Browsing by Subject "Cell death."

Now showing 1 - 4 of 4

Open Access
The ability to generate differentiated and senescent progeny is a major determinant of breast cancer heterogeneity
(2009) Mumcuoğlu, Mine
Breast cancer displays distinct subtypes, such as luminal A, luminal B, and basallike. The prognosis and therapeutic response of each subtype is different. The mechanisms involved in the generation of these tumor types are poorly understood. Our aim was to test whether the ability to generate senescent progeny contributes to breast cancer heterogeneity. A panel of 12 breast cancer cell lines, 31 isogenic clones, and 12 breast tumors were used. We classified breast cancer cell lines into senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All ER+ cell lines tested and some ER-positive (ER+) breast tumors displayed senescence. Acute loss and tamoxifen-mediated inactivation of ER triggered a robust senescence response in SCP type T47D cell line. In contrast, ER-overexpression, estrogen treatment and p21Cip1 knockdown inhibited senescence. Neutralization of reactive oxygen species also abolished senescence. Breast cancer cell subtypes displayed divergent ability to produce differentiated progeny. The SCP subtype cells produced CD24+ or ER+ luminal-like and ASMA+ myoepithelial-like progeny, in addition to CD44+ stem/progenitor-like cells. In contrast, ICP cell lines acted as differentiation-defective stem/progenitor cells. Some cell lines generated only CD44+/CD24-/ ER-/ASMA- progenitor/stem-like cells, and others only CD24+/ERluminal-like, but not ASMA+ myoepithelial-like cells. SCP cell lines were less tumorigenic, and they clustered with luminal A/normal like tumors. In contrast, ICP subtypes were more tumorigenic, and they clustered together with basal/luminal B tumors. Our results show that breast cancer cell lines clustering with luminal A/normal-like and basal/luminal B tumors respectively, differ from each other by the ability to generate differentiated and senescence-arrested progeny.
Open Access
Genetic and epigenetic analysis of immortal and senescence arrested liver cancer cells
(2009) Bağışlar, G. Sevgi
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.
Open Access
Molecular analysis of senescence-associated protein phosphatases DUSP10 and MTMR11
(2009) Gülay, Suna Pelin
Liver cancer is the fifth most common cancer in the world. Until recently, tumor cells were thought to proliferate indefinitely. In a previous study, our group showed spontaneous induction of replicative senescence in p53- and p16INK4a-deficient HCC (hepatocellular carcinoma) cell clones. The gene expression profiling was later done for these different clones, in an attempt to find novel therapeutic targets in HCC. Since protein kinases are known to be very important in disease formation and carcinogenesis, their partners in signaling, protein phosphatases should also be important in these processes. Hence analysis and targeting of protein phosphatases genes with differential expression between immortal and senescent clones might prove beneficial for HCC therapeutics. Among the phosphatase genes with differential expression patterns, we focused on two most upregulated genes in senescent clones with respect to immortal clones, DUSP10 and MTMR11. After gathering detailed information on these genes and their products by bioinformatics analysis, we confirmed the upregulation of the two genes in our senescent clones compared to our immortal clones by semi-quantitative RT-PCR. We then checked DUSP10 and MTMR11 expression in HCC and breast cancer cell lines to see if a differential expression of these genes are observed in different subtypes of these cell lines. Other experiments on MTMR11 focused on discovery of novel transcripts of this gene in HCC and breast cancer cell lines and checking the amounts of different transcripts in different subtypes of these cell lines, to form a bridge between MTMR11 transcript variants and carcinogenesis, however we did not observe differential expression. Two microarray studies comparing non-tumor and HCC tissues have listed MTMR11 as upregulated in HCC. Hence, upregulation of this gene in senescent clones may not be significant in hepatocarcinogenesis or replicative senescence, and further experiments should be performed. Considering DUSP10, we checked the subcellular localization of this protein in HCC cell lines by immunostaining, to see if the two subtypes (well-differentiated and poorlydifferentiated) of HCC cell lines differed in DUSP10 localization. We observed some cell lines having only nuclear or only cytoplasmic DUSP10, whereas most had both nuclear and cytoplasmic DUSP10. This lead the way for us to explore the factors that may be important in changing this protein’s localization, as this may be a type of regulation on this protein, and may change during carcinogenesis or upon induction of senescence. For this purpose, we checked to see if DUSP10 changed its localization in aging MRC-5 cell passages compared to young, proliferating ones and in premature senescence-induced cells compared to normal ones. Interestingly, it was found that upon replicative senescence induction, but not premature senescence, DUSP10 localized more to the cell nucleus which indicated a connection between DUSP10 localization and replicative senescence. We also checked to see if DUSP10 changed its localization upon disruption of the MAPK pathways it participates in, by kinase inhibitor experiments. Interestingly, it was found that DUSP10 localized significantly more to the cell nucleus upon inhibition of JNK pathway but not p38 pathway, in well-differentiated subtype of HCC cell lines. DUSP10 localization did not change significantly in poorly-differentiated subtype of HCC cell lines. Although JNKs, which seem to regulate DUSP10 through its localization according to this study, act as oncogenes in HCC, the significance of the change in DUSP10 localization should be characterized further before stating that DUSP10 can be a putative tumor suppressor. However, our other results indicate a relationship between DUSP10 localization and replicative senescence, which is promising because DUSP10 has emerged from our group’s microarray data as a replicative senescence-associated gene, and this connection should be analyzed further.
Open Access
Molecular mechanisms of senescence response to transforming growth factor-beta in liver cancer
(2010) Şentürk, Şerif
Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. HCC is associated with several etiological factors including infections with hepatitis B and C viruses, heavy alcohol consumption and chronic aflatoxin B1 exposure. Due to its multi-step disease hallmark characterized with genetic heterogeneity, liver cancer has very limited therapeutic options. In light of many previous findings, cellular senescence acts as a barrier against immortalization and prohibits the proliferation of premalignant cells in various tumors including HCCs. However, implications of this anti-tumor mechanism in hepatic tissues are not wellknown. TGF-β is a multifunctional cytokine implicated in diverse cellular processes including senescence arrest as well as liver physiology and pathophysiology. Although TGF-β-induced senescence has been described in different cell types, this issue has never been addressed for hepatic cells. According to our recent data, TGF- β1 expression pattern in various HCC malignancies closely correlated with reported frequencies of SABG activities in these corresponding disease stages. Therefore, we hypothesized that TGF-β signaling might play key role in hepatocellular senescence. Well-differentiated (WD) five cell lines characterized with epithelial-like morphology displayed TGF-β-induced growth inhibition associated with SABG activity, with lack of evidence of apoptosis induction. Even a brief exposure to TGF- β was sufficient to trigger a massive senescence response. Senescence arrest in WD cell lines was linked to c-myc down-regulation and a reciprocal increase in p21Cip1 and p15Ink4b protein levels. In addition, TGF-β-induced senescence was correlated with Nox4 induction, intracellular accumulation of reactive oxygen species (ROS) and sustained 53BP1 foci formation as a mark of DNA-damage response. Moreover, intratumoral injection of TGF-β in human HCC tumors, generated subcutaneously in immunodeficient mice, induced expanded SABG that was associated with a strong anti-tumor response activity. On the other hand, poorly differentiated (PD) HCC cell lines with mesenchymal-like characteristics appeared to be resistant to TGF−β-induced senescence. However, PD cell lines had intact TGF-β signaling from cell membrane to nucleus. Resistance of PD cell lines was partially due to zeb2 overexpression, homozygous p15Ink4b deletion and lack of pRb expression. Besides, PD cells did not display Nox4 upregulation and also lacked ROS accumulation upon TGF-β stimulation. In addition, we demonstrated that sustained exposure to TGF-β established resistant Huh7 subclone. The resistance was partially attributed to deregulated Smad signaling, permanent epithelial-mesenchymal transition-like transformation. Surprisingly enough, removal of TGF-β from culture medium of continuously treated Huh7 subclone did not resolve the resistance phenotype in the rescued subclone. Epigenetic regulations mainly histone modifications are considered as candidate mechanisms responsible for irreversible TGF-β-resistance and maintenance of mesenchymal-like phenotype. Taken together, our results establish a close link between senescence arrest and anti-tumor activity of TGF-β signaling pathway in WD cell lines by delineating the mechanisms underlying TGF-β-induced growth arrest. Moreover, we propose partial explanation for the resistance to TGF-β- mediated growth arrest in PD cell lines and thoroughly signify the potential mechanisms of acquired resistance to TGF-β in continuously treated cultures. Further studies to enlighten our knowledge about implications of TGF-β signaling in less differentiated HCCs are necessary. As a conclusion, we identify TGF-β signaling as a potent therapeutic option for well-differentiated early HCCs.