Moleküler Biyoloji ve Genetik Bölümü Koleksiyonu
https://hdl.handle.net/20.500.12573/209
2024-03-28T16:48:17ZMatching variants for functional characterization of genetic variants
https://hdl.handle.net/20.500.12573/2041
Matching variants for functional characterization of genetic variants
Cevik-Kaplan, Sebiha; Zhao, Pei; Zorluer, Atiyye; Pir, Mustafa Samet; Bian, Wenyin
Rapid and low-cost sequencing, as well as computer analysis, have facilitated the diagnosis of many genetic diseases, resulting in a substantial rise in the number of disease-associated genes. However, genetic diagnosis of many disorders remains problematic due to the
lack of interpretation for many genetic variants, especially missenses, the infeasibility of high-throughput experiments on mammals, and
the shortcomings of computational prediction technologies. Additionally, the available mutant databases are not well-utilized. Toward
this end, we used Caenorhabditis elegans mutant resources to delineate the functions of eight missense variants (V444I, V517D, E610K,
L732F, E817K, H873P, R1105K, and G1205E) and two stop codons (W937stop and Q1434stop), including several matching variants
(MatchVar) with human in ciliopathy associated IFT-140 (also called CHE-11)//IFT140 (intraflagellar transport protein 140). Moreover,
MatchVars carrying C. elegans mutants, including IFT-140(G680S) and IFT-140(P702A) for the human (G704S) (dbSNP: rs150745099)
and P726A (dbSNP: rs1057518064 and a conflicting variation) were created using CRISPR/Cas9. IFT140 is a key component of IFT complex A (IFT-A), which is involved in the retrograde transport of IFT along cilia and the entrance of G protein-coupled receptors into cilia.
Functional analysis of all 10 variants revealed that P702A and W937stop, but not others phenocopied the ciliary phenotypes (short cilia,
IFT accumulations, mislocalization of membrane proteins, and cilia entry of nonciliary proteins) of the IFT-140 null mutant, indicating that
both P702A and W937stop are phenotypic in C. elegans. Our functional data offered experimental support for interpreting human variants, by using ready-to-use mutants carrying MatchVars and generating MatchVars with CRISPR/Cas9.
2023-01-01T00:00:00ZResveratrolün Ph+ Akut Lenfoblastik Lösemide Terapötik Potansiyeli ve Resveratrol Tarafından Tetiklenen Apoptozda Seramid Metabolizmasının Rolü
https://hdl.handle.net/20.500.12573/2016
Resveratrolün Ph+ Akut Lenfoblastik Lösemide Terapötik Potansiyeli ve Resveratrol Tarafından Tetiklenen Apoptozda Seramid Metabolizmasının Rolü
Adan, Aysun; Baran, Yusuf
Proje ile resveratrolün, Ph+ ALL hücreleri üzerindeki büyümeyi inhibe edici etkisinin
arkasında yatan mekanizmalar, seramid metabolizmasının hedeflenmesi ve BCR-ABL
ifadesindeki değişimler ile ilişkilendirilerek araştırılmıştır. Resveratrol, SK inhibitörü (SKI II),
GSS inhibitörü (PDMP), SPT inhibitörü (Miriosin, Myriocin) ve resveratrol: inhibitör
kombinasyonlarının in vitro olarak Ph+ ALL SD1 ve SUP-B15 hücreleri üzerindeki büyümeyi
durdurucu ve apoptotik etkileri MTT hücre çoğalması testi, Aneksin-V/PI boyaması, kaspaz3, PARP ifadelerinin ve sitokrom c salınımının belirlenmesi (western blot) ile, sitostatik etki
(hücre döngüsü üzerindeki) ise akım sitometresi (PI boyaması) ile araştırılmıştır. Resveratrol
ve sfingolipid metabolizması enzimlerini hedefleyen inhibitör kombinasyonlarının BCR-ABL
protein ifadesi üzerine etkisi western blot ile belirlenmiştir. Ayrıca, resveratrolün SPT, SK-1/2,
GSS protein ifadeleri üzerindeki etkisi western blot ile belirlenmiştir. Her iki hücre hattında
resveratrol ve SKI II ve PDMP ile kombinasyonları hücre büyümesini baskılamış, apoptozu
tetiklemiş ve hücre döngüsünü S fazında tutmuştur. Resveratrol:Miriosin kombinasyonu ise
hücre büyümesi ve hücre döngüsü üzerinde hücreye özgü etkiler gösterirken apoptozu her iki
hücrede tetiklemiştir. Her iki hücre tipinde resveratol ve kombinasyonları sitokrom-c
salınımını, kaspaz-3 kesimini ve PARP kesimini genel olarak arttırmakla beraber hücreye
özgü değişimler de saptanmıştır. Resveratrol her iki hücrede SK-1/SK2 ve GSS ifadesini
azaltırken SPT ifadesini arttırmıştır. Resveratrol, SKI II ve PDMP BCR-ABL ifadesini
azaltırken Miriosin arttırmıştır. Resveratrol: SKI II ve PDMP kombinasyonları BCR-ABL
üzerinde artışlara neden olurken Miriosin ile kombinasyon BCR-ABL ifadesini azaltmıştır.
Sonuç olarak, resveratrol seramid metabolizmasını ve BCR-ABL ifadesini düzenleyerek Ph+
ALL üzerinde hücre büyümesini baskılamış ve apoptozu tetiklemiştir.; In this project, it is aimed to identifiy potential mechanisms behind resveratol-mediated
growth inhibitory effects in association with targeting of sphingolipid metabolism and
regulation of BCR-ABL on Ph+ ALL cells. To investigate the effect of treatment
with resveratrol combined with the inhibitors of sphingolipid metabolizing enzymes, SK (SKI
II), GCS (PDMP) and SPT (Myriocin), on Ph+ ALL SD1 and SUP-B15 cell growth inhibition
and apoptosis in vitro, the cells were incubated with different concentrations
of resveratrol and SK, GCS, SPT inhibitors alone and with the combinations of resveratrol
and the inhibitors. The cell growth inhibition was evaluated using the MTT assay. Apoptosis
was determined using Annexin V/propidium iodide double staining and measuring protein
levels of caspase-3, PARP and cytochrome c release by western blot. Cytostatic effects
(effects on cell cycle) of each agent alone and combinations were evaluated by PI staining.
The effects of resveratrol on BCR-ABL, SPT, SK-1/2 and GCS protein expression, and its
combinations on BCR-ABL protein expression were checked by western blot. In both cell
lines, resveratrol and its combinations with SKI II and PDMP supressed cell growth, induced
apoptosis and caused cell cycle arrest at the S phase. However, resveratrol: Myriocin
combination showed cell type specific effects on the cell growth and cell cycle progression
while induced apoptosis in both cell lines. In general, resveratrol and its combinations
triggered cytochrome-c release, caspase-3 and PARP clevage, however, for some cases,
there were cell specific responses. Resveratrol supressed SK-1/2 and GCS protein
expression while increased SPT expression. BCR-ABL expression decreased after
resveratrol, SKI II and PDMP treatment, however, Myriocin caused increases. Resveratrol:
SKI II and PDMP combinations increased BCR-ABL expression while resveratrol: Myriocin
combination increased its expression level. In conclusion, resveratrol supressed cell
proliferation and induced apoptosis through targeting sfingolipid metabolism and modulating
BCR-ABL expression.
2019-01-01T00:00:00ZAkut Myeloid Lösemi Tedavisi için Hedgehog Ve Otofaji Yolaklarının Düzenlenmesi
https://hdl.handle.net/20.500.12573/2015
Akut Myeloid Lösemi Tedavisi için Hedgehog Ve Otofaji Yolaklarının Düzenlenmesi
EL KHATIB, Mona
Akut myeloid lösemi (AML) çeşitli moleküler aberasyonlar ve sinyal yolaklarındaki bozuklukları
içeren klonal hastalıklar ile karakterize edilen bir grup heterojen malignanttır. Hedgehog (HH)
sinyal yolağı birçok kanserde deregüle edilen evrimsel olarak korunan bir sinyal yolağıdır. HH
sinyal yolağı lizozomal degradasyon prosesi otofajinin temel regülatörü olan PI3K/AKT/mTOR
aksesini de içeren diğer sinyal yolakları ile karşılıklı iletişim halindedir. Bu sinyal yolakları
AML’de deregüle edilmiştir. Birçok çalışmada otofajinin AML için bir kaçış mekanizması
olabileceği ortaya konulmuştur. Bizim çalışmamızda, HH ve otofaji yolaklarının farklı AML
türleri üzerine etkileri incelenmiştir. Çalışmamızda KML hücresi olan K562 ve CMK, MV4-11,
MOLM-13 ve NB4 AML hücreleri GLI1 inhibitörü GANT61 ve farklı otofaji modülatörleri ile
muamele edilmiştir.MTT sonuçları NB4, MOLM-13 ve MV4-11hücre proliferasyonun GLI
inhibisyonu sonrasında düştüğünü ancak CMK’nin diğer AML hücre hatlarına kıyasla GLI
inhibisyonuna daha az sensitif olduğunu ortaya koymuştur. Daha sonra, otofaji
modülasyonunun farklı AML hücre hatlarının proliferasyonu üzerine etkileri incelenmiştir.
Otofajinin gerek otofagozom-lizozom füzyonu aşamasında gerekse otofagolizozomal
degradasyon aşamasında inhibisyonunun ilaç konsantrasyonu ve muamele süresine bağlı
olarak AML sağkalımını azalttığı gözlemlenmiştir. Otofaji modülatörleri ve GANT61’in
kombinasyonunun MOLM-13 hücre hattı üzerinde sinerjistik bir etkisinin olduğu fakat CMK
hücre hattı üzerinde sinerjistik etkisinin olmadığı gözlemlenmiştir. GANT61 muamelesinin AML
hücre hatlarında otofajiyi artırdığı LC3II ekspresyonu ile western blot yöntemi ile ortaya
konulmuştur. Buna ek olarak, kombinasyonun MOLM-13 hücresinde LC3II’yi artırdığı
gözlenirken, bu oran CMK hücre hattında daha düşüktür. AKT proteinin ekspresyonu ilaca ve
hücre hattına gore farklılık göstermektedir. Sonuç olarak, HH ve otofaji sinyal yolaklarının
hedeflenmesi MOLM-13 hücre hattı için umut vaatedici bir terapi iken, CMK hücre hattında
benzer sonuçlara ulaşılamamıştır.; Acute myeloid leukemia (AML) is a heterogeneous group of malignancies characterized by
clonal disorders with diverse molecular aberrations and dysregulation in signaling pathways.
Hedgehog (HH) signaling pathway is an evolutionary conserved signaling pathway that is
deregulated in many cancers. HH pathway crosstalks with other pathways among which is the
PI3K/AKT/mTOR axes, a main regulator of autophagy, a lysosomal degradation process.
These pathways are deregulated in AML. Several studies suggested that autophagy
modulation could be an escape mechanism in AML. In this study, we investigated the effect of
Hedgehog pathway and autophagy on different subsets of AML cell lines. We have treated
CMK, MV4-11, MOLM-13 and NB4 AML cell lines, in addition to K562, CML cells, with GANT
61, GLI1 inhibitor, and different autophagy modulators. MTT assay have showed that GANT61
resulted in a significant decrease in the proliferation of NB4, MOLM-13, and MV4-11, however,
CMK showed less sensitivity to GLI inhibition compared to other AML cell lines. Then, we
proceeded with checking the effect of autophagy modulation on the survival of the different
AML cell lines. Inhibiting autophagy whether at the autophagosome-lysosome fusion stage or
autophagolysosomal degradation stage led to a decrease in the survival of AML to a certain
degree based on the drug concentration and timepoint of treatment. Combination treatment of
autophagy modulators and GANT61 had a synergistic effect in MOLM-13 but not in CMK.
GANT61 treatment increased autophagy in AML cell lines that was correlated with an increase
in the expression of LC3II detected by western blotting. Also, combination treatment elevated
that increase in LC3II in MOLM-13 cell lines but less in CMK cell lines. AKT protein expression
changed depending on the type of treatment and cell lines. In conclusion, targeting of HH and
autophagy is a promising therapy against MOLM-13 cell line but not against CMK.
2019-01-01T00:00:00ZInhibition of PI3K-AKT-mTOR pathway and modulation of histone deacetylase enzymes reduce the growth of acute myeloid leukemia cells
https://hdl.handle.net/20.500.12573/1941
Inhibition of PI3K-AKT-mTOR pathway and modulation of histone deacetylase enzymes reduce the growth of acute myeloid leukemia cells
Şansaçar, Merve; Sağır, Helin; Gencer Akçok, Emel Başak
One of the most widespread forms of blood cancer is known as acute myeloid leukemia (AML) which has an incidence of 80% with poor prognosis. Although there are different treatment methods for AML in clinic, the heterogeneity and complexity of the disease show that new treatments are needed. The aim of this study is to investigate the anticancer effects of inhibition of PI3K and HDAC enzymes on CMK and MOLM-13 AML cells lines. We demonstrated that the combination of LY294002 with SAHA and Tubastatin A significantly decreased the cell viability of both cell lines. In contrast, the LY294002 and PCI-34051 combination did not show a significant difference compared to the single LY294002 administration. The combination treatment of LY294002 and HDAC inhibitors did not induce apoptosis significantly. However, LY294002 + SAHA and LY294002 + PCI-34051 resulted in G0/G1 and G2/M cell cycle arrest in CMK cells, respectively. On the other hand, compared to control cells, LY294002 + SAHA and LY294002 + PCI-34051 led to G0/G1 phase arrest in MOLM-13. Furthermore, the LY294002 + PCI-34051 combination elevated the expression rate of LC3BII/I, an autophagy marker, in CMK cells by 2.5-fold. Our study revealed that the combinations of PI3K inhibitor and HDAC inhibitors showed a synergistic effect and caused a reduction in cell viability and increased cell cycle arrest on MOLM-13 and CMK cell lines. In addition, the expression of LC3BII was elevated in the CMK cell line. In conclusion, although more mechanistic studies are required, a combinational inhibition of PI3K and HDAC could be a promising approach for AML.
2023-01-01T00:00:00Z