Genetic and epigenetic analysis of immortal and senescence arrested liver cancer cells
Author
Bağışlar, G. Sevgi
Advisor
Öztürk, Mehmet
Date
2009Publisher
Bilkent University
Language
English
Type
ThesisItem Usage Stats
104
views
views
32
downloads
downloads
Abstract
Genetic and epigenetic aspects of cellular senescence and immortality in hepatocellular
carcinoma (HCC) are poorly elucidated. The aim of our thesis was to characterize
senescence and immortality gene network (SIGN) involved in these cancers. We also
wished to explore epigenetic changes associated with senescence and immortality of
HCC cells. First, we identified differentially expressed genes in immortal, pre-senescent
and senesce-arrested Huh7 clones. Our microarray analysis revealed 6390 probesets
significantly changing among groups. Moreover, the significant gene signature could
successfully discriminate both replicative senescent cells, and oncogene-induced
senescent cells from their immortalized counterparts. E2F1 targets, stem-cell related
genes, DNA repair, RNA splicing and cell cycle related gene sets were enriched
specifically in immortal cells, whereas immune function, stress response, electron
transporter activity, protein modification, metabolism, chromatin biogenesis related gene
groups were significantly up-regulated in senescent clones. Next, we integrated gene
expression data from senescence-programmed and immortal HCC cells with the data
from cirrhosis and HCC tissues to generate a SIGN signature. This signature identified
several HCC classes, including one “normal-like”, and two with increased expression of
immortality genes. Senescence-to-immortality transition was accompanied by hepatic
dedifferentiation and increased expression of cell proliferation, chromosome modification
and DNA damage response genes. Finally, we identified a large set of upregulated DNA
damage checkpoint and DNA repair genes that showed significant associations with some
SIGN classes of HCC tumors. As retinoblastoma/E2F pathway plays a key role in cellular
senescence, we also analyzed E2F and DP family members in senescent and immortal
hepatocellular carcinoma cells. E2F1, E2F5, E2F7, E2F8 and DP1 were up-regulated in
immortal hepatocellular carcinoma (HCC) cell lines as compared to senescent cells,
whereas E2F3a and DP-2 expressions were downregulated. Upregulation of DP2 expression in senescent cells correlated with increased DP2 protein expression, as tested
with TGF-beta induced senescence models. Finally, we demonstrated important
epigenetic changes associated with hepatocellular immortality and senescence. Among
histone methyltransferases and demethylases, MLL3, FBXL11, SUV420H1, UTX,
SMYD2, SETD2, JMJD2B, JMJD3, JARID1B and ASH1L genes were up-regulated, and
EZH2 was down-regulated in senescent cells. These changes were accompanied with
changes in histone methylation patterns. Of particular interest, H3K27me1, H3K27me3,
H4K20me3, H3R2me2a and H4R3me2a forms of methylated histones displayed
increased expression in both Huh7 and MRC5 senescent cells, as compared to their
immortal forms. Finally, H3K27me3, H4K20me3, H3K36me3, H3R17me2a, H4R3me2a
also showed decreased expression in some cirrhotic liver and primary HCC tumors. In
conclusion, we demonstrated that a large set of senescence and immortailty genes were
dysregulated in HCC. This profound change in gene expression was associated with
differential expression of histone modifying enzymes, as well as histone methylation
status. Thus, the immortalization of hepatocytes during hepatocellular carcinogenesis is
accompanied with global gene expression changes probably mediated by a major
modification of their epigenetic program via histone demethylation.