differentiation. Although
genetic regulation of polyploidy has not been fully defined, it is noteworthy
that in p21 transgenic mice hepatocytes become polyploid and have smaller
liver lobules indicating the presence of fewer hepatocytes in the liver (Wu et
al., 1996
). Studies using
colon carcinoma cells showed that polyploidy, induced by p21, increases
spontaneous apoptosis rates, as well as susceptibility to ionizing radiation
(Waldman et al., 1997
).
Although we did not study regulation of p21 or other activities in our cells,
greater SABG activation in cultured hepatocytes and enhanced polyploidy after
multiple oxidative injuries, i.e., partial hepatectomy and radiation, were in
agreement with such models. We chose to study the effects of radiation in our
cells for two specific reasons. First, ionizing radiation is well known to
induce oxidative stress, through physical changes, without metabolic
activation and the possible subsequent perturbations in metabolic pathways as
with chemical inducers of oxidative stress. Secondly, radiation could be
administered equally to all cells, irrespective of their ploidy class
distributions, whereas use of chemical agents to induce oxidative stress could
potentially have been affected by differences in the metabolic properties of
various hepatocyte subpopulations (Rajvanshi et al.,
1998
). Of course, radiation
had the disadvantage of inducing some DNA damage directly rather than solely
through the activation of oxidative stress. Therefore, additional analysis of
polyploidy induction with drugs or chemicals known to cause oxidant stress
through specific mechanisms should be helpful.
Use of rats prepared with retrorsine-PH to induce proliferation in
transplanted hepatocytes established that polyploid hepatocytes from PH livers
were less capable of producing daughter cells. Moreover, cell proliferation
ceased almost completely when hepatocytes from PH livers were treated
additionally with radiation. It was noteworthy that some cells subjected to PH
alone, as well as radiated cells from the unperturbed normal liver, showed
proliferation in our in vivo assay. Previous work by Overturf and colleagues
(Overturf et al., 1997)
established that adult mouse hepatocytes may possess stem-cell-like properties
with indefinite proliferation in intact mice. The studies involved
transplantation of unfractionated hepatocytes from normal mice into the liver
of genetically diseased FAH mice, in which tyrosinemia leads to selective and
progressive loss of endogenous hepatocytes. However, it was unclear whether
indefinite replication capacity was a feature of all transplanted hepatocytes
or whether replication capacity was restricted to hepatocyte subsets. Our
studies suggest that polyploid hepatocytes were significantly less capable of
proliferating, although cellular proliferation capacity was lost only after
more than one injury (PH plus radiation) and presumably extensive DNA injury.
Attenuation of proliferative capacity in hepatocyte subpopulations is further
substantiated by studies using diploid and polyploid rat hepatocytes
fractionated from the normal liver (Rajvanshi et al.,
1998
). Also, studies using
`small hepatocytes' from the rat liver, which constitute diploid cell
populations, indicate that small cells proliferate far more than larger
hepatocytes (Mitaka et al.,
1992
; Tateno et al.,
2000
). An exception to these
findings was reported by Grompe and colleagues using mouse hepatocytes, in
which transplantation of diploid cells was less effective in clonogenic liver
repopulation assays (Overturf et al.,
1999
); however,
cotransplantation of unfractionated cells could have confounded their
analysis. Another set of studies from Sandgren and colleagues using mouse
hepatocytes fractionated with flow cytometry was interpreted as showing no
difference in the behavior of diploid and higher ploidy cells (Weglarz et al.,
2000
). However, these studies
did not exclude the possibility of DNA damage from laser energy during flow
cytometric cell separations, which could have altered cell proliferation
capacity, as suggested by limited proliferation in their diploid cells
subjected to this manipulation. Therefore, our studies offer new information
by clearly establishing restriction in the replication potential of polyploid
cells subjected to oxidative DNA injury.
We found it of interest that polyploidy was induced when mitogenically
stimulated hepatocytes were exposed to oxidative injury. We used TGF to
stimulate DNA synthesis in our cells, although other hepatic growth factors,
such as hepatocyte growth factor, epidermal growth factor, etc., could also
have been utilized (Gupta et al.,
1992
; Sigal et al.,
1999
). Hepatic stimulation
with TGF
increases hepatic polyploidy in intact transgenic animals (Lee
et al., 1992
). Costimulation
of these cells with soluble signals (NE and VP) that mediate `stress'
responses greatly amplified cellular polyploidization. These findings are
especially relevant in the setting of partial hepatectomy, because NE is
released after partial hepatectomy and VP is known to play significant roles
in liver regeneration after partial hepatectomy (Russell and Bucher,
1983
; Knopp et al.,
1999
). In view of the wide
distribution in tissues of NP, VP and related hormones and exposure of
virtually all cells to oxidizing events, it is likely that our findings will
be of physiological relevance to nonhepatic epithelia. Again, in
structure-proliferation models of acinar renewal, interactions with
extracellular matrix components, stromal cells, growth factors and ambient
soluble signals, are critical elements in activating cell renewal versus
terminal differentiation (Slack,
2000
).
Models were recently proposed to demonstrate how senescence-type changes in
polyploid hepatocytes would be relevant in diseases (Gupta,
2000). One consideration is
that if an organ contains excessive numbers of polyploid cells with depletion
of renewing cell units, organ failure may occur in the setting of continuing
liver injury because polyploid cells will exhibit survival disadvantage. For
instance, liver failure and death occur when hepatocytes fail to survive in
the setting of hepatic polyploidy, such as in mutant mice with impaired
nucleotide excision and repair (McWhir et al.,
1993
). Secondly, to escape
this fate, nonpolyploid cell clones with resistance to ongoing disease
processes may emerge and confer greater propensity for oncogenesis. Our
findings of impaired proliferation capacity in polyploid cells are in
agreement with these possibilities and should thus provide further conceptual
frameworks in areas concerning organ development, regeneration and
oncogenesis.
From a translational perspective, induction of polyploidy with oxidative
DNA injury will have potential for therapeutic liver repopulation with
transplanted cells. Work from our laboratory and other laboratories has shown
that selective proliferation of transplanted cells is needed for significant
liver repopulation (Rhim et al.,
1994; Overturf et al.,
1997
; Mignon et al.,
1998
; Laconi et al.,
1998
; Gupta et al.,
1999
; Guha et al.,
1999
). Extensive polyploidy is
induced in rats treated with retrorsine and PH (Gupta,
2000
), which as shown here,
permits proliferation of transplanted cells in the liver. Retrorsine can be
combined with repeated tri-iodothyronine (T3) instead of PH for inducing
transplanted cell proliferation (Oren et al.,
1999
). We consider that T3 may
be effective because thyroid hormones regulate polyploidy following PH (Torres
et al., 1999
). Similarly, the
combination of radiation and PH, which induces hepatic polyploidy, as shown
here, permits extensive transplanted cell proliferation (Guha et al.,
1999
). Finally, when we
extended our findings and conditioned the host rat liver with oxidative
hepatic injury using radiation and ischemia-reperfusion, it became possible to
repopulate virtually the entire liver (Malhi et al.,
2000
; manuscript in
preparation). Therefore, additional strategies to induce polyploidy in
endogenous cells by oxidative DNA injury should facilitate organ repopulation
with unaffected normal cells and help obtain further insights into biological
mechanisms in the context of cellular polyploidy.
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ACKNOWLEDGMENTS |
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