(American Journal of Pathology. 2002;160:31-35.)
© 2002 American Society for Investigative Pathology
Liver Repopulation by Bcl-xL Transgenic Hepatocytes
Claudia Mitchell,
Vincent O. Mallet,
Jacques E. Guidotti,
Cyril Goulenok,
Axel Kahn and
Hélène Gilgenkrantz
From the Department of Genetics, Development and Molecular
Pathology, Institut Cochin de Génétique Moléculaire,
Paris, France
 |
Abstract
|
|---|
Liver repopulation could constitute a potential therapeutic
alternative to liver transplantation in the future. Therefore,
the development of liver repopulation strategies is of major interest.
We have previously reported that Bcl-2-expressing hepatocytes
are resistant to Fas-mediated apoptosis and that these
hepatocytes, when transplanted into the spleen, are
able to repopulate the liver of normal mice submitted to Fas-mediated
apoptosis. We now show that Bcl-xL-overexpressing
hepatocytes are able to repopulate up to 10% of a normal mouse liver
treated with successive injections of anti-Fas antibody. We show that a
twofold overexpression of Bcl-xL is sufficient to confer a
selective advantage to hepatocytes submitted to anti-Fas antibody.
Moreover, repopulation percentages obtained here were
comparable to those obtained when Bcl-2 hepatocytes were
transplanted, suggesting that both proteins are equivalent in
conferring a selective advantage to hepatocytes submitted to anti-Fas
antibody.
The liver is a very attractive organ
for gene therapy as it is the site of many metabolic processes and also
because hepatocytes can be targeted to secrete proteins into the
circulation. However, until now, the use of in vivo hepatic
gene transfer has been limited by the low levels of hepatocyte
transduction.1-3
An alternative approach to in
vivo gene transfer consists of transplanting genetically modified
hepatocytes, but this technique is hampered by the need for large
numbers of transplanted hepatocytes to achieve a therapeutic effect.
One way to circumvent these difficulties is to selectively amplify
transduced or transplanted cells, in other words, to repopulate the
liver from a small proportion of genetically modified hepatocytes.
We have previously developed a mouse model in which induction of
hepatocyte apoptosis is used to create an environment in the liver for
selective amplification of cells resistant to this aggression. When
Bcl-2-expressing hepatocytes that are resistant to Fas-mediated
apoptosis4
are transplanted into a normal mouse, they
progressively repopulate the liver after successive injections of an
anti-Fas antibody, Jo2.5,6
Furthermore, using a
bicistronic retroviral vector encoding Bcl-2 and a reporter gene, we
have recently demonstrated that we were able to selectively expand
1.5% of initially transduced hepatocytes to 85% of the liver after 10
weekly injections of anti-Fas antibody.7
Transgenic mice overexpressing Bcl-xL in the
liver (two- to fivefold the normal level) have also been shown to be
protected against lethal injections of Jo2, even if to a lesser extent
than Bcl-2 transgenic mice.8
We therefore wondered if
Bcl-xL-overexpressing hepatocytes could, as Bcl-2
transgenic hepatocytes, repopulate a normal mouse liver submitted to
repeated sub-lethal Fas challenges, as an alternative strategy of liver
repopulation.
|
Materials and Methods
|
|---|
Animal Procedures
A liver biopsy (the caudate lobe) was taken from four homozygous
L-PK-hBcl-xL mice of B6D2
background8
and analyzed by Western blot for the
expression of human Bcl-xL. The animals with the
highest and the lowest hBcl-xL expression were
determined and their hepatocytes were isolated according to a standard
protocol.9
Viable hepatocytes were separated from other
cells through an iso-density percoll centrifugation.10
One
million hepatocytes (>95% viability) were then injected into the
spleen of 7-week-old CBA mice. Mice in the experimental groups
received sub-lethal doses of Jo2, a hamster monoclonal anti-Fas
antibody (Pharmingen, San Diego, CA): 0.1 mg/kg was administered
intravenously once a week, beginning 48 hours after hepatocyte
transplantation. At this dose, about 40% of normal hepatocytes die of
apoptosis.11
The control groups were constituted of mice
not injected with Jo2. All mice were immunosuppressed with 2.5 mg/kg of
FK506 injected daily by intramuscular route (kindly provided by
Fujisawa GmbH, Munich, Germany).
Semiquantitative and Real-Time Polymerase Chain Reaction Analysis
Liver genomic DNA was extracted according to standard
protocols.12
Polymerase chain reaction (PCR) primers for
the murine Sry gene were: 5'-GAGTACAGGTGTGCAGCT-3' and 5'-
GTGGTGAGAGGCACAAGT-3'. The conditions for amplification were as
follows: 94° for 1 minute, 57° for 1 minute, and 72° for 1 minute
for 30 cycles. PCR products were hybridized with an internal probe
(5'-CTGTGTAGGATCTTCAATC-3') labeled with
[
-32]P-adenosine triphosphate (ATP).
Quantitation of Sry amplification was done in a PhosphorImager
(Molecular Dynamic, Sunnyvale, CA). A fragment of the
hBcl-xL transgene was amplified by real-time PCR
in a Light Cycler (Roche, Mannheim, Germany). A SYBR Green kit (Roche,
Mannheim, Germany) was used to amplify liver DNA in the Light Cycler
using the following primers: 5'-CCAGGAGAAATCAAACAGAG-3' and
5'-ACGGTGGTGGAGGAGCTCTT-3', under the following conditions: 95° for
15 seconds, 55° for 5 seconds, and 72° for 10 seconds.
Histology
Liver samples fixed in 10% phosphate-buffered formalin were
embedded in paraffin. Sections measuring 3 µm each were stained with
hematoxylin and eosin for standard microscopy.
Fluorescent in Situ Hybridization
Fresh frozen liver sections (12 µm thick) were fixed with 4%
paraformaldehyde. Fluorescent in situ hybridization (FISH)
was performed as described previously.13
Briefly, the Y
chromosome was detected using a 1.5 kb RNA probe, pY3531B, that was
generated against a repeat sequence of the mouse Y chromosome and
labeled with dioxigenin-uridine 5'-triphosphate. After several washes,
the dioxigenin was developed using an antibody against dioxigenin
conjugated to peroxidase. The antibody to dioxigenin was visualized
with tyramide-fluorescein isothiocyanate as substrate. Representative
sections were double-stained with a mouse monoclonal antibody to
cytokeratins 8, 18, and 19 (Affiniti Research Products Ltd., Exeter,
UK) developed with a Texas red anti-mouse secondary antibody. Cell
nuclei were counterstained with 4',6-diamindino-2-phenylindole (DAPI).
Statistical Analysis
Repopulation values were analyzed statistically using
Student-t and Mann-Whitney tests. Values are expressed as
mean ± SEM.
|
Results
|
|---|
Transgenic animals overexpressing a human
Bcl-xL transgene under the control of a
liver-specific promoter have been previously described.8
Bcl-xL expression in these transgenic mice has
been shown to be heterogenous among animals. To verify whether this
difference of expression could interfere with the resistance to
apoptosis, and consequently with the selective advantage of
Bcl-xL hepatocytes, a liver biopsy was taken from
four male Bcl-xL transgenic mice and a Western
blot against Bcl-xL was performed (data not
shown). The animals with the highest and lowest human
Bcl-xL expression levels (five- or twofold the
endogenous level, respectively) were chosen as donors of hepatocytes
(animals Bcl-xL[5x] and
Bcl-xL[2x], respectively). In group I, 12
normal CBA female mice received 1 million male
Bcl-xL[5x] hepatocytes by injection into the
spleen, while in group II, 12 other CBA female mice received male
Bcl-xL[2x] hepatocytes. Nine animals from each
group were submitted to eight weekly injections of anti-Fas antibody
Jo2 at sub-lethal doses and to daily immunosuppressive treatment with
FK506. The other three transplanted mice in each group served as
controls and received only the daily immunosuppressive treatment.
One week after the last Jo2 injection, all animals were killed and
their livers analyzed. To estimate the percentage of repopulation,
liver DNA was analyzed by two different PCR approaches: a
semiquantitative PCR for the Sry gene and a real-time PCR for the
Bcl-xL transgene. We compared the signals
obtained from our animals with those from serial dilutions of liver DNA
of a male Bcl-xL transgenic mouse. Actually, this
method underestimates the repopulation percentage, as only 
60% of total liver DNA originates from hepatocytes.14
Therefore, to obtain the real percentage of transgenic hepatocytes
present in the livers, the values corresponding to the signals obtained
by PCR were multiplied by 1.6. Figure 1
illustrates results obtained for the Sry gene. In group I, repopulation
percentages of the animals that received Jo2 ranged from 2 to 6% of
the hepatocytes (mean of 4.3 ± 0.6%), while the control animals
not submitted to Jo2 share a mean repopulation of 0.4% (±0.05%). In
group II, the repopulation varied from 1.5 to 10% in the animals that
received Jo2 (mean of 3.9 ± 0.8%), while the mice not submitted
to it showed a mean percentage of 0.2% (±0.06%). Equivalent results
were obtained using real-time PCR for the Bcl-xL
transgene (Figure 2)
. There was no
significant difference between the animals treated with Jo2 in groups I
and II, suggesting that the hepatocytes with a lower expression of
Bcl-xL were as resistant to the sub-lethal doses
of Jo2 used as the hepatocytes with a higher level of
Bcl-xL.
View larger version (21K):
[in this window]
[in a new window]
|
Figure 1. Semiquantitative PCR analysis of Sry DNA in transplanted mouse livers.
A: Serial dilutions of male Bcl-xL
transgenic mouse liver DNA in female non-transgenic DNA. B:
Liver DNA of five representative animals which received the weekly Jo2
injections (A1 and A2: group I; A3 to A5: group
II) and of one animal that did not receive Jo2
(C1: group 1).
|
|
View larger version (55K):
[in this window]
[in a new window]
|
Figure 2. Real-time PCR quantification of liver repopulation. Results obtained
from amplification of the Bcl-xL transgene using a Light
Cycler are represented as graphs. Error bars indicate SEM.
The number of mice used in each group is shown below the graph.
|
|
To verify that the liver morphology remained intact at the end of the
treatment, we performed a routine histological staining. Repopulated
livers appeared morphologically and histologically normal (Figure 3A)
. Moreover, to visualize male
Bcl-xL hepatocytes, a fluorescent in
situ hybridization against the Y chromosome was performed.
Y-chromosome-positive Bcl-xL hepatocytes could be
identified forming repopulation clusters in the liver parenchyma of
female recipients (Figure 3, B–F)
.
View larger version (90K):
[in this window]
[in a new window]
|
Figure 3. Histology and fluorescent in situ hybridization of a
Bcl-xL-repopulated liver. A: Liver section
examined by routine hematoxylin and eosin staining. Original
magnification, x100. B: Y chromosome staining in a liver
section of the same animal. Arrows indicate
repopulating-hepatocyte clusters. Original magnification, x100.
C–F: Identical fields showing a detailed view of
a hepatocyte cluster. Original magnification, x400. C:
Staining for the Y chromosome FISH
(green). D:
Immunostaining with an antibody to cytokeratins 8, 18, and 19
(red). E: DAPI
nuclei staining (blue).
F: Overlays of the Y chromosome, cytokeratins and DAPI
fluorescence.
|
|
|
Discussion
|
|---|
We have previously shown that Bcl-2-expressing hepatocytes were
able to repopulate a normal mouse liver5
and that these
repopulating hepatocytes could restore a physiological function of the
liver.6
However, our strategy seems hardly transposable to
a clinical setting since Bcl-2 has been associated with various
oncogenic processes; although never in the liver8,15
and
since it does not seem to play a role in hepatocyte
physiology.16
Indeed, the expression of Bcl-2 in the liver
seems restricted to bile duct cells.15
In the present paper we showed that Bcl-xL,
another anti-apoptotic member of the Bcl-2 family, is also able to
confer a selective advantage to hepatocytes submitted to anti-Fas
antibody. Bcl-xL is normally expressed in
hepatocytes and its expression stimulated during liver regeneration
after partial hepatectomy.16
This upregulation of
Bcl-xL 4 hours after partial hepatectomy could be
involved in the greater resistance of hepatocytes to Fas-mediated
apoptosis detected after partial hepatectomy.17
The fact
that hepatocytes overexpressing Bcl-xL are
resistant to sub-lethal doses of anti-Fas antibody corroborates this
hypothesis.
We show here that hepatocytes expressing high or low levels of a human
Bcl-xL transgene were able to be selectively
expanded and repopulate a normal mouse liver up to 10%. Moreover, the
repopulation percentages were not significantly different between the
two experimental groups. We are aware that our strategy is not directly
applicable in a clinical setting, mainly because of the involvement of
Fas pathway in fulminant hepatitis in humans.18
Nevertheless, our present results indicate that a slight overexpression
(about twofold) of a physiologically expressed protein is sufficient to
confer a selective advantage to hepatocytes. When compared with the
results obtained with the allogenic transplantation of Bcl-2 transgenic
hepatocytes,5
our present results are not statistically
different, suggesting that both proteins are equivalent in conferring a
selective advantage to hepatocytes submitted to sub-lethal doses of
anti-Fas antibody. The divergence between this result and the one
showing a slightly differential protective effect of Bcl-2 and
Bcl-xL in Fas-mediated apoptosis induced by
lethal injections of Jo28
may be due to a difference in
the strength of the death stimulus, since here sub-lethal doses of the
anti-Fas antibody were used to allow the repopulation to occur. In our
prior studies on liver repopulation by allogenic Bcl-2 hepatocytes, we
have discussed the reasons why repopulation extent was limited to about
10%. This is most likely due to the persistent immune rejection of
allogenic hepatocytes despite the use of an immunosuppressive regimen.
Indeed, endogenous hepatocytes transduced with a Bcl-2 retrovirus can
almost totally repopulate the liver.7
In conclusion, our results show that
Bcl-xL-overexpressing hepatocytes are able to
repopulate a normal mouse liver to a similar extent to Bcl-2
hepatocytes. This approach could represent an alternative model of
liver repopulation, in which a low overexpression of a physiological
protein confers a selective advantage to hepatocytes.
|
Acknowledgements
|
|---|
We thank Éva Mezey for her help with FISH. We also
thank M. Lambert for technical assistance and Fujisawa GmbH, Germany,
for kindly providing us with FK506. C. Mitchell is recipient of a
fellowship from National Council for Scientific and Technological
Development (CNPq)-Brazil.
|
Footnotes
|
|---|
Address reprint requests to Hélène Gilgenkrantz, U129 INSERM, 24, rue du Faubourg Saint Jacques, 75014 Paris, France. E-mail:
gilgenkrantz{at}cochin.inserm.fr
Supported by Association Française contre la Myopathie.
Accepted for publication September 20, 2001.
|
References
|
|---|
-
Miao CH, Snyder RO, Schowalter DB, Patijn GA, Donahue B, Winther B, Kay MA: The kinetics of rAAV integration in the liver. Nat Genet 1998, 19:13-15[Medline]
-
Yant SR, Meuse L, Chiu W, Ivics Z, Izsvak Z, Kay MA: Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nat Genet 2000, 25:35-41[Medline]
-
Park F, Ohashi K, Chiu W, Naldini L, Kay MA: Efficient lentiviral transduction of liver requires cell cycling in vivo. Nat Genet 2000, 24:49-52[Medline]
-
Lacronique V, Mignon A, Fabre M, Viollet B, Rouquet N, Molina T, Porteu A, Henrion A, Bouscary D, Varlet P, Joulin V, Kahn A: Bcl-2 protects from lethal hepatic apoptosis induced by an anti-Fas antibody in mice. Nat Med 1996, 2:80-86[Medline]
-
Mignon A, Guidotti JE, Mitchell C, Fabre M, Wernet A, De La Coste A, Soubrane O, Gilgenkrantz H, Kahn A: Selective repopulation of normal mouse liver by Fas/CD95-resistant hepatocytes. Nat Med 1998, 4:1185-1188[Medline]
-
Mitchell C, Mignon A, Guidotti JE, Besnard S, Fabre M, Duverger N, Parlier D, Tedgui A, Kahn A, Gilgenkrantz H: Therapeutic liver repopulation in a mouse model of hypercholesterolemia. Hum Mol Genet 2000, 9:1597-1602[Abstract/Free Full Text]
-
Guidotti JE, Mallet VO, Mitchell C, Fabre M, Schoevaert D, Opolon P, Parlier D, Lambert M, Kahn A, Gilgenkrantz H: Selection of in vivo retrovirally transduced hepatocytes leads to efficient and predictable mouse liver repopulation. FASEB J 2001, 15:1849-1851[Abstract/Free Full Text]
-
de la Coste A, Fabre M, McDonell N, Porteu A, Gilgenkrantz H, Perret C, Kahn A, Mignon A: Differential protective effects of Bcl-xL and Bcl-2 on apoptotic liver injury in transgenic mice. Am J Physiol 1999, 277:G702-G708[Abstract/Free Full Text]
-
Berry MN, Friend DS: High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J Cell Biol 1969, 43:506-520[Abstract/Free Full Text]
-
Kreamer BL, Staecker JL, Sawada N, Sattler GL, Hsia MT, Pitot HC: Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations. In Vitro Cell Dev Biol 1986, 22:201-211[Medline]
-
Guidotti JE, Mallet VO, Parlier D, Mitchell C, Fabre M, Jaffray P, Lambert M, Kahn A, Gilgenkrantz H: Fas/CD95 pathway induces mouse liver regeneration and allows for highly efficient retrovirus-mediated gene transfer. Hepatology 2001, 33:10-15[Medline]
-
Sambrook J, Fritsch E, Maniatis T: Molecular Cloning: A Laboratory Manual. 1989 New York, Cold Spring Harbor Laboratory Press, Cold Spring Harbor
-
Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR: Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000, 290:1779-1782[Abstract/Free Full Text]
-
Daoust R, Cantero A: The numerical proportion of cell types in rat liver during carcinogenesis by 4-dimethylaminoazobenzene (DAB). Cancer Res 1959, 19:757-762
-
Vail ME, Pierce RH, Fausto N: Bcl-2 delays and alters hepatic carcinogenesis induced by transforming growth factor alpha. Cancer Res 2001, 61:594-601[Abstract/Free Full Text]
-
Tzung SP, Fausto N, Hockenbery DM: Expression of Bcl-2 family during liver regeneration and identification of Bcl-x as a delayed early response gene. Am J Pathol 1997, 150:1985-1995[Abstract]
-
Takehara T, Hayashi N, Mita E, Kanto T, Tatsumi T, Sasaki Y, Kasahara A, Hori M: Delayed Fas-mediated hepatocyte apoptosis during liver regeneration in mice: hepatoprotective role of TNF alpha. Hepatology 1998, 27:1643-1651[Medline]
-
Kanzler S, Galle PR: Apoptosis and the liver. Semin Cancer Biol 2000, 10:173-184[Medline]
Top
Abstract
Materials and Methods
