Eiman Aleem | London South Bank University (original) (raw)

Papers by Eiman Aleem

Research paper thumbnail of Resistance Associated with a Novel MAP 2 K 1 Mutation in a Patient with Langerhans Cell Histiocytosis

Patients with Langerhans cell histiocytosis (LCH) harbor BRAF V600E and activating mutations of M... more Patients with Langerhans cell histiocytosis (LCH) harbor BRAF V600E and activating mutations of MAP2K1/MEK1 in 50% and 25% of cases, respectively. We evaluated a patient with treatment refractory LCH for mutations in the RAS-RAF-MEK-ERK pathway and identified a novel mutation in the MAP2K1 gene resulting in a p.L98_K104>Q deletion and predicted to be auto-activating. During treatment with the MEK inhibitor trametinib, the patient's disease showed significant progression. In vitro characterization of the MAP2K1 p.L98_K104>Q deletion confirmed its effect on cellular activation of the ERK pathway and drug resistance.

Research paper thumbnail of Abstract 2705: Functional proteomics to reveal distinct signature for acute myeloid leukemia subtypes

Cancer Research, 2018

Patients with relapsed/ refractory AML have an overall survival rate <30%. Genomic approaches ... more Patients with relapsed/ refractory AML have an overall survival rate <30%. Genomic approaches are being used for patients' stratification, but they have not been sufficient at predicting response to therapy. We propose an integrative approach to identify distinctive phosphorylated protein biomarkers in AML, map them to signaling networks, and compare these to pathways identified through analysis of gene expression. This study aims at identifying distinct phosphoproteomic signatures by mass spectrometry (MS) in different AML subtypes. Methods: Four AML cell lines and 40 bone marrow samples from patients with AML representing different AML subtypes are used. Samples were prepared using a MOAC-TiO2 stationary phase for phosphopeptide enrichment, and analysis on a Thermo LTQ Orbitrap Velos MS. Identified phosphorylated STY sites were cross-referenced against iPTMnet. Phosphoproteins were further matched to potential pathways using Ingenuity Pathway Analysis (IPA). Gene-expression...

Research paper thumbnail of Abstract B06: Overcoming hypoxia-induced resistance is essential for effective survivin targeting in acute myeloid leukemia

Cancer Research, 2018

Background: Although the clinical outcome of pediatric acute myeloid leukemia (AML) has improved ... more Background: Although the clinical outcome of pediatric acute myeloid leukemia (AML) has improved over the past few decades, approximately 30% of patients relapse. The overall survival (OS) rate of pediatric patients with primary refractory or relapsed AML is about 30%. Resistance to therapy presents a great challenge for AML patients. Therefore, there is a critical need to understand mechanisms of resistance, as well as to identify novel therapies that will improve OS. The leukemia-initiating cells reside in special hypoxic niches in the bone marrow. Thus, studying drug response of AML cells within a hypoxic environment is essential for more accurate prediction of patients’ response to therapy. We performed functional drug screening using AML cell lines, which demonstrated a differential response of AML cell lines to the survivin-targeting agent YM155 in normoxia versus hypoxia. Survivin (BIRC5), an inhibitor of apoptosis, is overexpressed in a variety of pediatric blood cancers com...

Research paper thumbnail of Clinical resistance associated with a novel MAP2K1 mutation in a patient with Langerhans cell histiocytosis

Pediatric blood & cancer, Jan 16, 2018

Patients with Langerhans cell histiocytosis (LCH) harbor BRAF V600E and activating mutations of M... more Patients with Langerhans cell histiocytosis (LCH) harbor BRAF V600E and activating mutations of MAP2K1/MEK1 in 50% and 25% of cases, respectively. We evaluated a patient with treatment-refractory LCH for mutations in the RAS-RAF-MEK-ERK pathway and identified a novel mutation in the MAP2K1 gene resulting in a p.L98_K104 > Q deletion and predicted to be auto-activating. During treatment with the MEK inhibitor trametinib, the patient's disease showed significant progression. In vitro characterization of the MAP2K1 p.L98_K104 > Q deletion confirmed its effect on cellular activation of the ERK pathway and drug resistance.

Research paper thumbnail of Abstract B02: Bortezomib-resistant pediatric acute myeloid leukemia cell lines derived from Down syndrome patients are sensitive to disulfiram

Cancer Research, 2016

Background: Acute myeloid leukemia (AML) continues to have an overall poor prognosis of about 50%... more Background: Acute myeloid leukemia (AML) continues to have an overall poor prognosis of about 50% in children. Treatment options for relapsed or refractory AML are limited. Bortezomib, a well-established proteasome inhibitor, is used in AML therapy. It binds to the beta 5 subunit (PSMB5) of the 26S proteasome and inhibits the chymotrypsin (CT)-like activity. Although patients develop resistance to bortezomib, the mechanism is not fully elucidated. Disulfiram (DS) is an aldehyde dehydrogenase (ALDH) inhibitor used to treat alcoholism without major side effects. DS has anticancer cytotoxicity that is in part copper (Cu2+)-dependent (DS/Cu2+). One of the reported mechanisms of action of DS is through proteasome inhibition. We previously demonstrated that both DS/Cu2+ and bortezomib are cytotoxic in a panel of AML cell lines. In the present study, we focused on two acute megakaryoblastic leukemia cell lines derived from patients with Down syndrome (CMY and CMK), one of the cell lines is...

Research paper thumbnail of Abstract B16: The nuclear IGF-1R regulates DNA damage tolerance through tyrosine phosphorylation of PCNA in human embryonic stem cells

Molecular Cancer Research, 2016

Background: The type 1 insulin-like growth factor receptor (IGF-1R) and its signaling components ... more Background: The type 1 insulin-like growth factor receptor (IGF-1R) and its signaling components promote cell proliferation, survival, and development of the malignant phenotype. Recently, the IGF-1R has been shown to translocate to the nucleus; however its nuclear functions are not fully elucidated. The proliferating cell nuclear antigen (PCNA) is a nuclear protein that coordinates DNA replication, and is involved in DNA damage repair and in the DNA damage tolerance (DDT) pathways. DDT allows the cell to replicate over polymerase-blocking lesions. DDT mechanisms include translesion DNA synthesis (TLS), and template switching, and the ubiquitination status of PCNA is crucial in these processes. Mono-ubiquitination of PCNA by Rad6 (E2)-Rad18 (E3) activates TLS. The mono-ubiquitin can be further extended though K63-linkages and the non degradative K63 polyubiquitination of PCNA by Ubc13 (E2)-SHPRH/HLTF (E3) leads to template switching. Purpose: The purpose of the present work was to s...

Research paper thumbnail of Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage

Journal of Biological Chemistry, 2017

We have previously shown that the insulinlike growth factor 1 receptor (IGF-1R) translocates to t... more We have previously shown that the insulinlike growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In the present study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA coincubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono-and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases (e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G1 cell cycle arrest and S-phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT.

Research paper thumbnail of Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells

Journal of experimental & clinical cancer research : CR, Feb 1, 2017

Children with Down syndrome (DS) have increased risk for developing AML (DS-AMKL), and they usual... more Children with Down syndrome (DS) have increased risk for developing AML (DS-AMKL), and they usually experience severe therapy-related toxicities compared to non DS-AMKL. Refractory/relapsed disease has very poor outcome, and patients would benefit from novel, less toxic, therapeutic strategies that overcome resistance. Relapse/resistance are linked to cancer stem cells with high aldehyde dehydrogenase (ALDH) activity. The purpose of the present work was to study less toxic alternative therapeutic agents for relapsed/refractory DS-AMKL. Fourteen AML cell lines including the DS-AMKL CMY and CMK from relapsed/refractory AML were used. Cytarabine (Ara-C), bortezomib (BTZ), disulfiram/copper (DSF/Cu(2+)) were evaluated for cytotoxicity, depletion of ALDH-positive cells, and resistance. BTZ-resistant CMY and CMK variants were generated by continuous BTZ treatment. Cell viability was assessed using CellTiter-Glo®, ALDH activity by ALDELUOR(TM), and proteasome inhibition by western blot of ...

Research paper thumbnail of Abstract 2791: Hypoxia selectively confers resistance to the survivin-targeting compound YM155 in acute myeloid leukemia and Ewings sarcoma cell lines

Cancer Research, 2016

Background Hypoxia is characteristic of almost all types of solid tumors and is an important comp... more Background Hypoxia is characteristic of almost all types of solid tumors and is an important component of the bone marrow microenvironment. It is an adverse prognostic indicator in cancer as it is associated with tumor progression and chemoresistance. Cells may adapt to hypoxia in different ways; including growth arrest, stimulation of angiogenic factors, inhibition of apoptosis, induction of the ER stress program, or modification of the cellular energy metabolism. YM155 inhibits survivin gene expression by suppressing promoter activity and subsequent gene expression. The role of hypoxia and its clinical implications in childhood cancer remain largely unknown. Therefore, studying the drug response of cancer cells within a hypoxic environment is critical for the accurate prediction of patients’ response to therapy. The aims of the present study were (1) to perform screening of the effects of 5 molecularly targeted compounds on a large panel of pediatric cancer cell lines including ac...

Research paper thumbnail of Downregulation of IGF-1 receptor occurs after hepatic linage commitment during hepatocyte differentiation from human embryonic stem cells

Biochemical and biophysical research communications, Jan 30, 2016

The insulin-like growth factor 1 receptor (IGF-1R) has been suggested to be involved in hepatocyt... more The insulin-like growth factor 1 receptor (IGF-1R) has been suggested to be involved in hepatocyte differentiation. Human hepatocyte cancer cells and stem cells are known to express IGF-1R whereas normal hepatocytes do not. In the present study we optimized a differentiation protocol and verified the different stages by established markers. The expression levels of IGF-1R and major downstream signaling proteins during differentiation from human embryonic stem cells (hESC) to mature hepatocytes were investigated. We could only demonstrate a minor decrease in IGF-1R expression during endodermal differentiation compared to hESC, but declined substantially (>50%) after hepatic lineage commitment during the hepatocyte specification and maturation stages. This downregulation was paralleled by an upregulation of ERK 1/2, AKT and insulin substrate-1. Neither inhibition nor activation of IGF-1R had any essential effect on endoderm differentiation of human embryonic stem cells. Therefore, ...

Research paper thumbnail of Abstract 3766: Targeting promoter regions of c-Myc and Bcl-2 in AML cells using G-quadruplex interacting drugs

Cancer Research, 2016

Cancer is a disease that can be characterized by overexpression of key oncogenic drivers that sup... more Cancer is a disease that can be characterized by overexpression of key oncogenic drivers that support tumor development and maintenance. In many instances, these oncogenic drivers are ‘undruggable’ because of structural challenges, the inability to effectively inhibit high concentrations of overexpressed proteins, and the development of drug resistance mutations. An alternative therapeutic approach is to directly inhibit gene transcription by targeting unique secondary DNA structures, called G-quadruplexes, that are associated with subsets of promoters. We investigated the activity of a class of compounds termed G-quadruplex Interacting Drugs (GQIDs) that are able to shut down the expression of specific oncogenic drivers, such as c-Myc and Bcl-2, used by tumor cells for growth and survival. We tested the activity of two GQIDs, GQC-05 and GSA-1103, on cell growth of a panel of eight pediatric and eight adult acute myeloid leukemia (AML) cell lines. Drug dose response analysis showed ...

Research paper thumbnail of Abstract B24: Targeting promoter regions of oncogenic drivers in pediatric AML cells using G-quadruplex Interacting Drugs (GQIDs)

Cancer Research, 2016

Cancer is primarily a disease characterized by aberrant gene expression that is manifested by the... more Cancer is primarily a disease characterized by aberrant gene expression that is manifested by the overexpression of key genes that support tumor development and maintenance. In many instances, oncogenic drivers are frequently ‘undruggable’ because of structural challenges, the inability to effectively inhibit high concentrations of overexpressed proteins, and the development of drug resistance mutations. An alternative therapeutic approach is to directly inhibit gene transcription by targeting unique secondary DNA structures, called G-quadruplexes, that are associated with subsets of promoters. Our laboratory investigated the activity of a class of compounds termed G-quadruplex Interacting Drugs (GQIDs) that are able to shut down the expression of specific genes used by tumor cells for growth and survival. We tested the activity of two GQIDs GQC-05 and GSA-1103 on cell growth of a panel of eight pediatric and eight adult AML cell lines. Drug dose response analysis showed IC50 values...

Research paper thumbnail of Abstract 3982: Repurposing disulfiram for treatment of relapsed/refractory acute myeloid leukemia

Cancer Research, 2014

Background Despite toxic treatments, acute myeloid leukemia (AML) continues to have an overall po... more Background Despite toxic treatments, acute myeloid leukemia (AML) continues to have an overall poor prognosis of about 50% in children while significantly less in older adults. For patients with relapsed AML refractory to chemotherapy, there is little chance for survival. Therefore, the need for novel treatment modalities continues to be critically important. Disulfiram (DS) is an aldehyde dehydrogenase (ALDH) inhibitor that has been used for decades for the treatment of alcoholism without major side effects. While DS has been reported to have anticancer cytotoxicity that is in part copper (Cu2+)-dependent, the mechanisms are still not fully elucidated. The goal of the present study was to investigate whether DS has significant cytotoxicity against AML. Methods and Results In the present study the IC50 were determined for DS alone or in combination with 1 µM Cu2+ in a panel of 16 AML cell lines using Cell Titer Glo assay. DS/Cu2+ resulted in significant killing of all cell lines (IC...

Research paper thumbnail of Abstract 5583: Integrative genomic analyses on pediatric acute myeloid leukemia

Cancer Research, 2014

Background Acute myeloid leukemia (AML) is a complex and heterogeneous disease leading to a wide ... more Background Acute myeloid leukemia (AML) is a complex and heterogeneous disease leading to a wide range of response variability to conventional therapy, excessive treatment related toxicity, and an overall poor outcome. The NCI supported Therapeutically Applicable Research to Generate Effective Treatments (TARGET) Initiative provided initial support for large-scale genomics to identify novel disease-associated genomic and epigenomic alterations that could be used to develop novel, targeted therapies for pediatric AML. Patients Diagnostic (Dx), remission (Rm), and relapse (Rel) samples were obtained from over 200 children with AML treated on the most recent Children's Oncology Group clinical trials. Data analyses were performed using Partek Genomics Suite 6.6 and Ingenuity Pathway Analysis. Results Methylation profiling (Illumina HumanMethylation27) identified 603 CpG sites significantly differentially methylated between Dx and Rm. In addition, 514 CpG sites were significantly dif...

Research paper thumbnail of Picropodophyllin causes mitotic arrest and catastrophe by depolymerizing microtubules via Insulin-like growth factor-1 receptor-independent mechanism

Oncotarget, Jan 30, 2014

Picropodophyllin (PPP) is an anticancer drug undergoing clinical development in NSCLC. PPP has be... more Picropodophyllin (PPP) is an anticancer drug undergoing clinical development in NSCLC. PPP has been shown to suppress IGF-1R signaling and to induce a G2/M cell cycle phase arrest but the exact mechanisms remain to be elucidated. The present study identified an IGF-1-independent mechanism of PPP leading to pro-metaphase arrest. The mitotic block was induced in human cancer cell lines and in an A549 xenograft mouse but did not occur in normal hepatocytes/mouse tissues. Cell cycle arrest by PPP occurred in vitro and in vivo accompanied by prominent CDK1 activation, and was IGF-1R-independent since it occurred also in IGF-1R-depleted and null cells. The tumor cells were not arrested in G2/M but in mitosis. Centrosome separation was prevented during mitotic entry, resulting in a monopolar mitotic spindle with subsequent prometaphase-arrest, independent of Plk1/Aurora A or Eg5, and leading to cell features of mitotic catastrophe. PPP also increased soluble tubulin and decreased spindle-a...

Research paper thumbnail of Multiple antitumor effects of picropodophyllin in colon carcinoma cell lines: Clinical implications

International Journal of Oncology, 2011

Although colorectal cancer can be successfully treated by conventional strategies such as chemo/r... more Although colorectal cancer can be successfully treated by conventional strategies such as chemo/radiotherapy and surgery, a substantial number of cases, in particular those with liver metastases, remain incurable. Therefore, novel treatment approaches are warranted. The IGF-1R and its ligands, mainly IGF-1 and IGF-2, have been suggested to play pivotal roles in proliferation, survival and migration of adenocarcinoma cells of the colon/rectum. Therefore, interference with IGF-1Rmediated signaling may represent a therapeutic option for this malignancy. In this study, semi-quantitative RT-PCR analyses of 48 paired, colorectal cancer patient samples showed significant overexpression of tumor IGF-1R and IGF-2 mRNA. There was also an overexpression of MMP-7, which was significantly correlated with histopathological parameters. Based on these findings, the effect of the IGF-1R-inhibitory cyclolignan picropodophyllin (PPP) was assessed in the four colon carcinoma cell lines HT-29, HCT-116, DLD-1 and CaCO-2. PPP strongly and dose-dependently inhibited proliferation and migration in all cell lines. However, when exposed to 0.5 µM PPP, only HT-29 showed a net decrease of viable cells as compared with the cell number at the beginning of the experiment, a finding that coincided with decreased expression/phosphorylation of IGF-1R, AKT and ERK. This cell line also exhibited PPP-induced downregulation of MMP-7 and MMP-9. Similar to the DLD-1 and HCT-116 cell lines, HT-29 also showed substantial cell detachment in response to PPP. Although a net reduction of cells by PPP seems to require a synchronized downregulation of IGF-1R, AKT and ERK1/2, part of the antitumor effect may be explained by other, possibly IGF-1R-unrelated mechanism(s). Such a multitude of inhibitory effects of PPP in colon cancer cells together with its low toxicity in vivo makes it a promising drug candidate in the treatment of this disease.

Research paper thumbnail of Serum IGFBP-3 is a more effective predictor than IGF-1 and IGF-2 for the development of hepatocellular carcinoma in patients with chronic HCV infection

Oncology letters, 2012

Hepatocellular carcinoma (HCC) contributes to 14.8% of all cancer mortality in Egypt, which has a... more Hepatocellular carcinoma (HCC) contributes to 14.8% of all cancer mortality in Egypt, which has a high prevalence of hepatitis C virus (HCV). We have previously shown alterations in the insulin-like growth factor-1 (IGF-1) receptor signalling pathway during experimental hepatocarcinogenesis. The aim of this study was to determine whether serum levels of IGF-1, IGF-2 and IGFBP-3 can be used to discriminate between HCC and the stages of hepatic dysfunction in patients with liver cirrhosis assessed by the Child-Pugh (CP) score, and to correlate these levels with HCC stages. We recruited 241 subjects to the present study; 79 with liver cirrhosis, 62 with HCV-induced HCC and 100 age-matched controls. Results showed that serum levels of IGF-1, IGF-2 and IGFBP-3 were reduced significantly in cirrhosis and HCC patients in comparison to the controls, and that this reduction negatively correlated with the CP scores. However, only IGFBP-3 levels showed significant negative correlation with α-f...

Research paper thumbnail of Schizophyllan inhibits the development of mammary and hepatic carcinomas induced by 7,12 dimethylbenz(α)anthracene and decreases cell proliferation: comparison with tamoxifen

Journal of Cancer Research and Clinical Oncology, 2012

Breast cancer is one of the leading causes of cancer mortality among women. Some anticancer compo... more Breast cancer is one of the leading causes of cancer mortality among women. Some anticancer compounds have been isolated from mushrooms. The aim of the present work was to study the anticancer effects of schizophyllan (SCH), a β-D: -glucan extracted from the mushroom Schizophyllum commune alone or in combination with tamoxifen (TAM) on 7, 12 Dimethylbenz(α)anthracene (DMBA)-induced carcinomas in mice. We isolated SCH from S. commune. Female mice received DMBA, SCH, DMBA+SCH, DMBA+TAM or DMBA+TAM+SCH or vehicles. We studied mice survival, tumour incidence, histopathology, oestrogen receptor (ER) expression, cell proliferation by immunohistochemical detection of proliferating cell nuclear antigen (PCNA), apoptosis by TUNEL assay, as well as caspase-3 expression. DMBA treatment resulted in mammary and hepatocellular carcinomas (HCC). Both SCH and TAM reduced the incidence of DMBA-induced mammary tumours by 85 and 75 %, respectively, and equally decreased the PCNA labelling index relative to DMBA. TAM treatment increased the incidence of- and PCNA index in HCCs relative to DMBA, while SCH suppressed these effects. TAM was more effective than SCH in the induction of apoptosis in both mammary and hepatic carcinomas. Caspase-3 levels correlated with the apoptotic index in most experimental groups. Only one dose of SCH had similar therapeutic effects against DMBA-induced mammary carcinomas as 4 weeks of TAM treatment. This coupled with the ability of SCH to suppress hepatic lesions associated with TAM treatment provides the rationale for further investigating the combined therapeutic effects of TAM+SCH in preclinical models of ER-positive breast cancer, as well as in liver cancer.

Research paper thumbnail of Targeting cell cycle regulators in hematologic malignancies

Frontiers in Cell and Developmental Biology, 2015

Hematologic malignancies represent the fourth most frequently diagnosed cancer in economically de... more Hematologic malignancies represent the fourth most frequently diagnosed cancer in economically developed countries. In hematologic malignancies normal hematopoiesis is interrupted by uncontrolled growth of a genetically altered stem or progenitor cell (HSPC) that maintains its ability of self-renewal. Cyclin-dependent kinases (CDKs) not only regulate the mammalian cell cycle, but also influence other vital cellular processes, such as stem cell renewal, differentiation, transcription, epigenetic regulation, apoptosis, and DNA repair. Chromosomal translocations, amplification, overexpression and altered CDK activities have been described in different types of human cancer, which have made them attractive targets for pharmacological inhibition. Mouse models deficient for one or more CDKs have significantly contributed to our current understanding of the physiological functions of CDKs, as well as their roles in human cancer. The present review focuses on selected cell cycle kinases with recent emerging key functions in hematopoiesis and in hematopoietic malignancies, such as CDK6 and its role in MLL-rearranged leukemia and acute lymphocytic leukemia, CDK1 and its regulator WEE-1 in acute myeloid leukemia (AML), and cyclin C/CDK8/CDK19 complexes in T-cell acute lymphocytic leukemia. The knowledge gained from gene knockout experiments in mice of these kinases is also summarized. An overview of compounds targeting these kinases, which are currently in clinical development in various solid tumors and hematopoietic malignances, is presented. These include the CDK4/CDK6 inhibitors (palbociclib, LEE011, LY2835219), pan-CDK inhibitors that target CDK1 (dinaciclib, flavopiridol, AT7519, TG02, P276-00, terampeprocol and RGB 286638) as well as the WEE-1 kinase inhibitor, MK-1775. The advantage of combination therapy of cell cycle inhibitors with conventional chemotherapeutic agents used in the treatment of AML, such as cytarabine, is discussed.

Research paper thumbnail of Detection and quantification of protein phosphatase inhibitor-1 gene expression in total rat liver and isolated hepatocytes

Molecular and cellular biochemistry, 2001

The mRNA expression of protein phosphatase inhibitor-1 (inhibitor-1) in rat liver was demonstrate... more The mRNA expression of protein phosphatase inhibitor-1 (inhibitor-1) in rat liver was demonstrated using highly sensitive semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Quantification by real-time RT-PCR (LightCycler technology) yielded the same copy number of inhibitor-1 mRNA in total rat liver and isolated hepatocytes (12 copies per cell). This novel finding shows that rat liver expresses indeed inhibitor-1 mRNA, albeit in low amounts. The low copy number explains why the mRNA had not been detected by Northern blotting so far. For comparison, about 425 copies/cell were detected in brain and 2500 copies/cell in skeletal muscle from rat. The full-length coding sequence of rat liver inhibitor-1 was cloned and sequenced, 100% homology with the muscle cDNA was obtained, indicating the expression of the same gene in liver and muscle. In vitro transcription and translation yielded a protein (Mr approximately 30 kDa) which could be detected with a specific ant...

Research paper thumbnail of Resistance Associated with a Novel MAP 2 K 1 Mutation in a Patient with Langerhans Cell Histiocytosis

Patients with Langerhans cell histiocytosis (LCH) harbor BRAF V600E and activating mutations of M... more Patients with Langerhans cell histiocytosis (LCH) harbor BRAF V600E and activating mutations of MAP2K1/MEK1 in 50% and 25% of cases, respectively. We evaluated a patient with treatment refractory LCH for mutations in the RAS-RAF-MEK-ERK pathway and identified a novel mutation in the MAP2K1 gene resulting in a p.L98_K104>Q deletion and predicted to be auto-activating. During treatment with the MEK inhibitor trametinib, the patient's disease showed significant progression. In vitro characterization of the MAP2K1 p.L98_K104>Q deletion confirmed its effect on cellular activation of the ERK pathway and drug resistance.

Research paper thumbnail of Abstract 2705: Functional proteomics to reveal distinct signature for acute myeloid leukemia subtypes

Cancer Research, 2018

Patients with relapsed/ refractory AML have an overall survival rate <30%. Genomic approaches ... more Patients with relapsed/ refractory AML have an overall survival rate <30%. Genomic approaches are being used for patients' stratification, but they have not been sufficient at predicting response to therapy. We propose an integrative approach to identify distinctive phosphorylated protein biomarkers in AML, map them to signaling networks, and compare these to pathways identified through analysis of gene expression. This study aims at identifying distinct phosphoproteomic signatures by mass spectrometry (MS) in different AML subtypes. Methods: Four AML cell lines and 40 bone marrow samples from patients with AML representing different AML subtypes are used. Samples were prepared using a MOAC-TiO2 stationary phase for phosphopeptide enrichment, and analysis on a Thermo LTQ Orbitrap Velos MS. Identified phosphorylated STY sites were cross-referenced against iPTMnet. Phosphoproteins were further matched to potential pathways using Ingenuity Pathway Analysis (IPA). Gene-expression...

Research paper thumbnail of Abstract B06: Overcoming hypoxia-induced resistance is essential for effective survivin targeting in acute myeloid leukemia

Cancer Research, 2018

Background: Although the clinical outcome of pediatric acute myeloid leukemia (AML) has improved ... more Background: Although the clinical outcome of pediatric acute myeloid leukemia (AML) has improved over the past few decades, approximately 30% of patients relapse. The overall survival (OS) rate of pediatric patients with primary refractory or relapsed AML is about 30%. Resistance to therapy presents a great challenge for AML patients. Therefore, there is a critical need to understand mechanisms of resistance, as well as to identify novel therapies that will improve OS. The leukemia-initiating cells reside in special hypoxic niches in the bone marrow. Thus, studying drug response of AML cells within a hypoxic environment is essential for more accurate prediction of patients’ response to therapy. We performed functional drug screening using AML cell lines, which demonstrated a differential response of AML cell lines to the survivin-targeting agent YM155 in normoxia versus hypoxia. Survivin (BIRC5), an inhibitor of apoptosis, is overexpressed in a variety of pediatric blood cancers com...

Research paper thumbnail of Clinical resistance associated with a novel MAP2K1 mutation in a patient with Langerhans cell histiocytosis

Pediatric blood & cancer, Jan 16, 2018

Patients with Langerhans cell histiocytosis (LCH) harbor BRAF V600E and activating mutations of M... more Patients with Langerhans cell histiocytosis (LCH) harbor BRAF V600E and activating mutations of MAP2K1/MEK1 in 50% and 25% of cases, respectively. We evaluated a patient with treatment-refractory LCH for mutations in the RAS-RAF-MEK-ERK pathway and identified a novel mutation in the MAP2K1 gene resulting in a p.L98_K104 > Q deletion and predicted to be auto-activating. During treatment with the MEK inhibitor trametinib, the patient's disease showed significant progression. In vitro characterization of the MAP2K1 p.L98_K104 > Q deletion confirmed its effect on cellular activation of the ERK pathway and drug resistance.

Research paper thumbnail of Abstract B02: Bortezomib-resistant pediatric acute myeloid leukemia cell lines derived from Down syndrome patients are sensitive to disulfiram

Cancer Research, 2016

Background: Acute myeloid leukemia (AML) continues to have an overall poor prognosis of about 50%... more Background: Acute myeloid leukemia (AML) continues to have an overall poor prognosis of about 50% in children. Treatment options for relapsed or refractory AML are limited. Bortezomib, a well-established proteasome inhibitor, is used in AML therapy. It binds to the beta 5 subunit (PSMB5) of the 26S proteasome and inhibits the chymotrypsin (CT)-like activity. Although patients develop resistance to bortezomib, the mechanism is not fully elucidated. Disulfiram (DS) is an aldehyde dehydrogenase (ALDH) inhibitor used to treat alcoholism without major side effects. DS has anticancer cytotoxicity that is in part copper (Cu2+)-dependent (DS/Cu2+). One of the reported mechanisms of action of DS is through proteasome inhibition. We previously demonstrated that both DS/Cu2+ and bortezomib are cytotoxic in a panel of AML cell lines. In the present study, we focused on two acute megakaryoblastic leukemia cell lines derived from patients with Down syndrome (CMY and CMK), one of the cell lines is...

Research paper thumbnail of Abstract B16: The nuclear IGF-1R regulates DNA damage tolerance through tyrosine phosphorylation of PCNA in human embryonic stem cells

Molecular Cancer Research, 2016

Background: The type 1 insulin-like growth factor receptor (IGF-1R) and its signaling components ... more Background: The type 1 insulin-like growth factor receptor (IGF-1R) and its signaling components promote cell proliferation, survival, and development of the malignant phenotype. Recently, the IGF-1R has been shown to translocate to the nucleus; however its nuclear functions are not fully elucidated. The proliferating cell nuclear antigen (PCNA) is a nuclear protein that coordinates DNA replication, and is involved in DNA damage repair and in the DNA damage tolerance (DDT) pathways. DDT allows the cell to replicate over polymerase-blocking lesions. DDT mechanisms include translesion DNA synthesis (TLS), and template switching, and the ubiquitination status of PCNA is crucial in these processes. Mono-ubiquitination of PCNA by Rad6 (E2)-Rad18 (E3) activates TLS. The mono-ubiquitin can be further extended though K63-linkages and the non degradative K63 polyubiquitination of PCNA by Ubc13 (E2)-SHPRH/HLTF (E3) leads to template switching. Purpose: The purpose of the present work was to s...

Research paper thumbnail of Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage

Journal of Biological Chemistry, 2017

We have previously shown that the insulinlike growth factor 1 receptor (IGF-1R) translocates to t... more We have previously shown that the insulinlike growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In the present study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA coincubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono-and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases (e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G1 cell cycle arrest and S-phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT.

Research paper thumbnail of Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells

Journal of experimental & clinical cancer research : CR, Feb 1, 2017

Children with Down syndrome (DS) have increased risk for developing AML (DS-AMKL), and they usual... more Children with Down syndrome (DS) have increased risk for developing AML (DS-AMKL), and they usually experience severe therapy-related toxicities compared to non DS-AMKL. Refractory/relapsed disease has very poor outcome, and patients would benefit from novel, less toxic, therapeutic strategies that overcome resistance. Relapse/resistance are linked to cancer stem cells with high aldehyde dehydrogenase (ALDH) activity. The purpose of the present work was to study less toxic alternative therapeutic agents for relapsed/refractory DS-AMKL. Fourteen AML cell lines including the DS-AMKL CMY and CMK from relapsed/refractory AML were used. Cytarabine (Ara-C), bortezomib (BTZ), disulfiram/copper (DSF/Cu(2+)) were evaluated for cytotoxicity, depletion of ALDH-positive cells, and resistance. BTZ-resistant CMY and CMK variants were generated by continuous BTZ treatment. Cell viability was assessed using CellTiter-Glo®, ALDH activity by ALDELUOR(TM), and proteasome inhibition by western blot of ...

Research paper thumbnail of Abstract 2791: Hypoxia selectively confers resistance to the survivin-targeting compound YM155 in acute myeloid leukemia and Ewings sarcoma cell lines

Cancer Research, 2016

Background Hypoxia is characteristic of almost all types of solid tumors and is an important comp... more Background Hypoxia is characteristic of almost all types of solid tumors and is an important component of the bone marrow microenvironment. It is an adverse prognostic indicator in cancer as it is associated with tumor progression and chemoresistance. Cells may adapt to hypoxia in different ways; including growth arrest, stimulation of angiogenic factors, inhibition of apoptosis, induction of the ER stress program, or modification of the cellular energy metabolism. YM155 inhibits survivin gene expression by suppressing promoter activity and subsequent gene expression. The role of hypoxia and its clinical implications in childhood cancer remain largely unknown. Therefore, studying the drug response of cancer cells within a hypoxic environment is critical for the accurate prediction of patients’ response to therapy. The aims of the present study were (1) to perform screening of the effects of 5 molecularly targeted compounds on a large panel of pediatric cancer cell lines including ac...

Research paper thumbnail of Downregulation of IGF-1 receptor occurs after hepatic linage commitment during hepatocyte differentiation from human embryonic stem cells

Biochemical and biophysical research communications, Jan 30, 2016

The insulin-like growth factor 1 receptor (IGF-1R) has been suggested to be involved in hepatocyt... more The insulin-like growth factor 1 receptor (IGF-1R) has been suggested to be involved in hepatocyte differentiation. Human hepatocyte cancer cells and stem cells are known to express IGF-1R whereas normal hepatocytes do not. In the present study we optimized a differentiation protocol and verified the different stages by established markers. The expression levels of IGF-1R and major downstream signaling proteins during differentiation from human embryonic stem cells (hESC) to mature hepatocytes were investigated. We could only demonstrate a minor decrease in IGF-1R expression during endodermal differentiation compared to hESC, but declined substantially (>50%) after hepatic lineage commitment during the hepatocyte specification and maturation stages. This downregulation was paralleled by an upregulation of ERK 1/2, AKT and insulin substrate-1. Neither inhibition nor activation of IGF-1R had any essential effect on endoderm differentiation of human embryonic stem cells. Therefore, ...

Research paper thumbnail of Abstract 3766: Targeting promoter regions of c-Myc and Bcl-2 in AML cells using G-quadruplex interacting drugs

Cancer Research, 2016

Cancer is a disease that can be characterized by overexpression of key oncogenic drivers that sup... more Cancer is a disease that can be characterized by overexpression of key oncogenic drivers that support tumor development and maintenance. In many instances, these oncogenic drivers are ‘undruggable’ because of structural challenges, the inability to effectively inhibit high concentrations of overexpressed proteins, and the development of drug resistance mutations. An alternative therapeutic approach is to directly inhibit gene transcription by targeting unique secondary DNA structures, called G-quadruplexes, that are associated with subsets of promoters. We investigated the activity of a class of compounds termed G-quadruplex Interacting Drugs (GQIDs) that are able to shut down the expression of specific oncogenic drivers, such as c-Myc and Bcl-2, used by tumor cells for growth and survival. We tested the activity of two GQIDs, GQC-05 and GSA-1103, on cell growth of a panel of eight pediatric and eight adult acute myeloid leukemia (AML) cell lines. Drug dose response analysis showed ...

Research paper thumbnail of Abstract B24: Targeting promoter regions of oncogenic drivers in pediatric AML cells using G-quadruplex Interacting Drugs (GQIDs)

Cancer Research, 2016

Cancer is primarily a disease characterized by aberrant gene expression that is manifested by the... more Cancer is primarily a disease characterized by aberrant gene expression that is manifested by the overexpression of key genes that support tumor development and maintenance. In many instances, oncogenic drivers are frequently ‘undruggable’ because of structural challenges, the inability to effectively inhibit high concentrations of overexpressed proteins, and the development of drug resistance mutations. An alternative therapeutic approach is to directly inhibit gene transcription by targeting unique secondary DNA structures, called G-quadruplexes, that are associated with subsets of promoters. Our laboratory investigated the activity of a class of compounds termed G-quadruplex Interacting Drugs (GQIDs) that are able to shut down the expression of specific genes used by tumor cells for growth and survival. We tested the activity of two GQIDs GQC-05 and GSA-1103 on cell growth of a panel of eight pediatric and eight adult AML cell lines. Drug dose response analysis showed IC50 values...

Research paper thumbnail of Abstract 3982: Repurposing disulfiram for treatment of relapsed/refractory acute myeloid leukemia

Cancer Research, 2014

Background Despite toxic treatments, acute myeloid leukemia (AML) continues to have an overall po... more Background Despite toxic treatments, acute myeloid leukemia (AML) continues to have an overall poor prognosis of about 50% in children while significantly less in older adults. For patients with relapsed AML refractory to chemotherapy, there is little chance for survival. Therefore, the need for novel treatment modalities continues to be critically important. Disulfiram (DS) is an aldehyde dehydrogenase (ALDH) inhibitor that has been used for decades for the treatment of alcoholism without major side effects. While DS has been reported to have anticancer cytotoxicity that is in part copper (Cu2+)-dependent, the mechanisms are still not fully elucidated. The goal of the present study was to investigate whether DS has significant cytotoxicity against AML. Methods and Results In the present study the IC50 were determined for DS alone or in combination with 1 µM Cu2+ in a panel of 16 AML cell lines using Cell Titer Glo assay. DS/Cu2+ resulted in significant killing of all cell lines (IC...

Research paper thumbnail of Abstract 5583: Integrative genomic analyses on pediatric acute myeloid leukemia

Cancer Research, 2014

Background Acute myeloid leukemia (AML) is a complex and heterogeneous disease leading to a wide ... more Background Acute myeloid leukemia (AML) is a complex and heterogeneous disease leading to a wide range of response variability to conventional therapy, excessive treatment related toxicity, and an overall poor outcome. The NCI supported Therapeutically Applicable Research to Generate Effective Treatments (TARGET) Initiative provided initial support for large-scale genomics to identify novel disease-associated genomic and epigenomic alterations that could be used to develop novel, targeted therapies for pediatric AML. Patients Diagnostic (Dx), remission (Rm), and relapse (Rel) samples were obtained from over 200 children with AML treated on the most recent Children's Oncology Group clinical trials. Data analyses were performed using Partek Genomics Suite 6.6 and Ingenuity Pathway Analysis. Results Methylation profiling (Illumina HumanMethylation27) identified 603 CpG sites significantly differentially methylated between Dx and Rm. In addition, 514 CpG sites were significantly dif...

Research paper thumbnail of Picropodophyllin causes mitotic arrest and catastrophe by depolymerizing microtubules via Insulin-like growth factor-1 receptor-independent mechanism

Oncotarget, Jan 30, 2014

Picropodophyllin (PPP) is an anticancer drug undergoing clinical development in NSCLC. PPP has be... more Picropodophyllin (PPP) is an anticancer drug undergoing clinical development in NSCLC. PPP has been shown to suppress IGF-1R signaling and to induce a G2/M cell cycle phase arrest but the exact mechanisms remain to be elucidated. The present study identified an IGF-1-independent mechanism of PPP leading to pro-metaphase arrest. The mitotic block was induced in human cancer cell lines and in an A549 xenograft mouse but did not occur in normal hepatocytes/mouse tissues. Cell cycle arrest by PPP occurred in vitro and in vivo accompanied by prominent CDK1 activation, and was IGF-1R-independent since it occurred also in IGF-1R-depleted and null cells. The tumor cells were not arrested in G2/M but in mitosis. Centrosome separation was prevented during mitotic entry, resulting in a monopolar mitotic spindle with subsequent prometaphase-arrest, independent of Plk1/Aurora A or Eg5, and leading to cell features of mitotic catastrophe. PPP also increased soluble tubulin and decreased spindle-a...

Research paper thumbnail of Multiple antitumor effects of picropodophyllin in colon carcinoma cell lines: Clinical implications

International Journal of Oncology, 2011

Although colorectal cancer can be successfully treated by conventional strategies such as chemo/r... more Although colorectal cancer can be successfully treated by conventional strategies such as chemo/radiotherapy and surgery, a substantial number of cases, in particular those with liver metastases, remain incurable. Therefore, novel treatment approaches are warranted. The IGF-1R and its ligands, mainly IGF-1 and IGF-2, have been suggested to play pivotal roles in proliferation, survival and migration of adenocarcinoma cells of the colon/rectum. Therefore, interference with IGF-1Rmediated signaling may represent a therapeutic option for this malignancy. In this study, semi-quantitative RT-PCR analyses of 48 paired, colorectal cancer patient samples showed significant overexpression of tumor IGF-1R and IGF-2 mRNA. There was also an overexpression of MMP-7, which was significantly correlated with histopathological parameters. Based on these findings, the effect of the IGF-1R-inhibitory cyclolignan picropodophyllin (PPP) was assessed in the four colon carcinoma cell lines HT-29, HCT-116, DLD-1 and CaCO-2. PPP strongly and dose-dependently inhibited proliferation and migration in all cell lines. However, when exposed to 0.5 µM PPP, only HT-29 showed a net decrease of viable cells as compared with the cell number at the beginning of the experiment, a finding that coincided with decreased expression/phosphorylation of IGF-1R, AKT and ERK. This cell line also exhibited PPP-induced downregulation of MMP-7 and MMP-9. Similar to the DLD-1 and HCT-116 cell lines, HT-29 also showed substantial cell detachment in response to PPP. Although a net reduction of cells by PPP seems to require a synchronized downregulation of IGF-1R, AKT and ERK1/2, part of the antitumor effect may be explained by other, possibly IGF-1R-unrelated mechanism(s). Such a multitude of inhibitory effects of PPP in colon cancer cells together with its low toxicity in vivo makes it a promising drug candidate in the treatment of this disease.

Research paper thumbnail of Serum IGFBP-3 is a more effective predictor than IGF-1 and IGF-2 for the development of hepatocellular carcinoma in patients with chronic HCV infection

Oncology letters, 2012

Hepatocellular carcinoma (HCC) contributes to 14.8% of all cancer mortality in Egypt, which has a... more Hepatocellular carcinoma (HCC) contributes to 14.8% of all cancer mortality in Egypt, which has a high prevalence of hepatitis C virus (HCV). We have previously shown alterations in the insulin-like growth factor-1 (IGF-1) receptor signalling pathway during experimental hepatocarcinogenesis. The aim of this study was to determine whether serum levels of IGF-1, IGF-2 and IGFBP-3 can be used to discriminate between HCC and the stages of hepatic dysfunction in patients with liver cirrhosis assessed by the Child-Pugh (CP) score, and to correlate these levels with HCC stages. We recruited 241 subjects to the present study; 79 with liver cirrhosis, 62 with HCV-induced HCC and 100 age-matched controls. Results showed that serum levels of IGF-1, IGF-2 and IGFBP-3 were reduced significantly in cirrhosis and HCC patients in comparison to the controls, and that this reduction negatively correlated with the CP scores. However, only IGFBP-3 levels showed significant negative correlation with α-f...

Research paper thumbnail of Schizophyllan inhibits the development of mammary and hepatic carcinomas induced by 7,12 dimethylbenz(α)anthracene and decreases cell proliferation: comparison with tamoxifen

Journal of Cancer Research and Clinical Oncology, 2012

Breast cancer is one of the leading causes of cancer mortality among women. Some anticancer compo... more Breast cancer is one of the leading causes of cancer mortality among women. Some anticancer compounds have been isolated from mushrooms. The aim of the present work was to study the anticancer effects of schizophyllan (SCH), a β-D: -glucan extracted from the mushroom Schizophyllum commune alone or in combination with tamoxifen (TAM) on 7, 12 Dimethylbenz(α)anthracene (DMBA)-induced carcinomas in mice. We isolated SCH from S. commune. Female mice received DMBA, SCH, DMBA+SCH, DMBA+TAM or DMBA+TAM+SCH or vehicles. We studied mice survival, tumour incidence, histopathology, oestrogen receptor (ER) expression, cell proliferation by immunohistochemical detection of proliferating cell nuclear antigen (PCNA), apoptosis by TUNEL assay, as well as caspase-3 expression. DMBA treatment resulted in mammary and hepatocellular carcinomas (HCC). Both SCH and TAM reduced the incidence of DMBA-induced mammary tumours by 85 and 75 %, respectively, and equally decreased the PCNA labelling index relative to DMBA. TAM treatment increased the incidence of- and PCNA index in HCCs relative to DMBA, while SCH suppressed these effects. TAM was more effective than SCH in the induction of apoptosis in both mammary and hepatic carcinomas. Caspase-3 levels correlated with the apoptotic index in most experimental groups. Only one dose of SCH had similar therapeutic effects against DMBA-induced mammary carcinomas as 4 weeks of TAM treatment. This coupled with the ability of SCH to suppress hepatic lesions associated with TAM treatment provides the rationale for further investigating the combined therapeutic effects of TAM+SCH in preclinical models of ER-positive breast cancer, as well as in liver cancer.

Research paper thumbnail of Targeting cell cycle regulators in hematologic malignancies

Frontiers in Cell and Developmental Biology, 2015

Hematologic malignancies represent the fourth most frequently diagnosed cancer in economically de... more Hematologic malignancies represent the fourth most frequently diagnosed cancer in economically developed countries. In hematologic malignancies normal hematopoiesis is interrupted by uncontrolled growth of a genetically altered stem or progenitor cell (HSPC) that maintains its ability of self-renewal. Cyclin-dependent kinases (CDKs) not only regulate the mammalian cell cycle, but also influence other vital cellular processes, such as stem cell renewal, differentiation, transcription, epigenetic regulation, apoptosis, and DNA repair. Chromosomal translocations, amplification, overexpression and altered CDK activities have been described in different types of human cancer, which have made them attractive targets for pharmacological inhibition. Mouse models deficient for one or more CDKs have significantly contributed to our current understanding of the physiological functions of CDKs, as well as their roles in human cancer. The present review focuses on selected cell cycle kinases with recent emerging key functions in hematopoiesis and in hematopoietic malignancies, such as CDK6 and its role in MLL-rearranged leukemia and acute lymphocytic leukemia, CDK1 and its regulator WEE-1 in acute myeloid leukemia (AML), and cyclin C/CDK8/CDK19 complexes in T-cell acute lymphocytic leukemia. The knowledge gained from gene knockout experiments in mice of these kinases is also summarized. An overview of compounds targeting these kinases, which are currently in clinical development in various solid tumors and hematopoietic malignances, is presented. These include the CDK4/CDK6 inhibitors (palbociclib, LEE011, LY2835219), pan-CDK inhibitors that target CDK1 (dinaciclib, flavopiridol, AT7519, TG02, P276-00, terampeprocol and RGB 286638) as well as the WEE-1 kinase inhibitor, MK-1775. The advantage of combination therapy of cell cycle inhibitors with conventional chemotherapeutic agents used in the treatment of AML, such as cytarabine, is discussed.

Research paper thumbnail of Detection and quantification of protein phosphatase inhibitor-1 gene expression in total rat liver and isolated hepatocytes

Molecular and cellular biochemistry, 2001

The mRNA expression of protein phosphatase inhibitor-1 (inhibitor-1) in rat liver was demonstrate... more The mRNA expression of protein phosphatase inhibitor-1 (inhibitor-1) in rat liver was demonstrated using highly sensitive semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Quantification by real-time RT-PCR (LightCycler technology) yielded the same copy number of inhibitor-1 mRNA in total rat liver and isolated hepatocytes (12 copies per cell). This novel finding shows that rat liver expresses indeed inhibitor-1 mRNA, albeit in low amounts. The low copy number explains why the mRNA had not been detected by Northern blotting so far. For comparison, about 425 copies/cell were detected in brain and 2500 copies/cell in skeletal muscle from rat. The full-length coding sequence of rat liver inhibitor-1 was cloned and sequenced, 100% homology with the muscle cDNA was obtained, indicating the expression of the same gene in liver and muscle. In vitro transcription and translation yielded a protein (Mr approximately 30 kDa) which could be detected with a specific ant...