Ayten Kandilci

Overexpression of N-Myc Rapidly Causes Acute Myeloid Leukemia in Mice

N-MYC encodes a basic helix-loop-helix/leucine zipper (bHLH/LZ) transcription factor that is frequently overexpressed in human neuroblastoma. N-MYC overexpression has also been reported in human acute myeloid leukemias (AML), which we show here is a frequent event. Myeloid cells in N-Myc-overexpressing mouse bone marrow hyperproliferate but those in c-MYC-overexpressing bone marrow do not. The NH(2)-terminal transactivation domain, nuclear localization signal, and bHLH/LZ domain of N-Myc are essential for this effect. Microarray analysis revealed 969 differentially expressed genes between N-Myc- and c-MYC-overexpressing myeloid cells. N-Myc-overexpressing cells showed decreased transforming growth factor beta signaling and increased c-Jun-NH(2)-kinase signaling, both of which are associated with proliferation and leukemic transformation of myeloid cells. Mice transplanted with bone marrow expressing wild-type N-Myc developed clonal and transplantable AML after approximately 1 month; those transplanted with bone marrow expressing mutant N-Myc did not. Twist, a known suppressor of the p19Arf/p53 pathway, was up-regulated in all tumors. These results show that N-Myc overexpression is highly oncogenic in mouse myeloid cells and suggest that N-MYC up-regulation contributes to human myeloid leukemogenesis.

Differing DNA methylation patterns and gene mutation frequencies in colorectal carcinomas from Middle Eastern countries

Purpose: The epidemiology of colorectal carcinoma is well known to differ among countries but the molecular characteristics are usually assumed to be similar. International differences in molecular pathology have not been studied extensively but have implications for the management of patients in different countries and of immigrant patients. Experimental Design: We evaluated the CpG island methylator phenotype pathway characterized by concordant methylation of gene promoters that often silences transcription of the genes, the microsatellite instability pathway, and K-ras and p53 gene status in 247 colorectal carcinomas from the three selected Middle Eastern countries of Egypt, Jordan, and Turkey. Results: Colorectal carcinoma from Egypt had the lowest frequencies of methylation. In multinomial logistic regression analysis, Jordanian colorectal carcinoma more frequently had methylation involving the p16 tumor suppressor gene (odds ratio, 3.5; 95% confidence interval, 1.2-10.6; P = 0.023) and MINT31 locus (odds ratio, 2.3; 95% confidence interval, 1.0-5.1; P = 0.041). The K-ras proto-oncogene was more frequently mutated in colorectal carcinoma from Turkey (odds ratio, 2.9; 95% confidence interval, 1.2-6.7; P = 0.016), but p53 overexpression was more common in both Jordanian and Turkish colorectal carcinoma than in Egyptian cases (odds ratio, 2.5; 95% confidence interval, 1.2-5.5; P = 0.019; and odds ratio, 3.6; 95% confidence interval, 1.8-7.1; P = 0.0003, respectively). The findings in Turkish colorectal carcinoma were most similar to those reported for Western cases. Conclusions: Colorectal carcinoma from Middle Eastern countries have differing gene methylation patterns and mutation frequencies that indicate dissimilar molecular pathogenesis, probably reflecting different environmental exposures. These molecular differences could affect prevention strategies, therapeutic efficacy, and transferability of clinical trial results.

The Novel ETS Factor TEL2 Cooperates with Myc in B Lymphomagenesis

ABSTRACT The human ETS family gene TEL2/ETV7 is highly homologous to TEL1/ETV6, a frequent target of chromosome translocations in human leukemia and specific solid tumors. Here we report that TEL2 augments the proliferation and survival of normal mouse B cells and dramatically accelerates lymphoma development in Eμ-Myc transgenic mice. Nonetheless, inactivation of the p53 pathway was a hallmark of all TEL2/Eμ-Myc lymphomas, indicating that TEL2 expression alone is insufficient to bypass this apoptotic checkpoint. Although TEL2 is infrequently up-regulated in human sporadic Burkitts lymphoma, analysis of pediatric B-cell acute lymphocytic leukemia (B-ALL) samples showed increased coexpression of TEL2 and MYC and/or MYCN in over one-third of B-ALL patients. Therefore, TEL2 and MYC also appear to cooperate in provoking a cadre of human B-cell malignancies.

SET-induced calcium signaling and MAPK/ERK pathway activation mediate dendritic cell-like differentiation of U937 cells

Human SET, a target of chromosomal translocation in human leukemia encodes a highly conserved, ubiquitously expressed, nuclear phosphoprotein. SET mediates many functions including chromatin remodeling, transcription, apoptosis and cell cycle control. We report that overexpression of SET directs differentiation of the human promonocytic cell line U937 along the dendritic cell (DC) pathway, as cells display typical morphologic changes associated with DC fate and express the DC surface markers CD11b and CD86. Differentiation occurs via a calcium-dependent mechanism involving the CaMKII and MAPK/ERK pathways. Similar responses are elicited by interferon-γ (IFN-γ) treatment with the distinction that IFN-γ signaling activates the DNA-binding activity of STAT1 whereas SET overexpression does not. In addition, unlike IFN-γ signaling, SET generated stress-induced p38/MAPK activity. Interestingly, IFN-γ treatment transiently upregulated endogenous SET in a dose-dependent manner. These results suggest that SET is part of both IFN-γ-mediated and stress-mediated cellular responses and that SET induces cell differentiation via calcium and MAPK/ERK pathways.

Effects of SET and SET-CAN on the differentiation of the human promonocytic cell line U937

Human SET encodes a nuclear phosphoprotein with a highly acidic carboxyl-terminus, forming a SET-CAN fusion gene in a patient with acute undifferentiated leukemia. SET is highly conserved between species and is ubiquitously expressed, suggesting a widespread biological role. Even though SET is involved in chromatin remodeling and transcriptional activation, its precise role in hematopoietic cells and the contribution of SET-CAN to leukemogenesis remains unknown. We determined the effect of tetracycline-regulatable expression of SET, a deletion mutant of SET, and SET-CAN on the human promonocytic cell line U937T. The expression of SET and SET-CAN inhibited proliferation of these cells. SET accomplishes this through the induction of the differentiation program, an effect that depends on the presence of its acidic domain. SET-CAN most likely inhibits growth by interfering with hCRM1, but it also partially blocks differentiation. Our results are the first demonstration of a potential role of SET in hematopoietic differentiation.

Neutrophil-mediated injury in ischemic skin flaps : Amelioration of ischemic injury by cyclosporine in the rat

The role of neutrophils, their presence, and their degree of infiltration was examined in ischemic skin flaps. In a rat model, caudally based dorsal flaps were studied and neutrophils were manipulated by giving cyclosporine at two different doses (15 and 30 mg per kilogram), administrated either for 5 days as a pretreatment or 15 minutes before flap elevation. The presence of neutrophils and lymphocytes in both intravascular and extravascular space was assessed at 15, 30, and 60 minutes by skin biopsies, taken after elevation of the flap, by direct quantitative counting under the light microscope. The correlation between the counts and localization of the neutrophils, but not the lymphocytes, and the percentage of necrosis showed an early and definite role of neutrophils on skin flap survival during ischemic insult. Cyclosporine-treated flaps showed a 24% to 37% increase in viability when compared to control flaps. These data suggest that neutrophils, probably their interactions and/or products, play an important role in ischemic flap survival, and cyclosporine A is able to inhibit neutrophil accumulation and sequestration.Cetinkale O, Bilgic L, Ayan F, Kandilci A. Neutrophil-mediated injury in ischemic skin flaps: amelioration of ischemic injury by cyclosporine in the rat.

Reintroduction of CEBPA in MN1-overexpressing hematopoietic cells prevents their hyperproliferation and restores myeloid differentiation

Forced expression of MN1 in primitive mouse hematopoietic cells causes acute myeloid leukemia and impairs all-trans retinoic acid-induced granulocytic differentiation. Here, we studied the effects of MN1 on myeloid differentiation and proliferation using primary human CD34(+) hematopoietic cells, lineage-depleted mouse bone marrow cells, and bipotential (granulocytic/monocytic) human acute myeloid leukemia cell lines. We show that exogenous MN1 stimulated the growth of CD34(+) cells, which was accompanied by enhanced survival and increased cell cycle traverse in cultures supporting progenitor cell growth. Forced MN1 expression impaired both granulocytic and monocytic differentiation in vitro in primary hematopoietic cells and acute myeloid leukemia cell lines. Endogenous MN1 expression was higher in human CD34(+) cells compared with both primary and in vitro-differentiated monocytes and granulocytes. Microarray and real-time reverse-transcribed polymerase chain reaction analysis of MN1-overexpressing CD34(+) cells showed down-regulation of CEBPA and its downstream target genes. Reintroduction of conditional and constitutive CEBPA overcame the effects of MN1 on myeloid differentiation and inhibited MN1-induced proliferation in vitro. These results indicate that down-regulation of CEBPA activity contributes to MN1-modulated proliferation and impaired myeloid differentiation of hematopoietic cells.

Mapping of MN1 sequences necessary for myeloid transformation.

The MN1 oncogene is deregulated in human acute myeloid leukemia and its overexpression induces proliferation and represses myeloid differentiation of primitive human and mouse hematopoietic cells, leading to myeloid leukemia in mouse models. To delineate the sequences within MN1 necessary for MN1-induced leukemia, we tested the transforming capacity of in-frame deletion mutants, using retroviral transduction of mouse bone marrow. We found that integrity of the regions between amino acids 12 to 458 and 1119 to 1273 are required for MN1’s in vivo transforming activity, generating myeloid leukemia with some mutants also producing T-cell lympho-leukemia and megakaryocytic leukemia. Although both full length MN1 and a mutant that lacks the residues between 12–228 (Δ12–228 mutant) repressed myeloid differentiation and increased myeloproliferative activity in vitro, the mutant lost its transforming activity in vivo. Both MN1 and Δ12–228 increased the frequency of common myeloid progentiors (CMP) in vitro and microarray comparisons of purified MN1-CMP and Δ12–228-CMP cells showed many differentially expressed genes including Hoxa9, Meis1, Myb, Runx2, Cebpa, Cebpb and Cebpd. This collection of immediate MN1-responsive candidate genes distinguishes the leukemic activity from the in vitro myeloproliferative capacity of this oncoprotein.

SET oncogene is upregulated in pediatric acute lymphoblastic leukemia

AIMS AND BACKGROUND The SET gene is a target of chromosomal translocations in acute leukemia and encodes a widely expressed multifunctional phosphoprotein. It has been shown that SET is upregulated in BCR-ABL1-positive cell lines, patient-derived chronic myeloid leukemia CD34-positive cells, and some solid tumors. METHODS AND STUDY DESIGN We determined the expression level of SET in 59 pediatric acute lymphoblastic leukemia patients who were BCR-ABL-negative using quantitative real-time reverse-transcriptase-polymerase chain reaction. Results. We showed that SET expression was significantly upregulated in 96.5% of B-acute lymphoblastic leukemia (28 of 29; 16.6 fold) and 93% of T-acute lymphoblastic leukemia (28 of 30; 47.6 fold) patients. This upregulation was not associated with any clinical features or overall and relapse-free survival. CONCLUSIONS Our results showed that SET is significantly overexpressed in pediatric acute lymphoblastic leukemia samples, and an increased level of SET might contribute to leukemic process.

Upregulated heme biosynthesis, an exploitable vulnerability in MYCN-driven leukemogenesis

The increased heme biosynthesis long observed in leukemia was previously of unknown significance. Heme, synthesized from porphyrin precursors, plays a central role in oxygen metabolism and mitochondrial function, yet little is known about its role in leukemogenesis. Here, we show increased expression of heme biosynthetic genes, including UROD, only in pediatric AML samples that have high MYCN expression. High expression of both UROD and MYCN predicts poor overall survival and unfavorable outcomes in adult AML. Murine leukemic progenitors derived from hematopoietic progenitor cells (HPCs) overexpressing a MYCN cDNA (MYCN-HPCs) require heme/porphyrin biosynthesis, accompanied by increased oxygen consumption, to fully engage in self-renewal and oncogenic transformation. Blocking heme biosynthesis reduced mitochondrial oxygen consumption and markedly suppressed self-renewal. Leukemic progenitors rely on balanced production of heme and heme intermediates, the porphyrins. Porphyrin homeostasis is required because absence of the porphyrin exporter, ABCG2, increased death of leukemic progenitors in vitro and prolonged the survival of mice transplanted with Abcg2-KO MYCN-HPCs. Pediatric AML patients with elevated MYCN mRNA display strong activation of TP53 target genes. Abcg2-KO MYCN-HPCs were rescued from porphyrin toxicity by p53 loss. This vulnerability was exploited to show that treatment with a porphyrin precursor, coupled with the absence of ABCG2, blocked MYCN-driven leukemogenesis in vivo, thereby demonstrating that porphyrin homeostasis is a pathway crucial to MYCN leukemogenesis.