Browsing by Subject "Immunohistochemistry"

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Open Access
The ability to generate senescent progeny as a mechanism underlying breast cancer cell heterogeneity
(Public Library of Science, 2010) Mumcuoğlu, Mine; Bağışlar, Sevgi; Yüzügüllü, Haluk; Alotaibi, Hani; Şentürk, Şerif; Telkoparan, Pelin; Gür-Dedeoğlu, Bala; Cingöz, Burcu; Bozkurt, B.; Tazebay, Uygar H.; Yuluğ, Işık G.; Akçalı, Kamil Can; Öztürk, Mehmet
Background Breast cancer is a remarkably heterogeneous disease. Luminal, basal-like, "normal-like", and ERBB2+ subgroups were identified and were shown to have different prognoses. The mechanisms underlying this heterogeneity are poorly understood. In our study, we explored the role of cellular differentiation and senescence as a potential cause of heterogeneity. Methodology/Principal Findings A panel of breast cancer cell lines, isogenic clones, and breast tumors were used. Based on their ability to generate senescent progeny under low-density clonogenic conditions, we classified breast cancer cell lines as senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All SCP cell lines expressed estrogen receptor (ER). Loss of ER expression combined with the accumulation of p21Cip1 correlated with senescence in these cell lines. p21Cip1 knockdown, estrogen-mediated ER activation or ectopic ER overexpression protected cells against senescence. In contrast, tamoxifen triggered a robust senescence response. As ER expression has been linked to luminal differentiation, we compared the differentiation status of SCP and ICP cell lines using stem/progenitor, luminal, and myoepithelial markers. The SCP 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 ICP cell lines generated only CD44+/CD24-/ER-/ASMA- progenitor/stem-like cells, and others also produced CD24+/ER- luminal-like, but not ASMA+ myoepithelial-like cells. Furthermore, gene expression profiles clustered SCP cell lines with luminal A and "normal-like" tumors, and ICP cell lines with luminal B and basal-like tumors. The ICP cells displayed higher tumorigenicity in immunodeficient mice. Conclusions/Significance Luminal A and "normal-like" breast cancer cell lines were able to generate luminal-like and myoepithelial-like progeny undergoing senescence arrest. In contrast, luminal B/basal-like cell lines acted as stem/progenitor cells with defective differentiation capacities. Our findings suggest that the malignancy of breast tumors is directly correlated with stem/progenitor phenotypes and poor differentiation potential.
Open Access
Age related alterations of adult neurogenesis and astrocytes in Zebrafish (Danio Rerio)
(2019-09) Ardıç, Narin Ilgım
Brain aging is marked by a decline in cognitive abilities and associated with neurodegenerative disorders. In order to identify appropriate interventions to change the course of brain aging and age-related neurological disorders, we should first understand the normal age-related changes. Previous studies claimed that there was a correlation between cognitive capacities and number of neurons. However, recent studies have shown no statistically significant change in total neuron number during healthy aging. Therefore, further studies are required to understand the reasons behind these changes in the brain. One possibility could be the age-related alterations in neuronal lineage and glial markers. Thus, this study aims to show the protein levels, distributions, and localizations of key neuronal lineage and glial markers, which include neural progenitor, early neuronal, immature neuron, and mature neuron and glial markers during healthy aging of the zebrafish brain. For this aim, we measured NeuN (Fox-3, Rbfox3, or Hexaribonucleotide Binding Protein-3), MAP-2 (Microtubule-associated protein 2), HuC (ELAV like neuron-specific RNA binding protein 3), DCAMKL-1 (Doublecortin-like kinase 1), and GFAP (Glial fibrillary acidic protein) with immunohistochemistry and western blot techniques. First, the immunohistochemistry technique was applied on two specific proliferation areas, pallium and optic tectum, to detect the changes in the number of neuronal lineages and glial marker. The results indicated no statistically significant changes between young and old groups. Secondly, we performed whole-brain immunohistochemistry of all markers and quantified every image by manually counting the positive signal. We found that aging did not have an effect on the distribution and expression of the markers, even in the whole brain. Finally, Western-blot was performed in whole brain lysates to compare neuron number and protein level changes. Western-blot results indicated an age-related statistically significant decline in immature neuron marker for specifically males and glial marker for specifically females. The protein level of neural progenitor marker showed the significant decline in males during aging but no change between two age groups. Results of the mature neuron antibody revealed that the protein levels were consistent through aging and did not show variation. Our results overall support the finding that the number of neurons and glia do not change during aging since the numbers of markers were not show statistically significant changes during the aging process in the proliferation areas of the zebrafish brain. However, protein levels showed changes between age and gender groups. Thus, this study shows that understanding changes in the number of cells need to count; protein level is not representative, and zebrafish is an appropriate model for brain aging studies.
Open Access
Differential expression patterns of metastasis suppressor proteins in basal cell carcinoma
(Wiley-Blackwell Publishing Ltd., 2015) Bozdogan, O.; Yulug, I. G.; Vargel, I.; Cavusoglu, T.; Karabulut, A. A.; Karahan, G.; Sayar, N.
Background: Basal cell carcinomas (BCCs) are common malignant skin tumors. Despite having a significant invasion capacity, they metastasize only rarely. Our aim in this study was to detect the expression patterns of the NM23-H1, NDRG1, E-cadherin, RHOGDI2, CD82/KAI1, MKK4, and AKAP12 metastasis suppressor proteins in BCCs. Methods: A total of 96 BCC and 10 normal skin samples were included for the immunohistochemical study. Eleven frozen BCC samples were also studied by quantitative real time polymerase chain reaction (qRT-PCR) to detect the gene expression profile. Results: NM23-H1 was strongly and diffusely expressed in all types of BCC. Significant cytoplasmic expression of NDRG1 and E-cadherin was also detected. However, AKAP12 and CD82/KAI1 expression was significantly decreased. The expressions of the other proteins were somewhere between the two extremes. Similarly, qRT-PCR analysis showed down-regulation of AKAP12 and up-regulation of NM23-H1 and NDRG1 in BCC. Morphologically aggressive BCCs showed significantly higher cytoplasmic NDRG1 expression scores and lower CD82/KAI1 scores than non-aggressive BCCs. Conclusion: The relatively preserved levels of NM23-H1, NDRG1, and E-cadherin proteins may have a positive effect on the non-metastasizing features of these tumors.
Open Access
Heparin mimetic peptide nanofiber gel promotes regeneration of full thickness burn injury
(Elsevier Ltd, 2017) Yergoz, F.; Hastar, N.; Cimenci, C. E.; Ozkan, A. D.; Güler, Mustafa O.; Tekinay, A. B.; Tekinay, T.; Güler, Mustafa O.
Burn injuries are one of the most common types of trauma worldwide, and their unique physiology requires the development of specialized therapeutic materials for their treatment. Here, we report the use of synthetic, functional and biodegradable peptide nanofiber gels for the improved healing of burn wounds to alleviate the progressive loss of tissue function at the post-burn wound site. These bioactive nanofiber gels form scaffolds that recapitulate the structure and function of the native extracellular matrix through signaling peptide epitopes, which can trigger angiogenesis through their affinity to basic growth factors. In this study, the angiogenesis-promoting properties of the bioactive scaffolds were utilized for the treatment of a thermal burn model. Following the excision of necrotic tissue, bioactive gels and control solutions were applied topically onto the wound area. The wound healing process was evaluated at 7, 14 and 21 days following injury through histological observations, immunostaining and marker RNA/protein analysis. Bioactive peptide nanofiber-treated burn wounds formed well-organized and collagen-rich granulation tissue layers, produced a greater density of newly formed blood vessels, and exhibited increased re-epithelialization and skin appendage development with minimal crust formation, while non-bioactive peptide nanofibers and the commercial wound dressing 3M™ Tegaderm™ did not exhibit significant efficiency over sucrose controls. Overall, the heparin-mimetic peptide nanofiber gels increased the rate of repair of burn injuries and can be used as an effective means of facilitating wound healing.
Open Access
Metastasis suppressor proteins in cutaneous squamous cell carcinoma
(Elsevier, 2016-07) Bozdogan, O.; Vargel, I.; Cavusoglu, T.; Karabulut, A. A.; Karahan, G.; Sayar, N.; Atasoy, P.; Yulug, I. G.
Cutaneous squamous cell carcinomas (cSCCs) are common human carcinomas. Despite having metastasizing capacities, they usually show less aggressive progression compared to squamous cell carcinoma (SCC) of other organs. Metastasis suppressor proteins (MSPs) are a group of proteins that control and slow-down the metastatic process. In this study, we established the importance of seven well-defined MSPs including NDRG1, NM23-H1, RhoGDI2, E-cadherin, CD82/KAI1, MKK4, and AKAP12 in cSCCs. Protein expression levels of the selected MSPs were detected in 32 cSCCs, 6 in situ SCCs, and two skin cell lines (HaCaT, A-431) by immunohistochemistry. The results were evaluated semi-quantitatively using the HSCORE system. In addition, mRNA expression levels were detected by qRT-PCR in the cell lines. The HSCOREs of NM23-H1 were similar in cSCCs and normal skin tissues, while RGHOGDI2, E-cadherin and AKAP12 were significantly downregulated in cSCCs compared to normal skin. The levels of MKK4, NDRG1 and CD82 were partially conserved in cSCCs. In stage I SCCs, nuclear staining of NM23-H1 (NM23-H1nuc) was significantly lower than in stage II/III SCCs. Only nuclear staining of MKK4 (MKK4nuc) showed significantly higher scores in in situ carcinomas compared to invasive SCCs. In conclusion, similar to other human tumors, we have demonstrated complex differential expression patterns for the MSPs in in-situ and invasive cSCCs. This complex MSP signature warrants further biological and experimental pathway research.
Open Access
Mutations in RAD21 disrupt regulation of apob in patients with chronic intestinal pseudo-obstruction
(W.B. Saunders, 2015) Bonora, E.; Bianco, F.; Cordeddu, L.; Bamshad, M.; Francescatto, L.; Dowless, D.; Stanghellini, V.; Cogliandro, R. F.; Lindberg, G.; Mungan, Z.; Cefle, K.; Ozcelik, T.; Palanduz, S.; Ozturk, S.; Gedikbasi, A.; Gori, A.; Pippucci, T.; Graziano, C.; Volta, U.; Caio, G.; Barbara, G.; D'Amato, M.; Seri, M.; Katsanis, N.; Romeo, G.; De Giorgio, R.
Background Aims Chronic intestinal pseudo-obstruction (CIPO) is characterized by severe intestinal dysmotility that mimics a mechanical subocclusion with no evidence of gut obstruction. We searched for genetic variants associated with CIPO to increase our understanding of its pathogenesis and to identify potential biomarkers. Methods We performed whole-exome sequencing of genomic DNA from patients with familial CIPO syndrome. Blood and lymphoblastoid cells were collected from patients and controls (individuals without CIPO); levels of messenger RNA (mRNA) and proteins were analyzed by quantitative reverse-transcription polymerase chain reaction, immunoblot, and mobility shift assays. Complementary DNAs were transfected into HEK293 cells. Expression of rad21 was suppressed in zebrafish embryos using a splice-blocking morpholino (rad21a). Gut tissues were collected and analyzed. Results We identified a homozygous mutation (p.622, encodes Ala>Thr) in RAD21 in patients from a consanguineous family with CIPO. Expression of RUNX1, a target of RAD21, was reduced in cells from patients with CIPO compared with controls. In zebrafish, suppression of rad21a reduced expression of runx1; this phenotype was corrected by injection of human RAD21 mRNA, but not with the mRNA from the mutated p.622 allele. rad21a Morpholino zebrafish had delayed intestinal transit and greatly reduced numbers of enteric neurons, similar to patients with CIPO. This defect was greater in zebrafish with suppressed expression of ret and rad21, indicating their interaction in the regulation of gut neurogenesis. The promoter region of APOB bound RAD21 but not RAD21 p.622 Ala>Thr; expression of wild-type RAD21 in HEK293 cells repressed expression of APOB, compared with control vector. The gut-specific isoform of APOB (APOB48) is overexpressed in sera from patients with CIPO who carry the RAD21 mutation. APOB48 also is overexpressed in sporadic CIPO in sera and gut biopsy specimens. Conclusions Some patients with CIPO carry mutations in RAD21 that disrupt the ability of its product to regulate genes such as RUNX1 and APOB. Reduced expression of rad21 in zebrafish, and dysregulation of these target genes, disrupts intestinal transit and the development of enteric neurons.
Open Access
Naringenin inhibits neointimal hyperplasia following arterial reconstruction with interpositional vein graft
(Lippincott Williams & Wilkins, 2010) Cayci, C.; Wahlquist, T. C.; Seckin, S. I.; Ozcan, V.; Tekinay, A. B.; Martens, T. P.; Oz, M. C.; Ascherman, J. A.
Vessels respond to injury by a healing process that includes the development of neointima. Stenosis secondary to neointima formation is the main cause of failure following arterial reconstructions. Vessel wall homeostasis is regulated by proinflammatory cytokines that affect smooth muscle cell proliferation, growth, migration, and death. We assessed the hypothesis that naringenin, a flavinoid possessing anti-inflammatory, antioxidant, and antiproliferative activities, reduces neointimal hyperplasia (NIH) following vascular injury.Arterial injury was created by interposition grafting of autologous right superficial epigastric vein graft into the right femoral artery (FA) in 48 male Sprague-Dawley rats. Following injury, the rats were divided into 4 groups (n = 12). Two groups were treated with naringenin (100 mg/kg intraperitoneal q daily) for 2 and 4 weeks each while 2 control groups received normal saline for the same durations. For Sham group (n = 10), the FA and vein were isolated without any additional procedure. Rats were killed at the end of treatment regimen in all groups, and FAs were harvested. Thickness of intima was measured in histologic sections, and levels of platelet derived growth factor (PDGF)-BB, TNFα, and Ki67 labeling index (Ki67 LI) were quantified in immunohistochemical analyses to assess the amount of NIH and mechanisms underlying its formation.Although there was no significant difference between the groups at 2 weeks, neointima thickness was lower in the naringenin treated group at 4 weeks (23.7 ± 2.3 vs. 35.6 ± 2.6 μm in control group; P < 0.001). The levels of PDGF-BB, and TNFα were lower in naringenin treated groups at both 2 weeks (PDGF-BB [0.21% ± 0.03% versus 0.39% ± 0.05% in control group, P < 0.001), TNFα (21.2% ± 0.8% vs. 36.1% ± 1.9% in control group, P < 0.001]) and 4 weeks (PDGF-BB [0.25% ± 0.03% vs. 0.57% ± 0.09% in control group, P < 0.001], TNFα [25.5% ± 1.8% vs. 45.0% ± 2.9% in control group, P < 0.001]). Ki67 LI was lower in naringenin treated groups at 2 weeks (13.9% ± 2.8% vs. 18.7% ± 3.7% in control group, P < 0.05), and at 4 weeks (17.5% ± 2.6% vs. 31.1% ± 4.7% in control group, P < 0.001), indicating a lower level of cellular proliferation.Naringenin reduces NIH following arterial reconstruction. This may be mediated by a decrease in PDGF-BB and TNFα levels and the resulting down-regulation of smooth muscle cells' migration and proliferation.
Open Access
Novel monoclonal antibodies detect Smad-interacting protein 1 (SIP1) in the cytoplasm of human cells from multiple tumor tissue arrays
(Elsevier, 2010) Oztas, E.; Avci, M. E.; Ozcan, A.; Sayan, A. E.; Tulchinsky, E.; Yagci, T.
Smad-interacting protein 1 (SIP1, also known as ZEB2) represses the transcription of E-cadherin and mediates epithelial-mesenchymal transition in development and tumor metastasis. Due to the lack of human SIP1-specific antibodies, its expression in human tumor tissues has not been studied in detail by immunohistochemistry. Hence, we generated two anti-SIP1 monoclonal antibodies, clones 1C6 and 6E5, with IgG1 and IgG2a isotypes, respectively. The specificity of these antibodies was shown by Western blotting studies using siRNA mediated downregulation of SIP1 and ZEB1 in a human osteosarcoma cell line. In the same context, we also compared them with 5 commercially available SIP1 antibodies. Antibody specificity was further verified in an inducible cell line system by immunofluorescence. By using both antibodies, we evaluated the tissue expression of SIP1 in paraffin-embedded tissue microarrays consisting of 22 normal and 101 tumoral tissues of kidney, colon, stomach, lung, esophagus, uterus, rectum, breast and liver. Interestingly, SIP1 predominantly displayed a cytoplasmic expression, while the nuclear localization of SIP1 was observed in only 6 cases. Strong expression of SIP1 was found in distal tubules of kidney, glandular epithelial cells of stomach and hepatocytes, implicating a co-expression of SIP1 and E-cadherin. Squamous epithelium of the esophagus and surface epithelium of colon and rectum were stained with moderate to weak intensity. Normal uterus, breast and lung tissues remained completely negative. By comparison with their normal tissues, we observed SIP1 overexpression in cancers of the kidney, breast, lung and uterus. However, SIP1 expression was found to be downregulated in tumors from colon, rectum, esophagus, liver and stomach tissues. Finally we did nuclear/cytoplasmic fractionation in 3 carcinoma cell lines and detected SIP1 in both fractions, nucleus being the dominant one. To our best knowledge, this is the first comprehensive immunohistochemical study of the expression of SIP1 in a series of human cancers. Our finding that SIP1 is not exclusively localized to nucleus suggests that the subcellular localization of SIP1 is regulated in normal and tumor tissues. These novel monoclonal antibodies may help elucidate the role of SIP1 in tumor development. © 2010 Elsevier Inc.
Open Access
A web tool to explore, annotate and classify the Acıbadem Breast Cancer Cohort RNA-seq data with gene signatures and clinical/mutation data, according to molecular subtypes
(2022-06) Çalışır, Kübra
Transcriptomics-based approaches have revealed the molecular heterogeneity and distinct gene expression patterns across breast cancer subtypes since the early 2000s. This led to the usage of molecular subtypes in clinics and translational research in prognostic assessment, therapeutic efficacy prediction, and retrospec- tive analysis of cohort studies. In this thesis, breast cancer subtypes of Acıbadem Breast Cancer Cohort (ABCC) RNA-seq data were classified with immunohisto- chemistry (IHC), PAM50, and SCMOD1 as molecular subtype predictors. The results revealed the moderate concordance of the methods across ABCC and se- lected five other public datasets. In addition, it was shown that the classification of ABCC and TCGA-BRCA RNAseq data strongly depends on the gene sig- nature selection. Further, a machine learning model trained with TCGA-BRCA RNA-seq data and PAM50 genes as predictors showed moderate results for ABCC and MATADOR due to the imbalanced nature of datasets where feature impor- tance revealed a subset of PAM50 genes as predictors. Additionally, the R-Shiny- based classABCC app was developed to facilitate clustering of ABCC with six gene signatures, molecular subtyping of ABCC, and prediction of subtypes with TCGA-BRCA RNA-seq trained machine learning model.