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From the Division of Hepatology and Gene Therapy,*
Clinica Universitaria and Medical School, University of Navarra,
Pamplona; and the Centro de Investigaciones Biologicas,
CSIC,
Madrid, Spain
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During past years, the approach based on the transfer of so-called suicide genes to tumor cells has emerged as a promising strategy in cancer gene therapy.11 A commonly used suicide gene is herpes simplex virus (HSV) thymidine kinase (tk) which confers sensitivity to ganciclovir (GCV). HSV-tk transforms GCV into a phosphorylated derivative with potent cytotoxic activity. In a previous study we reported that intraportal injection of an adenoviral vector expressing HSV-tk (AdCMVtk) followed by GCV administration resulted in marked reduction of the tumor mass in an animal model of multifocal hepatocellular carcinoma, but this antitumoral effect was accompanied by high mortality due to severe hepatotoxicity.12 Although early toxic liver changes have been described after gene therapy with AdCMVtk plus GCV,13 the long term follow up of animals subjected to this treatment has not been previously reported. The present study was undertaken to characterize the evolution of the liver damage which occurs in rats after intravascular injection of AdCMVtk followed by GCV administration. Our results show that this treatment causes hepatotoxicity and stimulates oval cell proliferation. In animals with moderate hepatocellular damage activation of oval cells was inconspicuous and liver architecture was normal few weeks after injection of the adenovirus. However, in cases with more intense liver cell damage a marked proliferation of oval cells was found during the first 6 weeks. This reaction was followed by nodular regeneration and marked ductular proliferation. Some animals developed intestinal metaplasia and cholangiofibrosis. Cholangiocarcinoma was found in one rat sacrificed at week 36. Our data show that the AdCMVtk/GCV system is a useful model to activate the oval cell compartment, to explore the pathophysiology of the liver stem cells and to study the pathological changes which result from disturbances in oval cell differentiation. Furthermore, our data emphasize the need to adjust the dose of the adenoviral vector when considering treatment of liver neoplasms using suicide genes to avoid undue toxicity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-smooth muscle actin antibodies were from Dako A/S (Copenhagen,
Denmark). Monoclonal antibody against desmin was from Sigma Chemical
Company (St. Louis, MO). Anti-fibronectin was provided by Dr Angeles
Garcia (CSIC, Madrid, Spain). Polyclonal antibodies against TGF-ß,
HGF, and c-met were purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). HGF cDNA was a gift from Dr. Jiro Fujimoto
(University of Hyogo, Nishinomiya, Japan). Anti-mouse immunoglobulin
labeled with peroxidase or with fluorescein isothiocyanate (FITC) and
anti-rabbit immunoglobulin labeled with FITC and 3,3'-diaminobenzidine
tetrahydrochloride were from Sigma. Ganciclovir (Cymevene) was
purchased from Roche S.A. (Madrid, Spain) and incorporated into
time-released ALZET osmotic pumps supplied by Criffa S.A. (Barcelona,
Spain). Adenoviral Vectors
The recombinant adenoviral vectors AdCMVtk and AdCMVlacZ expressing the HSV-tk gene and the reporter gene lacZ under the control of the cytomegalovirus enhancer/promoter (CMV) were constructed as previously described.11 These vectors were expanded in 293 cells and purified by double cesium chloride ultracentrifugation. The purified virus was extensively dialyzed against 10 mmol/L Tris per 1 mmol/L of MgCl2 and stored in aliquots at -80°C. The virus titer was determined by spectrophotometry and plaque assay as described.11
Treatment Protocol
Young adult male Wistar rats weighing 250 to 290 g were in cages maintained at room temperature under a 12-hour light/dark illumination cycle in an approved biohazard animal room. Standard pelleted chow and drinking water were administered ad libitum throughout the entire experimental protocol. All of the animal experimentation described in this study was conducted according to institutionally approved protocol.
The rats were divided into two groups: 53 in the experimental group and 27 in the control group. Each rat in the experimental group received 2 ml of suspension containing 4 x 1010 pfu/kg of AdCMVtk which was slowly injected into the tail vein. At this moment an osmotic pump (2ML2 ALZET) with GCV solution was placed intraperitoneally to deliver the drug at the dose of 30 mg/kg/day for 14 days. Control rats received 2 ml of phosphate-buffered saline (PBS) into the vein tail and PBS was also placed into the pump. After 14 days the pumps were removed and checked for complete emptying.
Rats from the experimental and control group were sacrificed under
ether anesthesia at 1, 2, 3, 4, 5, 6, 7, 8, and 36 weeks after the
administration of the adenoviral vector or PBS (Table 1)
. To test the adenovirus-mediated gene
transfer efficiency to liver cells, a group of 6 rats were given an
intravenous injection of AdCMVlacZ at the same those as AdCMVtk. After
48 hours, livers were removed and
5-bromo-4-chloro-3-indolyl-ß-D-galactoside staining
(X-gal) was used to detect lacZ gene expression as previously
described.11
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Liver tissue was removed at each time point as indicated above and was divided and fixed in either 10% buffered neutral formalin or placed in OCT compound, frozen in cold 2-metylbutane (Merck, Darmstadt, Germany) and stored at -80°C. All of the staining procedures for light microscopy were performed on 4-µm-thick, paraffin-embedded sections or on 6-µm-thick, frozen sections. Routine histological examinations were made for all liver tissue samples on sections (paraffin and frozen) stained with hematoxylin-eosin. Histochemical demonstration of glucose-6-phosphatase (G6Pase) activity was performed according to the method of Wachstein and Meisel.14
Immunohistochemical staining was carried out in frozen tissue with
monoclonal antibodies against OV6, BD1, BD2, and desmin using as a
second antibody anti-mouse immunoglobulin labeled with peroxidase. The
chromogenic reaction was developed with diaminobenzidine. Fibronectin
immunoreactivity was detected with anti-rabbit immunoglobulin labeled
with FITC. Detection of PCNA, -smooth muscle, HGF, c-met and TFG-ß
was performed after heating formalin-fixed paraffin-embedded tissue
sections by ~800-W microwaving in 0.01 mol/L citric acid buffer pH
6.0, followed by incubation with the corresponding antibody and
anti-mouse (PCNA and -smooth muscle actin) or anti-rabbit (HGF,
c-met, and TFG-ß) immunoglobulin labeled with peroxidase. For each
antibody, negative controls were performed by omitting the primary
antibody from the protocol. Negative control sections were included
with c-met (SP260) antibody that had been neutralized by pretreatment
with the immunizing peptide sc-162P (Santa Cruz).
Aspartate aminotransferase (AST) levels were determined in serum by automatic calorimetric assays (Technicon RA-1000, Bayer).
Western Blot Analysis of c-met Protein Expression
Frozen liver tissue was homogenized in RIPA buffer (9.1 mmol/L Na2HPO4, 1.7 mmol/L NaH2PO4, 150 mmol/L NaCl, 1.0% Nonidet P-40, and 0.1% sodium dodecyl sulfate (SDS), 0.05% sodium deoxycholate, and 1.1 mmol/L phenylmethylsulfonyl fluoride) with protease inhibitor cocktail tablets (Boehringer Mannheim, Mannheim, Germany). The protein content was determined by the Bradford assay (Bio-Rad, München, Germany). A total of 30 µg of protein was dissolved in the sample buffer heated at 90°C for 5 minutes and analyzed by 7.5% SDS-polyacrylamide gel electrophoresis. Immunoreactivity was visualized by incubation with Supersignal CL-HRP chemiluminescence substrate (Pierce Chemical, Rockford, IL).
Northern Blot
Total RNAs were isolated by a modified acid/guanidine thiocyanate/phenol/chloroform method15 using ULTRASPEC (Biotecx Laboratories, Inc, Houston, TX) reagents according to the manufacturer’s instructions. RNA concentrations were determined spectrophotometrically.
For Northern blotting total RNA (20 µg) was electrophoresed under denaturing conditions, transferred onto nylon membranes positively charged (Boehringer Mannheim), and fixed by UV cross-linking. The quality and comparative quantity was estimated on gels by ethidium bromide staining. The 2.6 kb rat HGF cDNA and the cDNA probe for ß-actin was labeled by random priming (Amersham Pharmacia Biotech Inc. Buckinghamshire, UK) and used for Northern blot. Membranes were prehybridized for 1 hour and hybridized 2 hours with QuikHyb (Stratagene, La Jolla, CA) at 68°C. The membranes were stringently washed and exposed to Kodak XAR film.
In Situ Hybridization
The rat albumin cDNA used for in situ hybridization (ISH) corresponds to 700 bp fragment from the 3' end of the rat albumin gene cloned into pGEM1. After linearizing the vector with HindIII or EcoRI, a digoxygenin-labeled antisense and sense cRNA probe were synthesized with T7 or SP6 RNA polymerase (BRL, Gibco) respectively using the DIG RNA labeling kit (Boehringer Mannheim) according to manufacturer’s instructions. The cRNA probes were purified by ethanol precipitation and correct size verified by 1.5% agarose gel electrophoresis. Typical yields of 10 µg of DIG-labeled cRNA for 1 µg of DNA were obtained.
ISH was performed on paraffin-embedded tissue sections. After deparaffinization and rehydration, sections were immersed in 0.2% Triton/PBS pH 7, followed by incubation with proteinase K (80 µg/ml) for 30 minutes at 37°C. They were then acetylated in triethanolamine containing 0.25% acetic anhydride. The sections were hybridized in buffer containing 0.01 mol/L Tris/HCl, pH 7.5, 12.5% Denhardt’s solution (0.02% Ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone), 2X SSC, 50% formamide, 0.5% SDS, 250 µg/ml salmon sperm DNA, 5 mg/ml sodium pyrophosphate, and the cRNA probe for 18 hours at 45°C. The following day, the sections were washed twice in 2X SSC/0.1% SDS for 10 minutes at room temperature, twice in 0.1X SSC/0.1% SDS at 43°C, and then treated with RNase A at 37°C for 15 minutes. Immunological detection of albumin mRNA was performed by reaction with anti-DIG antibody coupled to alkaline phosphatase using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolphosphate in a dark box for 2 hours, after which the color reaction was stopped with TE buffer and the slides were mounted in glycerol/PBS. Negative controls included hybridization with the sense probe and omission of the RNA probe or anti-DIG antibody.
Statistical Analysis
Mann-Whitney U tests were used to compare variables between two groups. A value of P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After intravenous injection of AdCMVlacZ, about 75% of hepatocytes stained positively with X-gal. There was, however, a total absence of transduction of bile duct epithelial cells (data not shown). No staining was found in organs other than the liver except a faint signal in renal proximal tubules in one of the rats tested.
Rats which received AdCMVtk followed by GCV treatment suffered a chain
of morphological changes in the liver which are summarized in Table 1
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As shown in this table, groups of 6 animals were sacrificed at weeks 1,
2, 3, 4, 7, and 8. Fourteen rats were sacrificed at weeks 5 and 6 and
three at week 36. A moderate infiltration of mononuclear cells in the
portal tract was observed on the first week after administration of the
adenovirus. On the second week, in four of the six rats studied there
were abundant acidophilic bodies and apoptotic bodies (Figure 1A)
indicating severe hepatocellular
damage. In these rats a ductular proliferation was apparent in the
vicinity of the limiting plate where small oval cells forming duct-like
structures were identified (Figure 1B)
. These cells reacted positively
with OV-6 and BD2, two monoclonal antibodies directed against rat oval
cell markers.16
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. These small epithelial cells which
stained positively for BD2 and OV-6, were disposed as rows or clusters
and frequently formed ductular structures (Figure 1, C and D)
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Proliferating oval cells showed positive PCNA staining. Two rats of
this group showed less liver damage with few oval cells at the portal
tracts. A normal liver architecture was observed in two more rats
(Table 1)
.
On weeks 5 and 6, six rats showed massive substitution of the liver
tissue by proliferating small OV-6 and BD2 positive cells (Figure 2A)
which diffusely infiltrated the cords
of damaged and dysplastic hepatocytes. In two rats, oval cell
proliferation was much less intense and in six animals liver
architecture was normal. As shown in Figure 2B
, in animals with marked
activation of the stem cell compartment it was possible to observe
nodules of basophilic hepatocytes which showed a round nuclei and scant
cytoplasm and were frequently surrounded by small OV-6 and BD2 positive
cells (Figure 2C)
which stained positively for PCNA. Whereas oval cells
lacked G6Pase activity, this enzymatic activity was present in the
nodules of small hepatocytes (Figure 2D)
although at levels
considerably lower than those observed in hepatocytes of a normal liver
(data not shown). We also analyzed by in situ hybridization
the expression of albumin mRNA as an additional marker of
hepatocellular differentiation. We observed that the levels of albumin
transcripts were low in the old damaged hepatocytes while both oval
cells and the nodules of small basophilic hepatocytes showed strong
albumin mRNA expression (Figure 2E)
. Thus, the presence in these
nodules of an intense albumin signal (as in oval cells) and a G6Pase
activity which is intermediate between OV-6 positive cells (absent
G6Pase) and mature normal hepatocytes (high G6Pase activity) strongly
suggests that the small basophilic hepatocytes represent newly formed
liver cells which derive from oval cells and replace old damaged
hepatocytes at the portal areas.
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On week 7, two rats showed a nodular pattern of hepatocellular
regeneration and persistence of proliferating ductules with positive
oval cell markers at the edge of the nodules (Figure 2F)
. The rest of
the rats presented normal liver architecture. Similarly, four of six
rats sacrificed at week 8 showed nodular hepatocytic regeneration with
abundant OV-6 and BD2 positive ductular formations surrounding the
nodules (Figure 3A)
. These hyperplastic
ductules showed positive PCNA staining indicating proliferative
activity of the epithelial cells (Figure 3B)
. At this time point most
of the bile ductular cells of such lesions also stained positively with
BD1, an antibody which recognizes mature bile duct
epithelium.17
In one of these rats, proliferating bile
ducts were supported by a thin fibrotic stroma and some of the ductules
were filled with mucus and exhibited intestinal metaplasia and goblet
cells. In three rats, ductular proliferation was more intense and
periductular fibrosis was more prominent (Figure 3C)
. Some of the
glandular structures in these cholangiofibrotic lesions exhibited also
intestinal metaplasia with the presence of enterocyte differentiation
and goblet cells (Figure 3D)
. Expression of HGF was detected by
immunohistochemistry only in metaplastic glands (Figure 3E)
.
Immunohistochemical expression of the HGF receptor, c-met, was found in
metaplastic glands and dysplastic bile epithelium of proliferating
ductules (Figure 3F)
. Expression of c-met was less intense in
non-dysplastic proliferating ductules. These data were confirmed by
Western blot that demonstrated a 140-kd band which was more pronounced
in the liver from rats with cholangiofibrosis or with ductular
proliferation and intestinal metaplasia than in normal liver (Figure 4)
. A cholangiocarcinoma with glands
which displayed positivity for both c-met and PCNA was found in one of
the three rats sacrificed at week 36.
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Role of Hepatic Stellate Cells and Growth Factors in Proliferation of Oval Cells
In livers showing proliferation of oval cells irradiating from the
portal area into the hepatic lobule, we observed that the proliferating
cells were surrounded by fibronectin deposition (Figure 5A)
. Intermingled with this extracellular
matrix component, there were many desmin positive cells, indicating a
participation of HSC in this modality of liver cell regeneration
(Figure 5B)
. HSC showed positivity for -smooth muscle actin,
especially in cholangiofibrotic lesions found on the
8th week after AdCMVtk administration (Figure 5C)
. In these lesions abundant fibronectin deposition and TGF-ß were
found surrounding the proliferating bile ducts (Figure 5, D and E)
.
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, HGF mRNA was not detected in
normal liver. However, we found very high levels of HGF mRNA in the
liver at weeks 1 and 2 after administration of adenovirus by Northern
Blot. The amount of HGF transcripts decreased slightly by week 3 but
increased on subsequent weeks although only in animals with persistent
oval cell proliferation. In cholangiofibrosis HGF mRNA expression was
present but at levels which were lower than in cases with high number
of proliferating oval cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is evidence that liver stem cells reside in bile ducts20 and, since bile duct cells are not transduced when adenoviral vectors are administered systemically, the facultative stem cells present in the bile ducts might be responsible for the oval cell response when damaged hepatocytes cannot regenerate. However, it is also possible that stem cells are periductular cells10 which could also be protected from adenoviral infection.
Our data show that after the 7th week some rats developed a nodular regenerative pattern with abundant proliferating ducts surrounding the hepatocellular nodules. This type of lesion would be compatible with a differentiation of oval cells along both hepatocytic and biliary epithelium lineages. Three rats sacrificed at week 8 developed cholangiofibrosis and a cholangiocarcinoma was found in one rat examined at week 36. Cholangiofibrotic lesions showed features of intestinal-like differentiation (intestinal metaplasia) as reflected by the presence of goblet cells and columnar enterocyte-like cells. Similar findings were described by Sirica et al in rats treated with furan where they found increased c-met expression in cholangiofibrotic lesions and in later-developed cholangiocarcinoma.21-23 In our rats, as in the furan model,24 the increased expression of c-met in proliferating ductules and in cholangiofibrosis was also associated with features of dysplasia and intestinal metaplasia and with a strong PCNA staining indicating enhanced proliferative activity. These findings suggest a role of c-met in malignant transformation. Expression of c-met was less intense in non-dysplastic proliferating ductules a finding which is compatible with the morphogenetic role of this receptor in particular for structures with lumen formation.25
In the model of hepatic stem cell activation here described, as it has
also been demonstrated in other models,26
we show a
functional association between HSC and oval cells. In livers showing
proliferation of oval cells irradiating from the portal area into the
hepatic lobule, desmin positive HSC were observed accompanying the oval
cells invading the hepatic lobule, with an apparent parallelism between
oval cell proliferation and proliferation of desmin-positive HSC.
Similarly HSC strongly expressing -smooth muscle actin have also
been observed surrounding bile ducts in late cholangiofibrotic lesions.
Both reactive ductules and HSC27,28
synthesize TGF-ß
which stimulates HSC in an autocrine and paracrine manner to produce
extracellular matrix (ECM) components. We observed that the presence of
HSC was associated with increased deposition of fibronectin and with
the presence of collagen and TGF-ß around bile ducts in
cholangiofibrotic lesions.
The extracellular matrix, in addition to form a physical scaffold for liver cells, is also implicated in important biological functions including differentiation processes and growth regulation.29,30 HSC besides their role in the production of ECM also express high levels of HGF mRNA.29 Infusion of HGF has been shown to enhance proliferation not only of oval cells but also of HSC.19 Our study has shown that HGF mRNA is highly expressed in rats exhibiting either oval cells response or biliary duct hyperplasia but not in those animals with normal liver architecture. In cholangiofibrotic lesions the expression of HGF was detected but at lower levels than in biliary duct hyperplasia without fibrosis suggesting, as proposed by Nakayama et al in a different model of cholangiofibrosis,31 down-regulation of HGF mRNA by TGF-ß. The observed association of HGF mRNA expression with persistent ductular proliferation suggests a role of HGF in the pathogenesis of this lesion possibly by inhibiting apoptosis of epithelial cells.32 Seemingly this effect may favor the evolution of ductular proliferation to cholangiocarcinoma as it has been observed in one of our rats 9 months after the administration of the adenoviral vector. Interestingly HGF was detected by immunohistochemistry only in glands with intestinal metaplasia, a lesion which is thought to be associated with the development of cholangiocarcinoma in both humans and experimental animals.22,24,33 These findings are in agreement with previous reports by Sirica34 and by Lai et al35 which showed selective HGF immunoreactivity in intestinal metaplastic glands associated with cholangiofibrosis and in neoplastic glands of subsequently developed cholangiocarcinoma in the rat furan model.
In summary, we report that the AdCMVtk/GCV system constitutes a new model to stimulate liver regeneration based on a potent oval cell response. This system is simple, does not require partial hepatectomy or the use of carcinogens, and it is especially useful to generate ductular hyperplasia as a source of bile duct epithelium cells. Our results also illustrate the pathological effects in the liver of a type of gene therapy which is being considered as a potential treatment for primary and metastatic liver tumors.
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Acknowledgements |
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Footnotes |
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Supported by the Comision Interministerial de Ciencia y Tecnologia, Spain, SAF 96–0268.
Accepted for publication May 19, 2000.
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References |
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inhibit both apoptosis and proliferation of activated rat hepatic stellate cells. Hepatology 1999, 30:196-202[Medline]
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