Effect of Notch and PARP Pathways’ Inhibition in Leukemic Cells

Differentiation of blood cells is one of the most complex processes in the body. It is regulated by the action of transcription factors in time and space which creates a specific signaling network. In the hematopoietic signaling system, Notch is one of the main regulators of lymphocyte development. The aim of this study was to get insight into the regulation of Notch signalization and the influence of poly(ADP-ribose)polymerase (PARP) activity on this process in three leukemia cell lines obtained from B and T cells. PARP1 is an enzyme involved in posttranslational protein modification and chromatin structure changes. B and T leukemia cells were treated with Notch and PARP inhibitors, alone or in combination, for a prolonged period. The cells did not show cell proliferation arrest or apoptosis. Analysis of gene and protein expression set involved in Notch and PARP pathways revealed increase in JAGGED1 expression after PARP1 inhibition in B cell lines and changes in Ikaros family members in both B and T cell lines after γ-secretase inhibition. These data indicate that Notch and PARP inhibition, although not inducing differentiation in leukemia cells, induce changes in signaling circuits and chromatin modelling factors

Indukcija urokinaznog tipa plazminogenskog aktivatora i njegova inhibitora etopozidom u glioblastomskoj staničnoj liniji A1235

Urokinase plasminogen activator (uPA) and its inhibitors (PAI) are elements of plasminogen activation system, a proteolytic system involved in many physiological and patological processes. In this paper their induction by etoposide, a topoisomerase II inhibitor, in A1235 glioblastoma cell strain is described. Etoposide induced uPA and PAI production, in a dose- and time-dependent manner. Induction was based on the activation of their promoters and extracellular proteolysis was dependent on their equilibrium. Etoposide caused p53 activation and p21 and gadd45 induction, which could be responsible for the cell growth arrest. Our data indicate that several pathways could be involved in the uPA and PAI induction.Urokinazni tip plazminogenskog aktivatora (uPA) i njegovi inhibitori (PAI) dijelovi su plazminogenskog aktivacijskog sustava, proteolitičkog sustava uključenog u mnoge fiziološke i patološke procese. U ovom je radu opisana njihova indukcija etopozidom, inhibitorom topoizomeraze II, u stanicama glioblastoma A1235. Etopozid je inducirao uPA i PAI ovisno o koncentraciji i vremenu inkubacije. Indukcija je ovisila o aktivaciji njihova promotora, a ekstracelularna proteoliza o njihovoj ravnoteži. Etopozid je uzrokovao aktivaciju p53, kao i indukciju p21 i gadd45, te bi oni mogli biti odgovorni za zaustavljanje staničnog rasta. Rezultati upućuju da bi nekoliko signalnih puteva moglo sudjelovati u indukciji uPA i PAI

Analysis of Ikaros Family Splicing Variants in Human Hematopoietic Lineages

Transcription factors from the Ikaros family are involved in lymphocyte differentiation and have a critical role at specific check points of the haemopoietic pathway. However, how developmentally regulated changes are reflected in gene expression programs of lymphocyte differentiation is not well understood. It has been suggested that disregulation of transcription factors from the Ikaros family is associated with the development of different human leukemias. In this work we analyzed the state of Ikaros family members in different leukemic cells with the aim to explore the transcriptional control of human hematopoietic lineages and shed some new light on our understanding of transcription factor significance in human leukemias. By means of RT-PCR and specific primers we investigated the expression of Ikaros, Aiolos and Helios transcription factors and their splicing variants in seven leukemia cell lines derived from different types of leukemia (ALL, CML, AML) and lymphoma (histiocytic lymphoma, Burkitt lymphoma and anaplastic large cell lymphoma). In all of the cell lines examined Ikaros was present in dominant Ik1 to Ik4 isoforms and small Ik6 isoform was absent. Aiolos was expressed in the majority of the cell lines, of both, B and T origin, in the form of the full length Aio1. Helios was also present only in two long isoforms Hel1 and Hel2, and was absent in one third of the lines. Similar distribution of positive and negative expression of Aiolos and Helios found in various types of leukemias could implicate common pathways of their regulation

MiR-7 in Cancer Development

MicroRNAs (miRNAs) are short non-coding RNA involved in the regulation of specific mRNA translation. They participate in cellular signaling circuits and can act as oncogenes in tumor development, so-called oncomirs, as well as tumor suppressors. miR-7 is an ancient miRNA involved in the fine-tuning of several signaling pathways, acting mainly as tumor suppressor. Through downregulation of PI3K and MAPK pathways, its dominant role is the suppression of proliferation and survival, stimulation of apoptosis and inhibition of migration. Besides these functions, it has numerous additional roles in the differentiation process of different cell types, protection from stress and chromatin remodulation. One of the most investigated tissues is the brain, where its downregulation is linked with glioblastoma cell proliferation. Its deregulation is found also in other tumor types, such as in liver, lung and pancreas. In some types of lung and oral carcinoma, it can act as oncomir. miR-7 roles in cell fate determination and maintenance of cell homeostasis are still to be discovered, as well as the possibilities of its use as a specific biotherapeutic

Humani kromosom 1 u mišjim imortalnim stanicama

Telomeres are specialized structures at the ends of linear chromosomes and are essential for normal cellular function. Telomeres prevent degradation and aberrant recombination of chromosome termini and facilitate appropriate replication of chromosome ends. In this work, the telomere dynamics was followed in the immortal mouse cell strain A9 in comparison with A9+1. The latter is derived from A9 cells by introduction of human chromosome 1. In spite of the telomerase presence, a great decrease in telomere lengths was noticed in A9+1 compared to A9 cells. Behavior of individual human and mouse telomeres was also followed under the conditions of the observed gross telomere shortening. Human chromosome 1 followed the overall telomere length in hybrid cells. It is suggested that telomere lengths are primarily determined by the cell protein background.Telomere su specijalizirane strukture na krajevima linearnih kromosoma i esencijalne su za normalnu staničnu funkciju. One sprečavaju degradaciju i pogrešnu rekombinaciju krajeva kromosoma i olakšavaju replikaciju kromosomskih krajeva. U ovom radu praćena je dinamika telomera u imortalnim mišjim staničnim linijama A9 i A9+1. A9+1 stanična linija dobivena je unošenjem ljudskoga kromosoma 1 u A9 stanice. Usprkos prisustvu telomeraze primijećeno je veliko skraćenje telomera kod A9+1 u usporedbi s A9 stanicama. Praćeno je i ponašanje ljudskoga kromosoma 1 u mišjim stanicama u uvjetima pod kojima je došlo do skraćivanja telomera. Raspon duljina telomera ljudskoga kromosoma 1 odgovarao je rasponu mišjih telomera stanica doma- ćina. Ovi rezultati ukazuju da na raspon duljina telomera najveći utjecaj ima ukupan sastav telomernih proteina stanice

miR-7 AND miR-34a sequence cloning and expression in a1235 glioblastoma cell line

miRNAs are small non-coding RNAs which have an important role in signalling circuits regulating different cell processes. miR-7 and miR-34a are known as tumour suppressors, and both of them can interfere with cell proliferation, differentiation, apoptosis and migration. We constructed plasmids containing pri-miRNA sequences for these two miRNAs and introduced them into the A1235 glioblastoma cell line. Clones containing increased expression of processed miR-7 and miR-34a were obtained. The proliferation and sensitivity to alkylation agent of transfected cells were similar to those of control cells. Our results indicate that an increase in miR-7 and miR34 expression alone in A1235 glioblastoma cells is not sufficient to change their proliferation or sensitivity to the influence of alkylating agents

Notch pathway connections in primary leukaemia samples of limited size

Background: The Notch pathway combined with other signalling molecules acts specifically for the development of each blood cell type and differentiation stage. A causative role of Notch dysfunction in leukaemia development has been found in many studies so, initially only for T- acute lymphoblastic leukaemia (T-ALL) but more recently also for B cell and myeloid leukaemia. The aim of our study is to introduce a method for multiple direct analysis of the Notch pathway partners in a population of only 500 or fewer cells. The notion of this method consists in gaining insight into gene expression at the level of the malignant clone population. A small number of cells is a significant limitation when working on primary cells either when freshly isolated or when analysed after several days in cocultures. Methods: The primers were designed to avoid genomic amplification through the selection of 3′ and 5′ primers that hybridise with different exons. Cell lines and primary cells were collected and multiplex quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) performed on a descending number of cells, ranging from 2, 500 cells up to 50 cells per sample, for the Notch pathway genes and other transcription factors important for cell differentiation. ImageJ program, STATISTICA 13.1 software package and Student’s t-test were used for statistical evaluation. We checked protein expression by western blot. Results: We characterised the gene expression levels of Notch, Ikaros and Parp genes in leukaemia cell lines of B and T origin and in primary leukaemia samples of limited size. We further compared our results to the cDNA analysis obtained by total RNA isolation from a large number of cells as routinely performed in clinical laboratories, and finally tested the method described on primary cells from leukaemia patients. Conclusions: This rapid multiple gene expression analysis of a small population of cells provides efficient cell classification determining malignant changes as an important additional information for clinical leukaemia diagnostics as well as for in vitro studies of primary cells

Urokinase-type Plasminogen Activator and Plasminogen Activator Inhibitor Induction by Etoposide in a Glioblastoma Cell Strain

Urokinase plasminogen activator (uPA) and its inhibitors (PAI) are elements of plasminogen activation system, a proteolytic system involved in many physiological and patological processes. In this paper their induction by etoposide, a topoisomerase II inhibitor, in A1235 glioblastoma cell strain is described. Etoposide induced uPA and PAI production, in a dose- and time-dependent manner. Induction was based on the activation of their promoters and extracellular proteolysis was dependent on their equilibrium. Etoposide caused p53 activation and p21 and gadd45 induction, which could be responsible for the cell growth arrest. Our data indicate that several pathways could be involved in the uPA and PAI induction