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Potential conflict of interest: Nothing to report.
In the retrorsine (RS)-based model of massive liver repopulation, preexposure to this naturally occurring alkaloid is sufficient to prime normal host parenchymal cells to be slowly replaced by transplanted normal hepatocytes. The basis for this striking effect is yet to be fully elucidated. In the present studies the possible involvement of cell senescence was investigated. Fischer 344 rats were treated according to the RS-based protocol for hepatocyte transplantation, i.e., two doses of RS, 2 weeks apart, and were killed at 4 or 8 weeks after treatment. Control groups were given saline. Expression of senescence-associated beta-galactosidase was greatly induced in hepatocytes exposed to RS. In addition, several other changes that have been related to cell senescence were observed: these included markers of persistent activation of a DNA damage response, an increased expression of mammalian target of rapamycin, and positive regulators of the cell cycle, together with the induction of p21 and p27 cyclin-dependent kinase inhibitors. Furthermore, RS treatment increased levels of interleukin-6 in the liver, consistent with the activation of a senescence-associated secretory phenotype. Conclusion: These findings indicate that RS induces hepatocyte senescence in vivo. We propose that cell senescence and the associated secretory phenotype can contribute to the selective growth of transplanted hepatocytes in this system. (HEPATOLOGY 2012)
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Hepatocyte transplantation is being intensely explored as a possible alternative to whole organ replacement for the treatment of endstage liver disease.1 A number of experimental models have been developed wherein massive repopulation of the host liver can be consistently obtained through the infusion of isolated hepatocytes into the portal circulation.2-4 In order to achieve this goal, a competitive growth and/or survival advantage must exist between resident parenchymal cells and the injected cell population.5 In many of the available models this advantage is based on a genetic differential, translating into a defective survival of hepatocytes in the host liver and allowing for their selective replacement by the injected cells carrying a normal genotype.2, 3, 6, 7
Almost two decades ago an alternative strategy was introduced whereby liver repopulation could be obtained in animals endowed with a normal genetic background.8 Phenotypically normal animals were exposed to a conditioning treatment based on the administration of naturally occurring pyrrolizidine alkaloids (PAs), followed by transplantation of normal hepatocytes immediately after 2/3 partial hepatectomy (PH). Under these conditions, donor-derived cells rapidly expanded, forming progressively larger clusters and eventually leading to massive repopulation of the host liver.4, 8
The biological and molecular bases underlying this remarkable phenomenon are yet to be fully elucidated. PAs, including the widely used compound retrorsine (RS), are known for their ability to block hepatocyte cell cycle.9, 10 Thus, a main working hypothesis is that PA-induced liver repopulation might be linked to differential mito-inhibition of resident hepatocytes, resulting in the selective proliferation of the donor-derived cells delivered after exposure to the alkaloid.5 However, there is evidence to suggest that additional mechanisms must be involved. For example, transplanted normal hepatocytes are able to repopulate RS-exposed liver even in the absence of exogenous growth stimuli11; moreover, at the end of the repopulation process the original, endogenous hepatocytes have largely disappeared from the host liver4; both these findings are indicative of a process of cell replacement that is difficult to reconcile with differential growth per se.
In light of these considerations, our aim is to further characterize the biology of liver repopulation in this system and to identify other possible driving forces for the growth of transplanted cells. In the present study the possible involvement of cell senescence was analyzed.
Cell senescence occurs when the cell cycle is blocked without inhibiting cell growth.12 As such, it is biologically distinct from a mere cell cycle arrest and represents a specific state of cell differentiation.12, 14, 15 It is characterized by a number of biological, biochemical, and molecular changes, including an increase in cell size (cell hypertrophy), expression of the enzyme senescence-associated β-galactosidase (SA-β-gal), irreversible growth arrest, up-regulation of inhibitory signals, and expression of a specific senescence-associated secretory phenotype (SASP).13 It is generally considered as a mechanism preventing the risk of neoplastic transformation in cells with damaged DNA.16 However, several lines of evidence indicate that senescent cells can also foster the neoplastic process,13 possibly by way of expression of SASP.14
In this report we present data to indicate that cell senescence is induced in rat hepatocytes in vivo following exposure to a preconditioning treatment that paves the way for liver repopulation by transplanted normal hepatocytes. The mechanistic implications of these findings are discussed in the context of regenerative medicine in the liver. It is suggested that RS-induced senescence of resident hepatocytes can contribute to the growth of transplanted normal and altered (preneoplastic) cells in this system.
Five-week-old male Fischer 344 rats (Charles River, Italy) were used. They were fed Rodent Chow diet (Mucedola, Italy) ad libitum. The experiments were approved by the University of Cagliari Ethical Committee for Animal Experimentation and were in accordance with National Institutes of Health (NIH) Guidelines for the Care and Use of Animals (NIH publication 86-23, revised 1985). Rats were divided into two groups of 12 animals each and given either two injections of RS (Sigma Chemical), 30 mg/kg each, intraperitoneally, 2 weeks apart, or saline, as described.4 Four weeks after the last injection six control and six RS-treated rats were killed; the remaining six animals in each group underwent PH and were killed four weeks thereafter. Both protocols are conducive to extensive liver repopulation when followed by hepatocyte transplantation.4, 11 Livers were excised and tissue samples were snap-frozen or fixed in formalin.
Staining for Senescence-Associated β-Galactosidase Activity and Cell Size Measurement.
Staining for SA-β-gal was performed according to published procedures.17 Experimental details are provided as Supporting Material.
Immunohistochemistry and Immunofluorescence.
Immunohistochemical staining for p21, p27, and mammalian target of rapamycin (mTOR) was performed on formalin-fixed sections. Slides were incubated with primary antibodies followed by alkaline phosphatase (AP)-conjugated secondary antibody and the avidin/biotin AP system (Vectastain ABC Kit; Vector Laboratories, Burlingame, CA). Immunofluorescent staining for the phosphorylated form H2A histone family, member X (γ-H2AX), p53 binding protein 1 (53BP1), ataxia-teleangiectasia-mutated (ATM), cyclin D1, and Ki67 was performed on frozen sections. Slides were fixed and incubated with primary antibodies for 1 hour at room temperature and then with fluorescent-conjugated secondary antibodies. Images were acquired with an IX71 fluorescence microscope with CCD camera (Olympus, Tokyo, Japan). The list of primary and secondary antibodies is reported in Table 1.
Table 1. Primary and Secondary Antibodies
γ-H2AX (phosphor S139)
Cyclin D1 (DCS-6)
Antirabbit IgG HRP-conj.
Antimouse IgG HRP-conj.
Antirabbit IgG Atto 550-conj.
Antimouse IgG Atto 550-conj.
Antirabbit IgG Dylight 488 conj.
Antimouse IgG Dylight 488 conj.
Expression of γ-H2AX, 53BP1 and ATM were estimated by evaluating nuclear labeling index (LI) in 10 randomly selected high-power fields from each sample; the results are reported as the range of variation of LI among different animals in the same group.
Samples were homogenized in RIPA lysis buffer containing protease inhibitors and centrifuged at 12,000 rpm for 30 minutes at 4°C. Protein concentration in supernatants was measured using the bicinchoninic acid method.18 Additional details are provided as Supporting Material.
RNA Isolation and Quantitative Polymerase Chain Reaction (q-PCR).
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. RNA integrity and purity were confirmed by 1% agarose gel electrophoresis and OD260/OD280 nm absorption ratio >1.8. Two grams of DNase-I-treated RNA of each sample were reverse-transcribed by PCR using Promega reagents. The resulting complementary DNA (cDNA) was analyzed by real-time q-PCR using specific TaqMan assays and TaqMan Gene Expression Master Mix on a StepOne System (all from Applied Biosystems, Carlsbad, CA). The rat-specific assays were: interleukin (IL)-6 (Rn01410330_m1); β2-microglobulin (Rn00560865_m1). For both assays the thermal profile was as follows: 50°C for 2 minutes, 95°C for 10 minutes, 45 cycles at 95°C for 15 seconds, and 60°C for 1 minute. Fold change was calculated by the 2-ΔΔCT method.19
Statistical analysis of results was performed with Student t test.
SA-β-gal is a marker enzyme described by Dmiri et al.,15 whose activity can be detected at pH 6 only in senescent cells. Although it does not appear to play any specific role in the induction of the senescent phenotype, it is still considered a useful marker of cell senescence,17 possibly reflecting the expansion of the lysosomal compartment in senescent cells.20
As reported in Fig. 1A,B, exposure to RS caused a prominent increase of SA-β-gal expression in hepatocytes, observed as late as 4 weeks after treatment. The enzyme activity was detected throughout the liver parenchyma, although hepatocytes in zone 3 of the liver acinus were relatively spared (Fig. 1B).
Given our previous observations that PH is able to accelerate the kinetics of liver repopulation in this model, we analyzed the expression of SA-β-gal in animals receiving RS followed by PH, according to the original protocol for hepatocyte transplantation.4 Animals were killed 4 weeks after surgery. The results indicated that SA-β-gal was strongly expressed in hepatocytes exposed to RS and PH, with no apparent zonal distribution, whereas very limited enzyme activity was detected in control animals receiving PH (Fig. 1C,D).
Hepatocyte Megalocytosis Induced by RS.
Cellular enlargement is considered one of the hallmarks of senescent cells, being the result of cell growth stimulation under conditions of replicative arrest. The presence of megalocytes in rat liver exposed to PAs, including RS, has long been reported in the literature as one of the typical effects of these compounds.21 In this study we measured the size of hepatocytes in rat liver exposed to RS under conditions that are conducive to liver repopulation, both in the absence and in presence of PH. In Fig. 2 the histological appearance of the liver is reported. Enlarged hepatocytes are readily evident in Fig. 2B (RS-treated) compared to Fig. 2A (untreated control).
In rats receiving RS+PH and killed 4 weeks after surgery, hepatocyte megalocytosis was greatly enhanced (Fig. 2D), in agreement with data reported in the literature,21 whereas no detectable change was seen in control animals after PH (Fig. 2A,C). When cell size was measured with the help of a computer-assisted image analyzer, highly significant differences were recorded between RS-treated and control groups, both prior to and post-PH, as well as between RS and RS+PH groups (Fig. 2E).
Cell Cycle Inhibitory Proteins in RS-Treated Rat Liver.
Progression of hepatocytes through the cell cycle is controlled by the activity of cyclin-dependent kinases (CDKs), regulated by cyclins and by other proteins including CDK-inhibitors (CDK-Is). In order to investigate the molecular bases of the RS-induced, persistent replicative block, we analyzed the expression of two CDK-Is of the Cip/Kip family, p21 and p27, which are known to interact with complexes of cyclins D and E and their CDKs targets.22 Western blot analysis of liver proteins revealed overexpression of p21 in animals exposed to RS compared to controls, and a further increase in p21 protein was seen in the group receiving RS+PH (Fig. 3A). This pattern of results was consistent with data obtained through immunohistochemical staining of liver sections with antibody specific to p21; the protein was found to be highly expressed in nuclei of RS-induced megalocytes, whereas it was rarely detected in control groups (Fig. 3B).
The results were similar when the expression of p27 was analyzed. Both western blotting and immunohistochemical staining revealed a prominent increase of p27 levels in the liver of rats exposed to RS, and the effect was further amplified following PH (Fig. 3A,C). Interestingly, increased expression of p27 was also observed in control rats as late as 4 weeks after PH; to our knowledge, this result has not been reported before.
DNA Damage Foci in RS-Treated Rat Liver.
One of the triggers of cell senescence is DNA damage. More specifically, a persistent activation of a DNA damage response (DDR) is frequently associated with the senescence phenotype.13 Cells that senesce with persistent DDR signaling harbor typical nuclear foci, which have been referred to as DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS).20 These persistent foci contain proteins involved in chronic DDR, such as the ataxia teleangiectasia-mutated (ATM) gene product. However, they are distinguishable from transient DDR complexes based on the presence of specific markers, including γ-H2AX and 53BP1.23
Based on the well-established genotoxic potential of RS,24 we tested whether RS-induced hepatocyte senescence was associated with signs of persistent DDR in rat liver in vivo. As presented in Fig. 4A, 30%-40% of hepatocytes from rats exposed to RS and killed 4 weeks later expressed ATM-positive nuclear foci, and this finding was even more frequent (60%-65%) in hepatocytes from animals treated with RS+PH and killed 4 weeks after surgery; by contrast, virtually no ATM-positive nuclear foci were found in control groups.
We further investigated RS-treated hepatocytes for the expression of γ-H2AX and 53BP1, markers considered indicative of persistent DDR. Both proteins were detected in hepatocyte nuclei of rats treated either with RS alone or with RS+PH. More specifically, γ-H2AX-positive hepatocytes were about 10% in both RS and RS+PH groups (Fig. 4B), whereas 53BP1-expressing nuclear foci were present in 40%-45% of hepatocytes in rats given RS and increased to 65%-70% in animals treated with RS+PH (Fig. 4C). Both markers were undetected (γ-H2AX) or rarely detected (53BP1) in hepatocytes from control rat liver (Fig. 4B,C).
Positive Regulators of Cell Growth and Cell-Cycle Progression in RS-Exposed Liver.
Stimulation of cell growth and cell cycle progression is considered the other side of the coin, opposite to cell cycle arrest, sustaining the emergence of a senescence phenotype.12 In an earlier report, we described the overexpression cell cycle-related gene products, including cyclin D1, CDK4, and proliferating cell nuclear antigen (PCNA), in rat liver exposed to RS.25 In the present study we extended the analysis to mTOR, which plays a pivotal role at the crossroads of cell metabolism, integrating energy balance and cell growth signals.26 There is in fact evidence to indicate that activation of mTOR under conditions of cell cycle arrest leads to cell senescence.12, 27
The results are presented in Fig. 5A: increased levels of mTOR protein are detected in the liver of rats exposed to RS compared to untreated controls; such a difference was still more prominent in groups receiving PH and killed 4 weeks later, despite the fact that PH caused a decrease in mTOR levels in both RS and control groups. We also confirmed the accumulation of cyclin D1 in rat liver treated with RS, as reported25; moreover, a further increase in this protein was seen in the group receiving RS+PH and killed 4 weeks later (Fig. 5A).
Expression of IL-6 in Rat Liver Exposed to RS.
A most intriguing feature of cell senescence is the associated secretory phenotype (SASP).28 This pattern of gene expression includes a series of cytokines, growth factors, and other products that are secreted by senescent cells and can exert a plethora of biological effects, mostly limited to the local tissue microenvironment.29 The specific composition of SASP is likely to vary depending on cell and tissue type. However, a consistent finding is the presence of the inflammatory cytokine IL-6, which has also been implicated in reinforcing the senescence phenotype through autocrine and/or paracrine mechanisms.30
Based on this evidence, it appeared reasonable to explore the possibility that alterations in IL-6 expression might also be present in rat liver exposed to RS. As documented in Fig. 5B,C, both IL-6 mRNA and protein levels were found to be increased in animals treated with RS; a further increase in both parameters was seen in RS+PH group, and this was particularly prominent for IL-6 mRNA.
Regenerative Nodules Do Not Express Markers of Cell Senescence.
Remarkably, no signs of phenotypic senescence were seen in regenerative nodules developing in rats given RS and PH. These nodules have long been described in the literature31 and are composed of small hepatocytes that are able to withstand the cell cycle block imposed by RS on surrounding parenchymal cells. They slowly expand and replace the entire liver, in a process unfolding over several months that we have referred to as endogenous liver repopulation.32 The cell of origin of small hepatocytes is still a matter of debate.33, 34
When regenerative nodules were probed for the presence of markers of cell senescence, they were found to express a phenotype that was in sharp contrast to that of surrounding megalocytes (Fig. 5A-F). In fact, regenerative nodules showed no significant SA-β-gal activity and only rare nuclear staining for p21 or p27 CDK-Is (Fig. 6A-C, respectively); in addition, mTOR expression was low in these nodules compared to surrounding liver (Fig. 5D); furthermore, cyclin D1-positive nuclei were common in RS-exposed megalocytes, as reported,25 whereas they were rare in regenerative nodules; conversely, numerous Ki67-expressing hepatocytes were found inside regenerative nodules, whereas they were rarely seen in the surrounding megalocytes (Fig. 5E); similarly, we found that CDK4 protein, which associates with cyclin D1, was frequently detected in the nuclei of megalocytes, whereas it was virtually absent inside regenerative nodules (Fig. 5F).
Taken together, the data presented in these studies are consistent with the conclusion that exposure to RS, under conditions related to liver repopulation, induces a senescence phenotype in rat hepatocytes in vivo.
According to a most comprehensive model proposed in the literature, the senescence phenotype emerges when a cell integrates two types of signals: one that reads for growth and one that imposes a block in the replicative cycle.12, 35 If these two signals persist for a sufficient length of time, one of the possible outcomes is cell senescence.12 For example, DNA-damaging agents do not induce senescence in quiescent cells; however, they do so if the presence of persistent DNA damage and cell cycle arrest is coupled with growth-promoting stimuli. Under these conditions, the cells express markers related to cell cycle block, such as p53, p16, p21, or p27, as well as markers associated with growth stimulation, including cell hypertrophy and up-regulation of cell cycle-related cyclins. Although this model is largely built on data derived from in vitro studies, our present results suggest that a similar scenario occurs in rat hepatocytes exposed to RS in vivo, according to a treatment protocol that is conducive to massive liver repopulation by transplanted hepatocytes.4, 11
This conclusion is based on the following evidence. RS and other related pyrrolizidine alkaloids have long been known for their ability to cause a persistent cell cycle block on hepatocytes,9 possibly by way of induction of nonrepairable DNA crosslinking.24 In a previous study, we reported that exposure to RS, according to the protocol developed for liver repopulation, is associated with a long-lasting delay in the regenerative response of the liver following PH. Enlarged hepatocytes emerging in RS-treated rats were unable to complete DNA synthesis or undergo mitotic division.10 We show in the present report that RS-exposed hepatocytes express chronically high levels of two CDK-Is, p21 and p27, thereby providing a mechanistic basis for the persistent cell cycle block associated with cell senescence in this system. Furthermore, treatment with RS induced the expression of ATM, 53BP1, and γ-H2AX, indicative of persistent DDR and DNA-SCARS.23
Considering the other side, i.e., the growth of stimulatory signals, RS-treated hepatocytes also express high levels of positive regulators of the cell cycle, including cyclin D1, CDK4, and PCNA,25 suggestive of a state of hyperstimulation imposed on a background of replicative arrest. In addition, a relevant finding of the present studies is that exposure to RS is also associated with a long-lasting increase in the levels of mTOR protein, which plays a central role in the integration of cell growth signals.25, 34 There is evidence to indicate that the mTOR pathway is critically involved in determining two possible cell fates, quiescence or senescence, depending on the conditions associated with the cell cycle block: inhibition of mTOR leads to cellular quiescence, whereas activated mTOR drives the cell to senescence.12, 27, 35
Direct support for the conclusion that RS induces hepatocyte senescence comes from the analysis of SA-β-gal expression. This enzyme is still considered a reliable marker of senescence.15, 19, 20 We found that a large proportion of hepatocytes expressed SA-β-gal 4 weeks after exposure to RS and the levels of the enzyme marker remained high in animals receiving RS followed by PH and killed 1 month later.
Cell senescence was initially interpreted as a fail-safe mechanism to avoid the risk of neoplastic transformation in cells harboring damaged DNA.13 However, its biological significance has been reconsidered to incorporate the overwhelming evidence that senescent cells can in fact foster the growth of premalignant and malignant cells.13 These effects are at least partly mediated by a specific secretory pattern, referred to as SASP,14, 28 which includes a growing list of factors secreted by senescent cells, comprising cytokines, growth factors, and proteases. A major component of SASP is the inflammatory cytokine IL-6, which is also able to reinforce the senescence phenotype through an autocrine loop mechanism.30 It is therefore of significance that liver tissue exposed in vivo to RS expressed persistent high levels of IL-6, further reinforcing the conclusion that this treatment induces cell senescence in the target tissue. Although being expressed by senescent cells, IL-6 is also a cytokine with an established role in liver regeneration and repair, thus making it a likely candidate contributing to the process of liver repopulation in RS-exposed animals.
Following the initial studies describing the RS-based model for liver repopulation, we reported that the RS-treated rat liver is also able to support the growth of transplanted preneoplastic/nodular hepatocytes, leading to their rapid evolution to hepatocellular carcinoma.36 This indicates that the senescence-associated microenvironment induced by RS is highly stimulatory to both normal and premalignant cells, adding to the biological complexity of cell senescence.37 Interestingly, the microenvironment of the physiologically aged rat liver is also supportive for the growth of both normal37, 38 and preneoplastic hepatocytes (Laconi et al., unpubl. obs.), although the magnitude of the effect is reduced compared to the RS-exposed liver.
It was intriguing to find that regenerative nodules emerging in rat liver exposed to RS+PH did not express markers related to cell senescence; in fact, their phenotype was in sharp contrast to that observed in surrounding tissue (Fig. 6). The cell of origin of these nodules is still controversial33, 34; however, it is well established that they slowly expand and eventually repopulate almost entirely the surrounding liver,32, 39 replacing megalocytic hepatocytes in a process that can be likened to endogenous repopulation.32 Such a sequence of events indicates that small hepatocytes in regenerative nodules have a competitive growth advantage compared to surrounding senescent hepatocytes, possibly driven, at least in part, by IL-6 secreted by the same surrounding megalocytes.41, 42 RS-exposed hepatocytes were also shown to be more prone to undergo apoptosis compared to normal counterparts.43 However, it is important to point out that, when isolated normal hepatocytes are transplanted into the liver of RS-treated animals, they are able to overrun the emergence of endogenous regenerative nodules, leading to massive repopulation by donor-derived cells.4, 11
In summary, these studies indicate that an RS-imposed preconditioning effect results in the widespread induction of hepatocyte senescence in the host liver. We suggest that senescent cells represent a driving force for the selective expansion of both normal and nodular/preneoplastic transplanted hepatocytes in this system, possibly by way of secretion of specific SAPS components such as IL-6. Our findings add to the mechanistic understanding of the biology of liver repopulation and may help devising general strategies to achieve this goal in a clinical setting.
We thank Anna Saba and Giovanna Porqueddu for excellent technical and secretarial assistance.