Screening of polymorphisms in the folate pathway in Turkish pediatric Acute Lymphoblastic Leukemia patients

Background and aim: Folate metabolic pathway plays a significant role in leukemogenesis because of its necessity for nucleotide synthesis and DNA methylation. Folate deficiency causes DNA damage. Thus polymorphisms of folate-related genes may affect the susceptibility to childhood Acute Lymphoblastic Leukemia (ALL). MTHFR (Methylenetetrahydrofolate Reductase), DHFR (Dihydrofolate reductase), CBS (Cystathionine b-synthase) and TYMS (Thymidylate Synthase) have an important role in folate pathway because their activated variants modulate synthesis of DNA and levels of folate. In this study, we aimed to investigate whether polymorphisms in genes related to folate metabolic pathway influence the risk to childhood ALL.Subject and methods: The patient groups who were diagnosed with childhood ALL at Losante Pediatric Hematology-Oncology Hospital and healthy control groups were included in the study. MTHFR 677 CT, MTHFR 1298 A-C, CBS 844ins68, DHFR 19-bp and TYMS 1494del6 polymorphisms were screened. Genotyping of these polymorphisms was performed by Restriction Fragment Length Polymorphism (RFLP) analysis and Real Time Polymerase chain Reaction (Real Time-PCR).Results: In total, we have screened 5 polymorphisms in the studied genes. The results were compared between childhood ALL patients and healthy groups. Genotype frequencies of MTHFR 677 C-T, MTHFR 1298 A-C, CBS 844ins68 and DHFR 19-bp del were similar for childhood ALL patients and healthy groups. However, statistical results showed that TYMS 1494del6 may be associated with ALL pathogenesis (p < 0.001).Conclusion: We showed that TYMS polymorphism (rs2853542) may be associated with ALL pathogenesis. In addition, our results demonstrated that MTHFR, DHFR and CBS do not affect development of leukemia. Our study displays also importance as it is the first screening results to identify association with the studied polymorphisms in Turkish patients with childhood ALL and determination of the frequency in Turkish population

The Ability to Generate Senescent Progeny as a Mechanism Underlying Breast Cancer Cell Heterogeneity

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. © 2010 Mumcuoglu et al

Ankaferd Blood Stopper induces apoptosis and regulates PAR1 and EPCR expression in human leukemia cells

Background: Ankaferd Blood Stopper (ABS) is a preparation of plant extracts originally used as a hemostatic agent. It has pleiotropic effects in many cellular processes such as cell cycle regulation, apoptosis, angiogenesis, signal transduction, inflammation, immunologic processes and metabolic pathways as well as hemostatic activity. This unique preparation has been widely investigated for its properties. However there are no studies investigating its action on leukemic cells. Aim: Aim of the study was to examine the ABS action on PAR1 and EPCR in leukemia cells. However, during the experiments, we observed the apoptotic effect of ABS on leukemic cells, particularly Jurkat cells. As a result the mechanism of apoptosis induced by ABS treatment was also explored in the study. Material and method: Two leukemia cell lines, K-562 and Jurkat, were utilized for the study. Expression analyses of PAR1, EPCR and p21 upon ABS treatment were performed by quantitative real time PCR. Annexin V method was used for apoptosis detection. Results: Our results demonstrated that ABS alters PAR1 and EPCR expression in K-562 and Jurkat cells in a time and dose dependent manner. Additionally it was found that ABS treatment induces apoptosis in leukemia cells. Possible involvement of PAR1 and p21 in this apoptotic process was observed in Jurkat cells. Conclusion: This study concludes that depending on the concentration and duration of the application, ABS causes apoptosis by regulating PAR1 and p53-independent p21 involvement in apoptosis stimulation in leukemia cells. The composition of ABS plant extracts might be responsible from the apoptotic effect that was observed. We think that our results could contribute to the development of new treatment for leukemia therapy

Retinoid N-(1H-benzo[d]imidazol-2-yl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-carboxamide induces p21-dependent senescence in breast cancer cells

Retinoids have been implicated as pharmacological agents for the prevention and treatment of various types of cancers, including breast cancers. We analyzed 27 newly synthesized retinoids for their bioactivity on breast, liver, and colon cancer cells. Majority of the retinoids demonstrated selective bioactivity on breast cancer cells. Retinoid 17 had a significant inhibitory activity (IC50 3.5 mu M) only on breast cancer cells while no growth inhibition observed with liver and colon cancer cells. The breast cancer selective growth inhibitory action by retinoid 17 was defined as p21-dependent cell death, reminiscent of senescence, which is an indicator of targeted receptor mediated bioactivity. A comparative analysis of retinoid receptor gene expression levels in different breast cancer cells and IC50 values of 17 indicated the involvement of Retinoid X receptors in the cytotoxic bioactivity of retinoid 17 in the senescence associated cell death. Furthermore, siRNA knockdown studies with RXR gamma induced decrease in cell proliferation. Therefore, we suggest that retinoid derivatives that target RXR gamma, can be considered for breast cancer therapies

Detection of TET2, KRAS and CBL variants by Next Generation Sequencing and analysis of their correlation with JAK2 and FLT3 in childhood AML

Background: Acute myeloid leukemia (AML) is a heterogeneous clonal disorder in terms of cytogenetic and molecular aberrations. Ten-Eleven-Translocation 2 (TET2), Kirsten rat sarcoma viral oncogene homolog (KRAS), and Casitas B-cell lymphoma (CBL) have an important role pathogenesis of acute myeloid leukemia (AML) and their activated mutations confer proliferative and survival signals. Aim: In this study, we aimed to find possible genetic markers for molecular analysis in childhood AML by screening hot-spot exons of TET2, KRAS, and CBL using Next Generation Sequencing (NGS) analysis. In addition, association between found variants and mutations of Januse Kinase-2 (JAK2) and Fms-Related Tyrosine Kinase (FLT3) were analyzed which are important prognostic risk factors for AML. Methods: Eight patients who were diagnosed with pediatric AML at Losante Pediatric Hematology–Oncology Hospital were included to the study. Hot-spot exons of TET2, KRAS and CBL genes were screened using the NGS method. Furthermore, FLT3-Internal Tandem Duplicate (FLT3-ITD) and JAK2-V617F were analyzed by Real Time Polymerase chain Reaction (Real Time-PCR). Results: In total, we identified 20 variants in studied genes by NGS. In our patient group, 16 variants in the TET2 (seven novel, seven missense and two silent), two variants in the KRAS (one missense and one intronic) and two variants in the CBL (two novel) were found. All of AML patients were found negative for JAK V617 F. Three of the eight patients (37.5%) showed mutations of both FLT3-ITD and TET2, KRAS, CBL. Conclusion: We found novel mutations for TET2, KRAS, and CBL. The detected variants in this article seem to be the first screening results of genes studied by NGS in childhood AML patients. Our results also showed some degree of association between FLT3-ITD and TET2, KRAS, CBL mutations

Retinoid N-(1H-benzo[d]imidazol-2-yl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-carboxamide induces p21-dependent senescence in breast cancer cells

Retinoids have been implicated as pharmacological agents for the prevention and treatment of various types of cancers, including breast cancers. We analyzed 27 newly synthesized retinoids for their bioactivity on breast, liver, and colon cancer cells. Majority of the retinoids demonstrated selective bioactivity on breast cancer cells. Retinoid 17 had a significant inhibitory activity (IC50 3.5 mu M) only on breast cancer cells while no growth inhibition observed with liver and colon cancer cells. The breast cancer selective growth inhibitory action by retinoid 17 was defined as p21-dependent cell death, reminiscent of senescence, which is an indicator of targeted receptor mediated bioactivity. A comparative analysis of retinoid receptor gene expression levels in different breast cancer cells and IC50 values of 17 indicated the involvement of Retinoid X receptors in the cytotoxic bioactivity of retinoid 17 in the senescence associated cell death. Furthermore, siRNA knockdown studies with RXR gamma induced decrease in cell proliferation. Therefore, we suggest that retinoid derivatives that target RXR gamma, can be considered for breast cancer therapies

Reprogramming of replicative senescence in hepatocellular carcinoma-derived cells

Tumor cells have the capacity to proliferate indefinitely that is qualified as replicative immortality. This ability contrasts with the intrinsic control of the number of cell divisions in human somatic tissues by a mechanism called replicative senescence. Replicative immortality is acquired by inactivation of p53 and p16(INK4a) genes and reactivation of hTERT gene expression. It is unknown whether the cancer cell replicative immortality is reversible. Here, we show the spontaneous induction of replicative senescence in p53-and p16(INK4a)-deficient hepatocellular carcinoma cells. This phenomenon is characterized with hTERT repression, telomere shortening, senescence arrest, and tumor suppression. SIP1 gene (ZFHX1B) is partly responsible for replicative senescence, because short hairpin RNA-mediated SIP1 inactivation released hTERT repression and rescued clonal hepatocellular carcinoma cells from senescence arrest