Toxicol Lett. 2016 Jan 22;241:19-31. doi: 10.1016/j.toxlet.2015.11.011.

Double staining of β-galactosidase with fibrosis and cancer markers reveals the chronological appearance of senescence in liver carcinogenesis induced by diethylnitrosamine.


Ruth Pacheco-Rivera, Samia Fattel-Fazenda, Jaime Arellanes-Robledo, Angelica Silva-Olivares. Leticia Alemán-Lazarini, Miguel Rodríguez-Segura, Julio Pérez-Carreón. Saúl Villa-Treviño, Mineko Shibayama, Jesús Serrano- Luna.

Departments of Cell Biology, Infectomics and Molecular Pathogenesis, Molecular Biomedicine, and Physics, Center for Research and Advanced studies of the National Polytechnic Institute, CP 07360, Mexico City Mexico.

CONACYT Research fellow, National Institute of genomic Medicine, CP 14610 Mexico City, Mexico.

National Institute of Genomic Medicine, CP 14610 Mexico City, Mexico.

Department of Biochemistry of the National School of Biological Sciences, National Polytechnic  Institute, CP  Mexico City Mexico.



Cellular senescence is characterized by irreversible cell arrest and is associated with the development of chronic diseases, including cancer. Here, we investigated the induction of cellular senescence during liver carcinogenesis. Liver cancer was induced in Fischer 344 rats with a weekly intraperitoneal injection of diethylnitrosamine (50mg/kg body weight) for 16 weeks. Double-detection of β-galactosidase with Ki67 for cell proliferation; a-SMA and Pdgfrb for cell specificity; p53, p21, p16, and cyclin D1, CDK2, and CDK4 for senescence-associated molecular pathways and γ-glutamyltranspeptidase (GGT) for hepatocarcinogenesis was assessed to determine the association of these markers with cellular senescence. DNA damage was measured through senescence-associated heterochromatin foci (SAHF) detection. Progressive cellular senescence was observed in both fibrotic septa and hepatocytes from week 10 to 18. The maximum peak of positive senescent and fibrotic cells was observed at week 16 and decreased at week 18, but cell proliferation remained high. Whereas the increased p16 expression and SAHF were concomitant with that of β-galactosidase, those of p53 and p21 were barely detected. Furthermore, β-galactosidase positive myofibroblast-like cells were mainly surrounding GGT-positive tumors. Our findings showed that in hepatocarcinogenesis by diethylnitrosamine, cellular senescence is associated with p16 pathway activation and is mainly localized in myofibroblast-like cells.

KEYWORDS: Fibrosis; Hepatocarcinogenesis; N-Diethylnitrosamine; Senescence

PMID: 26589970



Cellular senescence (CS) was described since 1961 by Leonard Hayflick and Paul Moorhead (1) in normal human fibroblasts and is characterized by irreversible proliferative cell arrest. This phenomenon is also characterized by specific changes in cell morphology and gene expression (Badiola et al, 2015 (2)). Until now we know that CS is a program activated by normal cells in response to various types of detrimental stimulus. These includes telomere uncapping (replicative senescence), DNA damage, oxidative stress, oncogene activity among others (premature senescence). Senescent cells differ from other non-dividing cells such as quiescent and terminally differentiated cells by the expression of several markers and morphological changes (cells become flat, large and vacuolated). These features include the absence of proliferative markers, senescence associated β-galactosidase (SA-β-gal), expression of tumor suppressor molecules such as p53, p21, p16, and pRB among others, and also DNA damage markers (γH2AX, H3K9met3).

On the other hand, hepatocellular carcinoma (HCC) accounts for more than 5% of all cancers and more than 500, 000 deaths per year worldwide. The majority of patients (85%) have pre-existing cirrhosis at the time they develop HCC. Several risk factors are involved in the occurrence of HCC such as infections with hepatitis B and C virus, chronic alcohol consumption, aflatoxin B, and non-alcoholic steatohepatitis (NASH).

Human ageing is associated with a gradual decline in the physiological functions and it is a key risk factor for many diseases including cancer. It was shown that ageing process is close related with widespread of CS. Tumor and senescent cells may be generated in response to the same stimuli, where both types of cells would constitute two opposite result of the same degenerative process.

In this work we hypothesized that the constant stimulus by diethylnitrosamine favors the presence of CS during the development of HCC. We used a liver injury model that mimic the sequential HCC progression, from fibrosis to cirrhosis and HCC, firstly described by Schiffer et al, 2005 (3). In this model we used male Fisher 344 rats weighting 180-200 g with weekly intraperitoneal administration of diethylnitrosamine (DEN) 50 mg/Kg body weight (4). Groups of five rats were sacrificed weekly from week 10 to 18 after  DEN intraperitoneal injection, then we exposed the peritoneal cavity to obtain the whole liver (Figure 1) and began the prosecution of the samples to perform different methodologies (4).




Figure 1. Male Fisher 344 rat sacrificed at 10 week after diethylnitrosamine injection. We observed the diaphragmatic face of the liver with white pink point lesions.


In this work we used a novel staining histological technique to detect the expression of SA-β-gal with other specific proteins in the same tissue, frozen liver tissues were treated for β-gal detection using an enzymatic assay and then incubated with different antibodies (immunohistochemistry) to evaluate proliferation (Ki67); cell specificity (a-SMA and Pdgfrb); senescence-associated molecular pathways (p53, p21, and p16). We also proved a double staining assay to detect two enzymatic assays (β-gal and γ-glutamyl transpeptidase (GGT)) (4).  We performed Western blots to evaluate cell specificity (a-SMA) and cell cycle molecules (cyclin D1, CDK2, and CDK4). DNA damage were measured by the presence of γH2AX and H3K9met.

We reported for the first time that the administration of DEN favored the presence of senescence in two different types of hepatic cells, the stellate cells and the hepatocytes. In Figure 2 we analyzed the presence of the myofibroblast-like cell marker a-SMA during DEN treatment in rats by Western blot assay. We observed a-SMA expression increased from week 10 to 16 and then decrease in week 18 corroborated the results with a-SMA in an immunohistochemistry assay (4); we also observed that some of these myofibroblast-like cells became in senescent myofibroblasts that surround liver tumor areas. These experiments show us that maybe myofibroblast-like cells could have a main role in the development of liver tumor cells.



fig2Figure 2. a-SMA expression and quantification during DEN treatment. A) Fibrotic area. B) Western blot of a-SMA. NT; non-treated livers; W: week.


Because DEN produces hepatocyte DNA-damage response (DDR), we also evaluated the possibility that senescence could be present in hepatocytes. Using β-gal detection assay we be able to see senescent hepatocytes from10 to 18 weeks of DEN treatment (4); at the same time we evaluated the detection of phosphorylated histone H2AX (γH2AX) and histone 3 lysine 9 trimethylation (H3K9me3). We found that γH2AX histone was present mainly in hepatocytes and the number of hepatocytes with this nucleus marker increased with the time of DEN treatment (4). In Figure 3 we also found a decrease in the label of H3K9me3 histone (an epigenetic nucleus marker of methylated histone in hepatocites) during DEN treatment.



Figure 3. Ten week of DEN treatment; positive H3K9me3 histone (FITC green) in hepatocyte nucleus cell stained with DAPI (blue) at 12 week of DEN treatment.


At last, we explored the possible pathway of senescence in liver carcinogenesis founding that p16 is the molecule that co-localized with SA-β-gal expression in both types of cells myofibroblast like cell and hepatocytes (4). We also found that cyclin D1 expression increased from week 10 to week 12 and then decreased consistently until week 18. Instead the cyclin dependent kinase Cdk4 was increased over DEN treatment. These results means that p16 regulates cyclin D1 expression through its association with Cdk4 (4).

In conclusion using this chemically induced neoplastic nodules in rat liver, we can mimic several morphological, biochemical, genetic and epigenetic changes that happens in human HCC. We believe that this work lays the foundation for a deeper study of the relationship between cellular senescence and hepatic pre-neoplastic and neoplastic lesions.



(1) Hayflick L, Moorehead PS (1961) The serial cultivation of human diploid cell strains. Exp Cell Res 25: 585-621.

(2) Badiola I, Santaolalla F, García-Gallastegui P, Del Rey AS, Unda F, Ibarretxe G (2015) Biomolecular bases of the senescence process and cancer. A new approach to oncological treatment linked to ageing. Ageing Res Rev 23: 125-138.

(3) Schiffer E, Housset C, Cacheux W, Wendum D, Desbois-Mouthon C, Rey C, Clergue F, Poupon R, Barbu V, Rosmorduc O (2005) Gefitinib, an EGFR inhibitor, prevents hepatocellular carcinoma development in the rat liver with cirrhosis. Hepatology 41: 307-314.

(4). Pacheco-Rivera R, Fattel-Fazenda S, Arellanes-Robledo J, Silva-Olivares A, Alemán-Lazarini L, Rodríguez-Segura M, Pérez-Carreón J, Villa-Treviño S, Shibayama M, Serrano-Luna J (2016) Double staining of β-galactosidase with fibrosis and cancer markers reveals the chronological appearance of senescence in liver carcinogenesis induced by diethylnitrosamine.


Acknowledgments: This study was supported by grant No. 83710 and fellowship No. 91672 from CONACYT.



Jesús Serrano-Luna Ph.D. and MD.

Department of Cell Biology

Center for Research and Advanced Studies of the National Polytechnic Institute

CP 07360 Mexico city, Mexico.  



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