Browsing by Subject "Cancer cells."

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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
Analysis of members of the SLIT-ROBO pathway as diagnostic and prognostic tools in hepatocellular carcinoma with special focus on ROBO2-associated cellular phenotype
(2009) Avcı, Mehmet Ender
Hepatocellular carcinoma is the sixth most common cancer in the world, with an annual incidence exceeding half a million. It is associated mainly with hepatitis B and C viral infections; and is the main cause of death among cirrhotic patients. Aflatoxin B1 exposure, chronic alcohol consumption and virtually all cirrhosis-inducing conditions are of the other etiologies. For early diagnosis of HCC, surveillance of the risk groups is a crucial task requiring the development of novel markers for HCC with stronger sensitivity and specificity. In addition, description of biomarkers specific to hepatocellular carcinoma subtypes could identify novel targets for therapy. In this study, we analyzed members of the SLIT-ROBO gene families as novel diagnostic and prognostic markers in hepatocellular carcinoma. We defined an expression signature for members of the SLIT-ROBO gene families in HCC cell lines and tissues by real-time quantitative RT-PCR analysis. We showed that ROBO1 was overexpressed as stage and differentiation of the HCC proceeds. Furthermore, ROBO4 downregulation and SLIT2 overexpression marked late stage and poorly differentiated HCCs. Our results suggest that the expression of ROBO1 and ROBO4 can be used in early diagnosis of HCC. As another focus, we stably knockdowned ROBO2 expression in a model AFP positive cell line Huh7 and characterized the associated cellular phenotype. ROBO2 downregulation caused a significant decrease in proliferation rate whereas in wound-healing assay no significant difference in migration rate was observed. In addition, we performed a microarray experiment and found the differentially expressed genes between stable ROBO2 knockdown and negative clones. In this analysis, we found an overexpression of CK19, CD44, ABCG2/ BCRP1 hepatic progenitor cell markers and CD133 that is also a putative cancer stem cell marker of HCC, in stable ROBO2 knockdown clones. In addition KLF4 expression was augmented in these ROBO2 knockdown clones. We propose a genetic association between SLIT-ROBO pathway and CD133 at transcriptional level.
Open Access
Characterization of cancer stem cells in hepatocellular carcinoma
(2014) Abdüsselamoğlu, Merve Deniz
Hepatocellular carcinoma (HCC) is the third most common cause of death from cancer worldwide due to the challenges in both its diagnosis and treatment. According to recent studies, HCC tumors, like many other solid tumors are initiated and maintained by a subpopulation of cells called “cancer stem cells (CSCs)” or "tumor-initiating cells (TICs)". HCC stem cells can be identified by the expression of cardinal CD markers such as CD133 (Prominin-1) and epithelial cell adhesion molecule (EpCAM). This study primarily focuses on the investigation of mechanisms involved in the generation of HCC stem cell sub-population using a panel of 15 HCC cell lines. Preliminary data indicates that four cell lines (27%) display CD133+ stem cell populations at frequencies ranging from 8 to 90% when tested by flow cytometry. Among these CD133 positive cell lines, two isogenic cell line with different positivity levels prompted us to focus on two specific cell lines;, i) parental HepG2 cell line and its clone, which was transfected with four copies of hepatitis B virus (HBV), namely ii) HepG2-2215. With tumorigenicity assay induced in atymic nude mice, data revealed that HepG2-2215 that had higher CD133+ ratio, showed higher and rapid tumor formation than parental HepG2 that had much lower CD133+ sub-cellular proportion. Microarray analyses were performed to underpin the mechanisms of in CD133+ cell number variations of these two cell lines. Our initial findings suggested that FGFR signaling pathway might have played a role. To investigate these findings, FGFR signaling pathway was inhibited via potent inhibitor as well as knock down with siRNA. However, preliminary data did not indicate these presumptions and further studies are needed to clarify the relationship between FGFR signaling and CSC formation in HCC. Also, role of suppressive oligodeoxynucleotide (ODN) was studied to see the effects of suppression of DNAdriven immunostimulation. Findings showed that suppressive ODN decreased CD133 levels, which indicates the difference between these two cell lines may arise from the HBV transfection of HepG2-2215 cell line which can produce HBV particles. However, further investigation is needed to understand the relationship between HBV infection and CSC population in HCC.
Open Access
Characterization of chemosensitivity profiles of breast cancer cell lınes, with and without stem cell like features = Kök-hücre özelliği olan ve olmayan meme kanseri hücre hatlarının ilaç hassasiyet profillerinin tanımlanması
(2014) Akbar, Muhammad Waqas
Breast cancer is the second most common cause of death worldwide from cancer due to complications with its diagnosis and resistance to therapy. Recent studies have shown that breast tumors when compared with other solid tumors also contain a subpopulation termed as cancer stem cells (CSCs). CSCs are hard to kill due to their therapy resistant capacities. These unharmed cells then result into relapse of tumor after treatment. Some established breast cancer cell lines also behave in similar fashion to CSCs in overall manner thus termed as CSC like cell lines. This study primarily focuses on characterizing CSC like cell lines from non CSC like cell lines based upon their gene expression and prediction of drugs which can target these groups separately. In this study two databases, Cancer Cell Line Encyclopedia (CCLE) and Cancer Genome Project (CGP), were used which contain gene expression data and drugs cytotoxicity data for most of the established cancer cell lines. Breast cancer cell lines gene expression data was used to predict two gene lists which can separate breast cancer cell lines into CSC like and non CSC like cell lines by in silico analysis. These gene lists were named as Patentable and Non Patentable. Additionally four drugs were predicted which can target CSC like group (Midostaurin and Elesclomol) and non CSC like group (Panobinostat and Lapatinib) separately. Later these findings were validated in vitro. Non Patentable gene list could not be validated due to low concordance with microarray data. On the other hand, Patentable gene list was validated and was found concordant with microarray data. Out of four selected drugs, Panobinostat and Lapatinib showed increased toxicity to non CSC like cell lines while only Midostaurin showed toxicity to CSC like cell lines. To investigate further that cell lines were grown in 3D cell culture conditions, to increase their stem cell like properties (stemness). But only one cell line MDA-MB-157 which was found as CSC like, showed expected behavior. Additionally this cell line increased resistance to Lapatinib and Panobinostat and became more sensitive to Midostaurin. Correlation analysis showed some genes as potential biomarkers for selected drugs. In conclusion, in this study various genes are proposed to differentiate CSC like cell lines from non CSC like cell lines. And Midostaurin can be potential drug to treat CSC like cells while Lapatinib and Panobinostat showed increased activity against non CSC like cell lines.
Open Access
Characterization of FAM134B in the context of hepatocellular carcinoma and endoplasmic reticulum protein stress
(2011) Yılmaz, Mustafa
Family with sequence similarity 134, member B (FAM134B) is a replicative senescence associated gene, previously identified in studies of our group as a result of microarray analysis in spontaneously senescent clones of Huh7 hepatocellular carcinoma cell line and their immortal counterparts. Originating from this finding, this study primarily focused on characterization of FAM134B in the context of hepatocellular carcinoma and endoplasmic reticulum stress. At the beginning, the relationship between senescence and FAM134B was experimented by inducing premature senescence in Huh7 cells. Adriamycin or TGF-β induced premature senescence did not result in amplification of FAM134B gene expression, suggesting that upregulation of FAM134B expression in spontaneous replicative senescence is not directly associated with a senescence phenotype. Then, FAM134B mRNA and protein levels were analyzed in both well- and poorly-differentiated HCC cell lines. Results showed that FAM134B expression is greater in poorly-differentiated cell lines, which represent advanced and metastatic HCC in vitro. On the other hand, our studies on the relationship between FAM134B and endoplasmic reticulum (ER) stress showed that FAM134B is an ER stress response gene, whose expression is upregulated by induction of ER stress with chemicals, such as thapsigargin, tunicamycin or DTT. Therefore, high protein and mRNA levels of FAM134B in poorly-differentiated cell lines are linked to the presence of a basal level ER stress response in this group of cell lines. Furthermore, overexpression studies in Huh7 cells indicated that FAM134B cannot trigger an ER stress response or autophagic response in these cells. However, FAM134B was detected as an effector in cellular response, when ER stress is artificially induced by thapsigargin or tunicamycin treatments. FAM13B4 overexpression in Huh7 resulted in increased sensitivity to thapsigargin or tunicamycin induced apoptosis. Moreover, increased FAM134B expression was also associated with decreased proliferative capacity in response to ER stress induction with the same chemicals. Consequently, FAM134B was suggested to affect the severity of stress in the ER when ER stress is started with an inducer. In addition, our tissue based experiments revealed that FAM134B is expressed in the brain and liver. Taken together, FAM134B might be an important protein contributing to the liver tissue damage and pathogenesis of HCC.
Open Access
Contribution of notch signaling on HCC stem cell status and utilizing TLR agonists and notch inhibition to improve HCC theraphy
(2014) Ertuna, Yusuf İsmail
Hepatocellular carcinoma (HCC) is the seventh most common cancer type worldwide, and ranked third place among cancer-related deaths within both sexes. As in many solid tumors, HCC shelters a cancer stem cell subpopulation, and is held responsible for the resistance developed during chemo-and-radio-therapy of HCC. The only option to cure HCC is liver transplantation, which is the bottleneck to provide a remedy to patients due to limited availability. Understanding the stem cell behavior of HCC would critically contribute to develop effective eradication strategies. In this study, a panel of 17 HCC cell lines was evaluated for their CSC status. Of these cell lines, six of them were determined to be positive for CD133 expression, a cardinal CSC marker. Next, HepG2, Huh7 and Hep3B-TR, (a desensitized TGF-beta-1 receptor clone) were selected and Notch activity vs. CSC fraction was investigated by analyzing CD133+/EpCAM+ levels. Our results revealed that DAPT (a notch inhibitor) led to a drop in CD133+/EpCAM+ levels in HepG2 and Huh7 by half, but not in Hep3B-TR cells, implicating a possible TGFβ1R involvement on CSC generation/maintenance. Treatment of cells with a notch ligand, Jagged-1, however, had little or no positive effect on CD133+/EpCAM+ expressions in all tested cells. Additionally, HCC cells' response to different TLR ligands and the resulting transcript expressions of TLRs were investigated by PCR. Of note, TLRs are widely used in immunotherapy of cancers. Here we aimed to combine Notch inhibitor along with selected TLR ligand, thereby improving tumor clearance in athymic mice xenografted with HCC. We found that in three selected cell lines upon TLR2 ligand stimulation, TLR5 and TLR7 were highly upregulated. Afterwards, treating these HCC cell lines with these ligands we observed that TLR3, TLR7/8 and TLR9 levels were activated. In the final part of this study, tumor-bearing mice with Huh7, were subjected to a combination therapy with TLR ligands +/- DAPT. We demonstrated that combination therapy comprising TLR3, 7/8 and 9 ligands and DAPT (only two injections, a week apart) induced significant tumor regression.
Open Access
Gene expression profiling of hedgehog pathway genes in epithelial cancers
(2006) Öztürk, Özlem Akıllı
The Hedgehog (Hh) signaling pathway has been investigated in many cancer types and shown to have important effects, but not effectively studied in breast, colon and liver cancers. In this study, gene expression profile of BCL2, SHH, SMO, IHH, PTCH1, GLI1, GLI2 and GLI3 were analyzed in 15 breast, 14 colon, and 15 hepatocellular carcinoma (HCC) cell lines and 29 primary breast tumor samples and three matched normal tissue sample pools by quantitative real-time RT-PCR. Breast cell lines have different levels of target gene expression in the Hh pathway, such that this pathway is activated only in some of the breast carcinoma cell lines possibly through a ligand-independent pathway. In all the breast cancer cell lines studied, PTCH1, SMO, GLI3 and BCl2 had high expression. Expression profiles of the target genes predicted the estrogen receptor status correctly in 93% of the breast cell lines studied. In HCC cell lines, Hh pathway gene expression profile differentiates the poorly differentiated HCC cell lines from well differentiated ones in Discriminant Function Analysis (DFA) perfectly. High SMO and IHH expressions have been found to be markers for aberrant Hh pathway activity in the well differentiated HCC cell lines. In colon cancer cell lines deregulated expression profile among the genes were observed. In primary breast tumor samples, there was a very strong prediction for ER status of the samples with the expression of the genes included in this study. This is the first comprehensive study that shows the transcriptional gene expression profiles of the main target genes of the Hh pathway in cancer cell lines and breast cancer tissue samples
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
Identification and characterization of two endoplasmic reticulum protein isoforms encoded by senescence-associated FAM134B gene
(2008) Taşdemir, Nilgün
Liver cancer is the fifth most common cancer in the world. Until recently, tumor cells were known to have the capacity to proliferate indefinitely. In a previous study, we showed the spontaneous induction of replicative senescence in p53- and p16INK4a-deficient HCC (hepatocellular carcinoma) cells. In a follow-up study, we have analyzed the Affymetrix expression profiling of the senescent and immortal HCC clones that we had established. Among the genes with differential expression pattern, in this study, we have focused on a novel gene, FAM134B (family with sequence similarity 134, member B), which is significantly up-regulated (p-value=1.097E-06) in our senescent clones with respect to their immortal counterparts. FAM134B gene is located on human chromosome 5p15.1 near a LOH region, and its protein product has not yet been characterized. To begin with, we confirmed the up-regulation of FAM134B in our senescent clones as compared to our immortal clones by RT-PCR analysis. As a next step, meta-analysis of HCC microarray data indicated that the expression of FAM134B gene is progressively down-regulated in non-metastatic and metastatic HCC as compared to normal liver. Thus, we decided to characterize the protein product of this gene. Two known forms of transcripts were used to construct FLAG-tagged expression plasmids (encoding two isoforms with predicted molecular weights of 30 and 55 kDa). Immuno-staining experiments performed after transient ectopic expression indicated that both short and long isoforms of FAM134B-encoded protein localize to the endoplasmic reticulum (ER). Both protein isoforms co-localized with calnexin, a well known ER-chaperon. Thus, it appears that senescent cells over-express FAM134B-encoded ER protein isoforms, while cancer cells are deficient in their expression. We have also performed gain-of-function studies by stable ectopic expression of these two protein isoforms in an HCC cell line and addressed the potential role(s) of these isoforms in senescence and ER-stress. Our studies indicated that over-expression of these proteins did not have a ‘causative’ role in induction of senescence and did not affect the rate of cell proliferation. We also did not observe any changes in the responses of cells over-expressing these two protein isoforms to ER-stress induced via tunicamycin treatment. Therefore, FAM134B gene may be performing a yet unidentified function in senescent cells. All in all, we have identified two FAM134B-encoded proteins that localize to the ER, the function and the senescence association of which need further investigation.
Open Access
Identification of putative protein kinase inhibitors acting on liver cancer cells
(2012) Güven, Ebru Bilget
Hepatocellular carcinoma (HCC) as a, heterogeneous, multi-step, slowprogressing disease, has very limited treatment options due to its chemoresistant nature and late diagnosis. According to World Health Organization (WHO) reports HCC is a major public health problem. Each year, 748,000 new cases appear and 696,000 people lose their lives, all due to liver cancer alone. Sorafenib, the multi-tyrosine kinase inhibitor, is still the only (Food and Drug Administration) FDA-approved drug available for treatment. Therefore, there is an urgent need for novel, target-specific drugs based on the underlying molecular mechanisms of liver carcinogenesis. In this Ph.D. dissertation, synthetic purine, purine nucleoside analogs, aminotriazole and thiadiazine derivatives were evaluated for their cytotoxic activities and mechanisms of action against liver cancer. These novel molecules were selected because of their potential kinase inhibitory activity. Protein kinases, involved in signaling pathways, are the main enzymes of target-specific drug discovery; therefore, discovery of novel, putative protein kinase inhibitors as drug candidates can be promising for the treatment of primary liver cancer. Initially, sulforhodamine B (SRB) assay was used to screen the novel smallmolecules for their cytotoxic activities against breast, colon and liver cancer cell lines. Active molecules, then, were further exploited on a panel of HCC cell lines. The differential IC50 (half-maximal inhibitory concentration) values obtained, might indicate that these small-molecules interfere with cell signaling; since, these cell lines have individual characteristics of cell signaling activities. Further investigations are envisaged for the identification of the molecular mechanisms that these putative kinase inhibitors are involved in. Among the 228 newly synthesized putative kinase inhibitors, 3 smallmolecules were identified as promising anti-cancer agents against liver cancer with druggable cytotoxic activities and remarkable kinase inhibition potentials. The purine analogue, AUM32, and the purine nucleoside analogue, AUM42, were revealed as pro-senescence therapeutic agents in liver cancer. Both drug candidates were shown to initiate senescence-induced cell death and the underlying mechanism was confirmed for AUM42 as the induction of p15(INK4b) and the correlated decrease in Rb phosphorylation. Synthetic 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine derivative, ClAT-TM, is the other effective small-molecule with kinase inhibition potential. Upon ClATTM treatment, liver cancer cells experience a growth inhibition accompanying with dramatic morphological changes. Rounded, swollen and eventually detached cells were shown to be arrested in the G2/M stage of the cell cycle and eventually undergo apoptosis. Moreover, ROS (reactive oxygen species) accumulation and the activation of JNK signaling pathway were found as associated with the mechanism of action ClAT-TM cytotoxicity.
Open Access
Identification of the interacting domain of p53 family members with P33ING1 protein
(2002) Dinçel, Deniz
p53 is a tumor suppressor gene, which is mutated, in about 50% of human cancers. The product of p53 gene encodes a sequence specific transcription factor. The genes transactivated by p53 code for proteins that are implicated in the negative regulation of cell proliferation and DNA damage repair. Two proteins, p63 and p73 members of p53 family, show striking homology to p53. p53 protein interacts with several viral and cellular proteins and these interactions are important in the regulation and dysregulation of the functions of p53. Another gene named, ING1 was identified as a candidate tumor suppressor gene due to its functions in apoptosis and cell cycle arrest. p24ING1, one of the protein product of ING1, was shown to enhance the growth suppressor functions of p53. Furthermore a physical association between p53 and p33ING1, another ING1 transcript, proteins has been detected by immunoprecipitation. In this study, we investigated the physical interaction between p53 family proteins and p33ING1 using in vitro techniques in order to determine the region(s) of p53 family proteins and p33ING1 that enabled this interaction. As a preliminary step for the study, the wild-type p53 cDNA and its several deletion mutant constructs were used in GST pulldown assays and Far Western assays with purified GST-p33 protein to map the interacting region on p53 protein. New deletion mutant constructs of p53 protein were created and cloned into expression vectors for the detailed analysis of the interacting domain of p53 protein. Also the other members of p53 protein family, p63 and p73 were examined in vitro for interaction with p33ING1. Deletion mutants of these proteins were created and cloned into expression vectors for protein-protein interaction assays. The results of this study shows that p53 protein interacts with p33ING1 and suggests that oligomerization domain of p53 protein is needed for this interaction. In addition, for the first time, it was showed that p63 and p73 proteins interact with p33ING1 and in p63 the C-terminus region is the primary determinant region, involved in these interactions with p33ING1.
Open Access
Identification of the role of p33ING1 protein in the cellular activities of p53 tumor suppressor protein
(1999) Emre, N. C. Tolga
p53 is a tumor suppressor gene which is mutated in about 50% of human cancers. The product of p53 gene encodes a sequence-specific transcription factor. The genes are transactivated by p53 code for proteins that are implicated in the negative regulation of cell proliferation (via apoptosis or cell cycle arrest) and DNA damage repair. p53 protein interacts with several viral and cellular proteins and these interactions are important in the regulation and dysregulation of the functions of p53. Another gene, named ING1 (for "Inhibitor of Growth 1"), was identified as a candidate tumor suppressor gene due to its functions in apoptosis and cell cycle arrest. p33ING1, the protein product of ING1, was shown to enhance the growth suppressive functions of p53. Furthermore, a physical association between p53 and p33ING1 proteins has been detected by immunoprecipitation. In this study, we investigated the physical interaction between p53 and p33ING1 using in vitro methods in order to determine the region of p53 that enabled this interaction. As a preliminary step for the study, the ING1 cDNA was amplified from total cDNA of a cell line by PCR and cloned into expression vectors. The recombinant p33ING1 protein was overexpressed in E.coli and purified as a fusion protein with GST. The wild-type p53 cDNA and its several deletion mutant constructs were subcloned into a suitable expression vector to enable subsequent in vitro transcription-translation reactions. In vitro transcription-translation products of these constructs were used in GST pulldown assays with purified GST p53 protein to map the interacting region on the human p53 protein. The results of the study suggest that the primary determinant on p53 protein in its interaction with p33ING1, is the C-terminal domain while there may be other regions that are involved in the interaction.
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
Senescence and immortality genes as markers of hepatocellular carcinogenesis
(2009) Arslan Ergül, Ayça
Cellular senescence is a tumor-suppression mechanism, and immortalization facilitates neoplastic transformation. Both mechanisms may be highly relevant to hepatocellular carcinoma (HCC) development. We worked on two major aspects of cellular senescence and immortality in HCC. First, we analyzed the role of ZEB2 (Smad-interacting protein SIP1, ZFXH1B) gene for a senescence-related role in HCC. Then, we extended our work on the identification and analysis of a senescence and immortality gene network (SIGN) in relation to hepatocellular carcinogenesis. ZEB2 is a transcriptional repressor of E-cadherin, and induces epithelial-mesenchymal transition (EMT), a key process involved in tumor metastasis and progression. However, ZEB2 is also a repressor of telomerase reverse transcriptase (TERT) gene, which encodes a key enzyme required for telomere maintenance and tumor cell immortality. We performed in-vivo, in-silico and in-vitro studies to explore potential implications of ZEB2 in hepatocellular carcinoma (HCC). Tissue expression of ZEB2 transcripts displayed stepwise decreases in HCC lesions, as compared to liver cirrhosis. This inverse correlation suggested that sustained ZEB2 expression is not compatible with HCC progression. Next, vekt¨or¨u ile transfekte edilen Hep3B h¨ucrelerinin, ya¸slanma ili¸skili !-galaktozidaz aktivitesi ile, kalıcı h¨ucre d¨ong¨us¨u hapsine girdiklerini g¨ord¨uk. ZEB2 ile ind¨uklenen ya¸slanma hapsi, TERT ifadesinin baskılanması ve e¸slik eden siklin-ba˘gımlı kinaz engelleyici p21Cip1’in ifadesindeki artı¸s ile ba˘gıntılı idi. ZEB2’nin ge¸cici ifadesi, p21Cip1 ifadesindeki artı¸sı ind¨uklemedi. Son olarak, ZEB2’yi a¸sırı ifade eden Hep3B ve A431 klonlarının, in vitro k¨ult¨urde dereceli olarak azalması ile ZEB2 a¸sırı ifadesinin, kanser h¨ucrelerinin in vitro ya¸samı ile uyumlu olmadı˘gı sonucuna varıldı. Bu g¨ozlemler, ZEB2 geninin, EMT’deki rol¨un¨un dı¸sında, HCC h¨ucre b¨uy¨umesi ve ya¸samasında negatif rol oynadı˘gını d¨u¸s¨und¨urd¨u. Di˘ger ¸calı¸smamızda SIGN imzasını olu¸sturmak i¸cin, ya¸slanmaya programlanmı ¸s ve ¨ol¨ums¨uz HCC h¨ucrelerinden gelen gen ifade datasını, siroz ve HCC dokularından gelen data ile birle¸stirdik. SIGN imzası normal karaci˘ger, siroz, displazi ve HCC lezyonlarını do˘grulukla sınıflandırdı ve ya¸slanmadan ¨ol¨ums¨uzl¨u˘ge d¨on¨u¸s¨um¨un, ilk olarak displaziden erken HCC’ye ge¸ci¸ste ger¸cekle¸sti˘gini belirledi. Bu d¨on¨u¸s¨um, t¨um¨or ilerlemesine de katkıda bulunur. Ya¸slanmadan ¨ol¨ums¨uzl¨u˘ge d¨on¨u¸s¨ume, hepatik geri ba¸skala¸sım ile ve h¨ucre ¸co˘galması, kromozom de˘gi¸simi ve DNA hasar yanıtı genlerindeki ifade artı¸sı e¸slik etti. Bu nedenle, HCC ¨ol¨ums¨uzl¨u˘g¨u, k¨ok h¨ucre/¨onc¨u h¨ucre benzeri ¨ozellikler ile yakından ili¸skilidir. Son olarak, DNA hasar kontrol noktası ve DNA tamir genlerindeki artı¸s ile, t¨um¨or ba¸slangıcı ve ilerlemesi arasında ili¸ski bulduk. Bu genler, HCC’nin engellenmesinde ve terapisinde potansiyel hedefler olabilirler.