Senescent Hepatocytes Research Articles

Immune surveillance of senescent cells--biological significance in cancer- and non-cancer pathologies

Cellular senescence, a state of stable growth arrest, can occur in response to various stress stimuli such as telomere shortening, treatment with chemotherapeutic drugs or the aberrant activation of oncogenes. Senescent cells communicate with their environment by secreting various cytokines and growth factors, and it has become clear that this 'secretory phenotype' can have pro- as well as anti-tumorigenic effects. Recent work from our laboratory showed that premalignant, senescent hepatocytes are recognized and cleared through an antigen-specific immune response and that this immune response, designated as 'senescence surveillance' is crucial for tumor suppression in the liver [(Kang,T.W. et al. (2011) Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature, 479, 547-551]. It is an emerging concept that immune responses against senescent cells have a broader biological significance in cancer- as well as non-cancer pathologies and current data suggest that distinct immune responses are engaged to clear senescent cells in different disease settings. In this review article, we will discuss different examples how immune responses against senescent cells are involved to restrict disease progression in cancer- and non-cancer pathologies.

Impaired antioxidant defence and accumulation of oxidative stress in caspase-2-deficient mice

Caspase-2 has been implicated in apoptosis and in non-apoptotic processes such as cell cycle regulation, tumor suppression and ageing. Using caspase-2 knockout (casp2(-/-)) mice, we show here that the putative anti-ageing role of this caspase is due in part to its involvement in the stress response pathway. The old casp2(-/-) mice show increased cellular levels of oxidized proteins, lipid peroxides and DNA damage, suggesting enhanced oxidative stress. Furthermore, murine embryonic fibroblasts from casp2(-/-) mice showed increased reactive oxygen species generation when challenged with pro-oxidants. Reduced activities of antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were observed in the old casp2(-/-) mice. Interestingly, in the old casp2(-/-) animals expression of FoxO1 and FoxO3a was significantly reduced, whereas p21 levels and the number of senescent hepatocytes were elevated. In contrast to young wild-type mice, the casp2(-/-) animals fed an on ethanol-based diet failed to show enhanced GSH-Px and SOD activities. Thus, caspase-2, most likely via FoxO transcription factors, regulates the oxidative stress response in vivo.

Senescence Surveillance Clears Premalignant Hepatocytes

Abstract Major finding: Clearance of senescent hepatocytes by liver-infiltrating immune cells prevents HCC. Mechanism: RAS-induced senescence activates a CD4+ T-cell–mediated adaptive immune response. Impact: These findings may explain increased rates of HCC observed in immunosuppressed patients.

Senescence surveillance of pre-malignant hepatocytes limits liver cancer development

Upon the aberrant activation of oncogenes, normal cells can enter the cellular senescence program, a state of stable cell-cycle arrest, which represents an important barrier against tumour development in vivo. Senescent cells communicate with their environment by secreting various cytokines and growth factors, and it was reported that this 'secretory phenotype' can have pro- as well as anti-tumorigenic effects. Here we show that oncogene-induced senescence occurs in otherwise normal murine hepatocytes in vivo. Pre-malignant senescent hepatocytes secrete chemo- and cytokines and are subject to immune-mediated clearance (designated as 'senescence surveillance'), which depends on an intact CD4(+) T-cell-mediated adaptive immune response. Impaired immune surveillance of pre-malignant senescent hepatocytes results in the development of murine hepatocellular carcinomas (HCCs), thus showing that senescence surveillance is important for tumour suppression in vivo. In accordance with these observations, ras-specific Th1 lymphocytes could be detected in mice, in which oncogene-induced senescence had been triggered by hepatic expression of Nras(G12V). We also found that CD4(+) T cells require monocytes/macrophages to execute the clearance of senescent hepatocytes. Our study indicates that senescence surveillance represents an important extrinsic component of the senescence anti-tumour barrier, and illustrates how the cellular senescence program is involved in tumour immune surveillance by mounting specific immune responses against antigens expressed in pre-malignant senescent cells.

Final act of senescence

Damaged cells can initiate cancer. To avert this, faulty cells disable their own propagation by undergoing senescence. But for full protection against liver cancer, the senescent cells must be cleared by the immune system. See Letter p.547 Oncogene-induced senescence has been shown to function as an intrinsic tumour suppressor mechanism. Lars Zender and colleagues now introduce the concept of 'senescence surveillance' by showing that premaligant senescent hepatocytes are cleared through a tumour antigen-directed immune response. This requires CD4+ T cells and suppresses the development of liver tumours in mouse models. The authors also provide evidence that senescent hepatocytes accumulate in the livers of immune-suppressed patients, demonstrating that senescence surveillance may also operate in humans. Strategies designed to harness antigen-specific immune surveillance of premalignant senescent cells may have potential in cancer prevention and therapy, and antigen-specific immune responses of this type may be relevant in vaccine production.

Short term treatment of old-aged recipient rats with growth hormone improves liver repopulation by transplanted hepatocytes

Hepatocyte transplantation is an alternative to whole liver transplantation. Yet, the cellular and molecular mechansims prompting integration are largely unknown. We previously reported that the poor liver repopulation efficacy by transplanted hepatocytes in adult recipient livers is probably due to the host microenviroment and caused by the inefficient regulation of the growth hormon-IGF1-axis. The aim of this study was to investigate the effects of growth hormone on liver repopulation by transplantated hepatocytes. The age-dependent proliferative potential of wild type F344 rat hepatocytes was investigated after transplantation and administration of recombinant human growth hormon (rhGH) into old CD26-deficient F344 rat host livers. Repopulation rates were verified by flow cytometry and assay of CD26 enzyme activity. Cell cycle-dependent protein expression of C/EBPalpha and PP2A was determined. Detection of serum levels of rhGH indicated the efficient increase after systemic administration before and during hepatocyte transplantation. Hepatocytes isolated from either young or old donor rats formed bigger clusters of donor-derived hepatocytes in senescent host livers treated with rhGH as compared to untreated livers. Repopulation of senescent host livers by old or young hepatocytes after treatment with rhGH was 10 times higher as compared to untreated hosts. No functional difference between transplanted juvenile and senescent hepatocytes was observed in the host liver tissue as assessed by glycogen storage. The protein expression of C/EBPalpha and PP2A revealed the recovery of the senescent host liver microenviroment after treatment with rhGH favouring growth of transplanted hepatocytes, derived from both young and old donors, in the recipient senescent liver. Thus, in rats, the age of the recipient liver and the regulation along the growth hormone-IGF1-axis impacts repopulation by transplanted hepatocytes.

Metabolism, Genomics, and DNA Repair in the Mouse Aging Liver

The liver plays a pivotal role in the metabolism of nutrients, drugs, hormones, and metabolic waste products, thereby maintaining body homeostasis. The liver undergoes substantial changes in structure and function within old age. Such changes are associated with significant impairment of many hepatic metabolic and detoxification activities, with implications for systemic aging and age-related disease. It has become clear, using rodent models as biological tools, that genetic instability in the form of gross DNA rearrangements or point mutations accumulate in the liver with age. DNA lesions, such as oxidized bases or persistent breaks, increase with age and correlate well with the presence of senescent hepatocytes. The level of DNA damage and/or mutation can be affected by changes in carcinogen activation, decreased ability to repair DNA, or a combination of these factors. This paper covers some of the DNA repair pathways affecting liver homeostasis with age using rodents as model systems.

Bile ductular cell reaction with senescent hepatocytes in chronic viral hepatitis is lost during hepatocarcinogenesis

Cellular senescence is defined as irreversible cell arrest and could work as a safeguard against tumorigenesis. This mechanism was examined in chronic viral hepatitis-related hepatocarcinogenesis. By using surgical resected or wedge biopsied liver specimens from 87 chronic viral hepatitis patients in whom 35 neoplastic nodules (dysplastic nodules and hepatocellular carcinoma) were complicated, P21 expression and senescence-associated beta galactosidase activity, a marker of senescence, were examined. All of these neoplastic nodules harbored portal tracts within the tumors. Hepatocytes expressing senescence markers and cytokeratin (CK)7-positive bile ductules including hepatic progenitor-like cells were increased in periseptal areas in cirrhosis. Interestingly, these cells appeared to form an anatomical complex that was completely lost in the periportal areas within the neoplastic nodules. In one-third of the neoplastic nodules, CK7-positive small neoplastic hepatocytes resembling hepatic progenitor cells proliferated zonally around the portal tracts. In conclusion, loss of a complex of senescent hepatocytes and ductular cell including hepatic progenitor-like cells in the periportal or periseptal areas may be associated with emergence of neoplastic hepatocytes and their proliferation followed by neoplastic nodules arising in liver cirrhosis. Zonal proliferation of CK7-positive small neoplastic hepatocytes resembling hepatic progenitor cells may develop during early hepatocarcinogenesis.

Age‐dependent liver repopulation by transplanted hepatocytes

The low liver repopulation efficacy of transplanted hepatocytes is probably due to their poor proliferative capacity linked to the age of the liver donors. The age‐dependent proliferative potential of wild type F344 rat hepatocytes was investigated after transplantation into old and young CD26‐deficient F344 rat host livers. Repopulation rates were determined by flow cytometry and assay of CD26 enzyme activity.Hepatocytes isolated from either young or old donor rats formed small and rare clusters of donor‐derived hepatocytes in senescent host livers. However, both hepatocytes from juvenile and from senescent donor livers transplanted into juvenile host livers developed cell clusters significantly larger and more frequently as compared with senescent host livers. Repopulation of juvenile host livers by old or young hepatocytes amounted to 20 % as compared 2 % in senescent hosts. No functional difference between transplanted juvenile and senescent hepatocytes was observed in the host liver tissue in terms of glycogen storage. As compared to senescent host animals, serum levels of IGF1 were significantly higher in juvenile rats. Thus, in rats, the age of the recipient liver seems to be critical for the efficient repopulation by transplanted hepatocytes involving regulation along the growth hormone‐IGF1‐axis.

Oval cells compensate for damage and replicative senescence of mature hepatocytes in mice with fatty liver disease.

Hepatic steatosis may have a generally benign prognosis, either because most hepatocytes are not significantly injured or mechanisms to replace damaged hepatocytes are induced. To determine the relative importance of these mechanisms, we compared hepatocyte damage and replication in ethanol-fed and ob/ob mice with very indolent fatty liver disease to that of healthy control mice and PARP-1(-/-) mice with targeted disruption of the DNA repair enzyme, poly(ADP-ribose) polymerase. Compared to the healthy controls, both groups with fatty livers had significantly higher serum alanine aminotransferase values, hepatic mitochondrial H(2)O(2) production, and hepatocyte oxidative DNA damage. A significantly smaller proportion of the hepatocytes from fatty livers entered S phase when cultured with mitogens. Moreover, this replicative senescence was not reversed by treating cultured hepatocytes with agents (i.e., betaine or leptin) that improve liver disease in intact ethanol-fed or leptin-deficient mice. Hepatocytes from PARP1(-/-) mice also had more DNA damage and reduced DNA synthesis in response to mitogens. However, neither mice with fatty livers nor PARP-1-deficient mice had atrophic livers. All of the mice with senescent mature hepatocytes exhibited hepatic accumulation of liver progenitor (oval) cells and oval cell numbers increased with the demand for hepatocyte replacement. Therefore, although hepatic oxidant production and damage are generally increased in fatty livers, expansion of hepatic progenitor cell populations helps to compensate for the increased turnover of damaged mature hepatocytes. In conclusion, these results demonstrate that induction of mechanisms to replace damaged hepatocytes is important for limiting the progression of fatty liver disease.

RNA Synthesis by Hepatocytes Isolated from Adult and Senescent Wistar Rat Liver

The rates of synthesis of total RNA, poly(A)+ RNA and poly(A)+ RNA are decreased by 41, 49 and 42%, respectively, in hepatocytes isolated from senescent (25 months) compared to adult (12 months) female Wistar rats. Results from transcription run-on assays indicate that an impaired ability to initiate transcription largely accounts for the decreased synthesis of RNA by the senescent hepatocytes.

Cellular theories of aging as related to the liver.

The age-associated decline in function of several organs, including the liver, may be caused by mechanisms operating on the cellular level. Fibroblasts and several other cell types derived from normal individuals have limited lifespans in culture, and several abnormalities described for senescent cultured fibroblasts also apply to hepatocytes and other cell types obtained from aged organisms. Cellular theories of aging can be divided into two broad and overlapping categories: (a) those that view cell death as an actively programmed developmental process, and (b) those that consider cellular aging to result from a passive accumulation of errors in macromolecules. These theories are not necessarily mutually exclusive, and many of the phenotypic changes in senescent hepatocytes, fibroblasts and other cells are compatible with several different theories. The challenge for the future is to distinguish primary causes from secondary consequences of cellular aging so that rational attempts to intervene in the aging process are possible.