Gene Expression In Leukemia Cells Research Articles

The super elongation complex (SEC) and MLL in development and disease

Transcriptional regulation at the level of elongation is vital for the control of gene expression and metazoan development. The mixed lineage leukemia (MLL) protein and its Drosophila homolog, Trithorax, which exist within COMPASS (complex of proteins associated with Set1)-like complexes, are master regulators of development. They are required for proper homeotic gene expression, in part through methylation of histone H3 on Lys 4. In humans, the MLL gene is involved in a large number of chromosomal translocations that create chimeric proteins, fusing the N terminus of MLL to several proteins that share little sequence similarity. Several frequent translocation partners of MLL were found recently to coexist in a super elongation complex (SEC) that includes known transcription elongation factors such as eleven-nineteen lysine-rich leukemia (ELL) and P-TEFb. Importantly, the SEC is required for HOX gene expression in leukemic cells, suggesting that chromosomal translocations involving MLL could lead to the overexpression of HOX and other genes through the involvement of the SEC. Here, we review the normal developmental roles of MLL and the SEC, and how MLL fusion proteins can mediate leukemogenesis.

Design of Ad5F35 vectors for coordinated dual gene expression in candidate human hematopoietic stem cells

Adenoviral vector-mediated gene expression is an attractive approach to manipulate or report gene expression in human hematopoietic stem cells (HSC) when transient gene expression is preferred. Previous studies have demonstrated that fiber-retargeted Ad5F35 vectors can mediate efficient gene transfer into human HSCs. In this study, we investigated the potential of bi-directional promoter-controlled Ad5F35 vector for coordinated dual gene expression in candidate HSCs. We have engineered Ad5F35-DeltaLNGFR-BiDp encoding kinase domain deleted low-affinity NGF receptor (DeltaLNGFR) and green fluorescent protein (GFP) expression cassette controlled by a synthetic bi-directional promoter, which is composed of human phosphoglycerate kinase promoter and minimal core promoter from human cytomegalovirus. The expression pattern of DeltaLNGFR and GFP following Ad5F35-DeltaLNGFR-BiDp gene transfer in various cell types, including candidate HSCs, was compared to Ad5F35-DeltaLNGFR-IRES vector encoding phosphoglycerate kinase promoter-controlled bicistronic expression cassette for DeltaLNGFR and GFP. Using Ad5F35-DeltaLNGFR-BiDp, we demonstrated a coordinated, high-level dual gene expression in leukemic cells and cord blood CD34(+) cells. However, the ability of Ad5F35-DeltaLNGFR-BiDp-GFP for coordinated dual gene expression varied significantly between repopulating progenitor cells. In nonobese diabetic severe combined immune deficient mice bone marrow transplantation assay, sorted CD34(+)/DeltaLNGFR(+)/GFP(+) cells following infection with Ad5F35-DeltaLNGFR-BiDp showed predominantly myeloid lineage reconstitution with limited lymphoid lineage differentiation capacity, whereas the CD34(+)/DeltaLNGFR(+)/GFP(-) cells exhibited both myeloid and lymphoid reconstitution. This study indicates that bi-directional promoter-controlled Ad5F35 vector, such as Ad5F35-DeltaLNGFR-BiDp, can be particularly useful for manipulation of myeloid progenitor cells and potentially in myeloid lineage leukemic cells as well.

Gene Expression Patterns Associated with Cytarabine Pharmacology and Outcome in Pediatric Acute Myeloid Leukemia.

Abstract 114To identify genes that influence responses to cytarabine (ara-C) treatment, we explored the association of gene expression in leukemic cells at diagnosis with multiple pharmacological and clinical end-points in children with acute myeloid leukemia (AML) treated with ara-C on the St. Jude AML97 clinical trial. We applied a novel statistical procedure, PRojection Onto the Most Interesting Statistical Evidence (PROMISE; PR), to identify genes with expression levels associated with clinical and pharmacological endpoints. To do this, we first defined the following values of the clinical and pharmacological variables as “therapeutically beneficial” :higher leukemic cell ara-C triphosphate levels, lower DNA synthesis values on days 1 and 2 of treatment relative to baseline, decreases in leukocyte counts on day 2 of treatment, improved response and decreased risk of relapse, death, or second malignancy. We considered a gene to show a therapeutically beneficial pattern of association if its expression was positively correlated with ara-CTP levels, negatively correlated with DNA synthesis levels, negatively correlated with decrease in leukocyte counts on day 2, positively correlated with better treatment response, and negativelycorrelated with the risk of relapse or death. A gene showed a therapeutically detrimental pattern of association if its expression had the opposite correlations with the clinical and pharmacological variables. We performed five variable (PR5 using early pharmacologically interesting phenotype measures) or seven variable (PR7 all the above indicated phenotypes) PROMISE analyses. PR5 identified 275 beneficial probe sets and 69 detrimental probe sets (p ≤ 0.005). PR7 analysis identified 112 beneficial probe sets and 115 detrimental probe sets (p ≤ 0.005). To confirm these results, we performed a PROMISE for a cohort of patients treated with ara-C and other agents on the AML02 protocol. Gene expression in leukemic cells at diagnosis was analyzed for a beneficial or detrimental pattern of association with three phenotypes (PR-3); diagnostic blast ara-C cytotoxicity, minimal residual disease (MRD) and event-free survival (EFS). Eighty-one probe sets identified by PR5 or PR7 analyses in the initial cohort were confirmed in the PR-3 analysis of AML02 data. Genes identified in the present study may serve as predictive markers of response and candidates for future drug development. Disclosures:No relevant conflicts of interest to declare.

MLL Fusion Proteins Directly Regulate a Small Set of Wild Type MLL Target Genes.

Abstract 1279Poster Board I-301The MLL (mixed-lineage leukemia) gene at chromosome band 11q23 is rearranged frequently in AML and ALL, and associated with poor prognosis. The consequence of these translocations is the formation of a chimeric oncogenic transcription factor that specifies a unique expression signature distinct from other subtypes of acute leukemia. However, it is poorly understood, which changes in gene expression in leukemic cells are under the direct control of MLL fusion proteins (fusion), nor is it clear what is the potential overlap between MLL wild type (WT) and fusion target genes. In the present study, we used genome-wide location analysis to determine the genomic loci that are specifically bound by MLL fusion proteins. Combining the binding analysis with expression profiling, we further defined the subset of MLL fusion-bound genes whose expression is regulated by the presence of MLL fusion proteins. Using ChIP-chip (Chromatin Immunoprecipitation coupled with micro-array), we determined the MLL-bound regions in 5 myeloid leukemic cell lines using a custom array containing the entire genomic region of 200 genes previously found to have altered expression in MLL-rearranged leukemias. Examination of these 200 genomic loci revealed a largely overlapping set of genes bound by MLL (wild type and/or fusion proteins) in WT/WT (U937: 110 genes, HL60: 79 genes) and WT/Fusion cells (MV4;11: 62 genes, THP-1: 89 genes). Surprisingly, the MLL-bound genes in fusion/fusion (ML-2) cells (25 genes) are a small subset of that found in each of the other 4 cell lines, despite comparable levels of detected MLL binding signal across all lines examined. These data suggest that the MLL fusion protein is likely only localized to a limited portion of genomic loci occupied by the MLL wild type protein. To test this hypothesis in a more systematic way, we examined an inducible MLL-ENL–ER transformed cell line (Slany et al, MCB 2004), which grow as myeloblastic cells in the presence of MLL-ENL, and differentiate into neutrophils upon inactivation of the fusion protein. As MLL-ENL promotes histone H3 lysine 79 (H3K79) methylation, we determined both MLL binding and H3K79 methylation using a genome-wide location analysis. We anticipated that MLL-fusion bound genomic regions would exhibit a significant drop in either MLL and/or K79 signal upon inactivation of MLL-ENL. Unexpectedly, among thousands of genes that are bound by MLL, only 10% of them (222 genes) showed a pattern of binding increase between MLL-ENL induced and un-induced conditions. To explore the impact of MLL fusion protein on gene activation, we performed whole genome expression profiling in the presence or absence of MLL-ENL. Increased levels of either MLL binding or H3K79 methylation are significantly associated with differential gene expression. Among 222 MLL fusion target genes, 12 of them are differentially up-regulated in the presence of MLL-ENL, indicating that a large fraction of MLL fusion bound genes do not exhibit significant changes in mRNA expression. The identified 12 genes include key regulators in cellular differentiation and cell cycle regulation, as well as Meis1, Hoxa9 which are known to be essential for the development of MLL leukemia. To explore the apparent discrepancy between the massive expression changes in MLL rearranged leukemia and the small number of direct fusion target genes we identified, we tested the hypothesis that a significant portion of the MLL fusion protein expression signature was derived from its direct fusion target genes Meis1 and Hoxa9. Using publicly available data, we compared the MLL leukemia associated expression profile with the set of genes that were down-regulated upon knock-down of Meis1 and Hoxa9. We found significant enrichment of Hoxa9/Meis1 downstream targets in the expression profile defined by MLL fusion proteins. Altogether, our data suggest that MLL fusion proteins are likely to contribute to the development of acute leukemia through direct activation of a very small set of genes. The results have important implications in understanding the mechanisms of target gene specificity involving oncogenic transcription factors. DisclosuresNo relevant conflicts of interest to declare.

A Genomic Approach to Improve Prognosis and Predict Therapeutic Response in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy characterized by a variable clinical course. Several parameters have prognostic capabilities but are associated with altered response to therapy in only a small subset of patients. We used gene expression profiling methods to generate predictors of therapy response and prognosis. Genomic signatures that reflect progressive disease and responses to chemotherapy or chemoimmunotherapy were created using cancer cell lines and patient leukemia cell samples. We validated and applied these three signatures to independent clinical data from four cohorts, representing a total of 301 CLL patients. A genomic signature of prognosis created from patient leukemic cell gene expression data coupled with clinical parameters significantly differentiated patients with stable disease from those with progressive disease in the training data set. The progression signature was validated in two independent data sets, showing a capacity to accurately identify patients at risk for progressive disease. In addition, genomic signatures that predict response to chlorambucil or pentostatin, cyclophosphamide, and rituximab were generated and could accurately distinguish responding and nonresponding CLL patients. Thus, microarray analysis of CLL lymphocytes can be used to refine prognosis and predict response to different therapies. These results have implications for standard and investigational therapeutics in CLL patients.

Experimental identification of drugs with function of targeted up-regulating ID4 expression: bioinformatics-based prediction and preliminary validation

To screen new candidate molecular-targeted anti-leukemia compounds with potential functions of targeted up-regulating ID4 gene expression. Promoter region of ID4 gene including the upstream - 3000 bp sequence of transcriptional start site and message RNA sequence were fished out. Online promoter analysis tools of TESS and Genomax were used to search possible sequence of transcriptional start site and message RNA sequence were fished out. Online promoter analysis tools of TESS and Genomax were used to search possible cis-acting structure from human transcription factor database. The activity of related drugs with potential effects upon ID4 gene expression was analyzed using SAGE database. GEO database was applied to search the gene expression profiling regulated by ID4 gene. Finally, similar analysis between gene expression profiling by ID4 and genome-wide profiling regulated by 163 known drugs or active compounds was manipulated to screen the drugs and candidate compounds with similar gene expression profiling with ID4 gene. MOLT4 cell line was treated with the above candidate active compounds to investigate the ID4 gene expression by RT-PCR assay. ID4 gene had a type II promoter with a typical TATA box in upstream -45 bp of transcription start site. The 1300 bp-length promoter of ID4 gene contained a few cis-acting structures classified into two function types, i. e. positive regulatory type, including transcription factors Spl and c-Myb, cAMP, glucocorticoid receptor (GR) and estrogen receptor (ER), and negative regulatory type, including Wilms tumor-1 (WT1) and early growth response-2 (EGR2). The similarity of gene expression profiling was identified between cAMP and ID4 gene. ID4 gene expression was induced in MOLT4 cell line after treatment with calcium dibutyryladenosine cyclophosphate at the concentration of 0.1 mmol/L. The comprehensive bioinformatic analysis, based upon the combination of regulatory sequence prediction of promoter, similarity analysis of gene expression profiling and literature review, can be considered as a practical tool in screening the candidate drugs with the activity of targeted regulating functional genes. Calcium dibutyryladenosine cyclophosphate can induce ID4 gene expression in leukemic cells.

PU.1 Epigenetically Represses the Expression of Metallothionein-1 Gene in Leukemia Cells

Recently, it was reported that reduced level of PU.1 expression developed acute myeloid leukemia (AML) in mice. To clarify the mechanisms of AML, first we established PU.1 knockdown K562 (K562PU.1KD) cells, that express reduced level of PU.1 by stably transfected PU.1 siRNA. Flow cytometric analysis demonstrated that among other lineage markers, expressions of myeloid CD13/CD33 markers are significantly decreased in K562PU.1KD cells compared to control cells. We next tried to identify PU.1 target genes to elucidate the mechanisms of AML. To solve this, microarray and real time PCR analyses were performed. We revealed that several genes including metallothionein (MT) s (MT-1G, MT-1A) were markedly induced in K562PU.1KD cells. MTs are a group of low-molecular weight, cysteine rich intracellular proteins. A number of studies have shown an increased expression of MTs in various human solid tumors. To verify the relevance, we established PU.1 over-expressed K562 (K562PU.1OE) cells and found the significant suppression of these genes. Furthermore, we revealed a negative correlation between PU.1 and MTs mRNA expression in 43 primary AML specimens (MT-1G; R=−0.50, p<0.001, MT-1A; R=−0.57, p<0.0005). These results suggest that MTs are bona fide targets of PU.1. To clarify MTs regulation by PU.1, chromatin immunoprecipitation assay was employed and demonstrated an increased association of acetylated histone H3 and H4 with the MT-1G and MT-1A promoter in K562PU.1KD cells, compared to control cells. As there are 38 CpG sites in approximately −430 to +170 bp from transcription start site in MT-1G and MT-1A promoter, we next performed bisulfite DNA sequencing analysis to examine the methylation status of these CpG sites. We revealed that the proportion of the methylated CpG sites in MT- 1G, as well as MT-1A promoter were significantly decreased in K562PU.1KD cells (MT- 1G: control cells, 29–41% vs K562PU.1KD cells, 16–29%; MT-1A: control cells, 12% vs K562PU.1KD cells, 4%). In contrast, methylations of these sites were markedly increased in K562PU.1OE cells (MT-1G: control cells, 36% vs K562PU.1OE cells, 59%; MT-1A: control cells, 12% vs K562PU.1OE cells, 18%). These suggest that downregulation of PU.1 leads to the impairment of the recruitment of DNA methyltransferase (DNMT) and histone deacetylase, results in the aberrant gene expression in leukemia cells. Therefore, we next hypothesized that there is some specific effect of epigenetic drugs on PU.1 transgenic cells. From WST assay and trypan blue dye exclusion assay, we observed that K562PU.1KD cells were resistant to DNMT inhibitor 5-aza-2′-deoxycytidine (5-Azadc). Viable cells were approximately 20% increased in K562PU.1KD cells in the presence of 5-Azadc, and 20% decreased in K562PU.1OE cells compared to control cells respectively. These suggest that expression of PU.1, MTs and proportion of methylated CpG sites in MTs' promoter may useful therapeutic marker to predict the effect of 5-Azadc on leukemia cells.

Concordant Gene Expression in Leukemia Cells and Normal Leukocytes Is Associated with Germline cis-SNPs

The degree to which gene expression covaries between different primary tissues within an individual is not well defined. We hypothesized that expression that is concordant across tissues is more likely influenced by genetic variability than gene expression which is discordant between tissues. We quantified expression of 11,873 genes in paired samples of primary leukemia cells and normal leukocytes from 92 patients with acute lymphoblastic leukemia (ALL). Genetic variation at >500,000 single nucleotide polymorphisms (SNPs) was also assessed. The expression of only 176/11,783 (1.5%) genes was correlated (p<0.008, FDR = 25%) in the two tissue types, but expression of a high proportion (20 of these 176 genes) was significantly related to cis-SNP genotypes (adjusted p<0.05). In an independent set of 134 patients with ALL, 14 of these 20 genes were validated as having expression related to cis-SNPs, as were 9 of 20 genes in a second validation set of HapMap cell lines. Genes whose expression was concordant among tissue types were more likely to be associated with germline cis-SNPs than genes with discordant expression in these tissues; genes affected were involved in housekeeping functions (GSTM2, GAPDH and NCOR1) and purine metabolism.

Thymosin β4 and AcSDKP inhibit the proliferation of HL‐60 cells and induce their differentiation and apoptosis

Our previous works have shown that bone marrow stromal cells secrete thymosin beta4 (Tbeta4) and AcSDKP. Tbeta4 and AcSDKP are existed in the conditioned medium of bone marrow endothelial cells. They exerted inhibitory effects on hematopoietic cells and then had protective effect on the early hematopoietic cells, which were cultured in the presence of hematopoietic stimulators. Thymosin beta4 consists of 43 peptides with a molecular weight of 4963. It contains at its N-terminal end the sequence of the acetylated tetrapeptide Ac-N-Ser-Asp-Lys-Pro (AcSDKP). This study was performed to evaluate the effect of Tbeta4 and AcSDKP on the growth of HL-60 cells. It was showed that Tbeta4 (10(-11)-10(-7)mol/L) and AcSDKP (10(-11)-10(-7)mol/L) had the dose-dependent inhibitory effect on the proliferation of HL-60 cells. Based on cell morphology and NBT reduction, Tbeta4 and AcSDKP induced differentiation of HL-60 cells. Morphologic and DNA fragment analysis proved that Tbeta4 and AcSDKP induced apoptosis of HL-60 cells. In order to analyze the mechanism of the effects of Tbeta4 and AcSDKP, intracellular free Ca(2+) concentration ([Ca(2+)](i)) of HL-60 leukemic cells was tested and Atlas cDNA Expression Array was performed. The results showed that Tbeta4 and AcSDKP could increased [Ca(2+)](i) by stimulating the release of Ca(2+) from intracellular Ca(2+) pool. Moreover, AcSDKP could also elicit a potent extracelluar calcium influx in HL-60 cells. Tbeta4 could also change apoptotic-related gene expression in leukemic cells, and resulted in the inhibition of proliferation and induction of differentiation and apoptosis of leukemic cells.

Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region

Although the bcr-abl translocation has been shown to be the causative genetic aberration in chronic myeloid leukemia (CML), there is mounting evidence that the deregulation of other genes, such as the transcription factor interferon regulatory factor 4 (IRF-4), is also implicated in the pathogenesis of CML. Promoter methylation of CpG target sites or direct deletions/insertions of genes are mechanisms of a reversible or permanent silencing of gene expression, respectively. Therefore, we investigated whether IRF-4 promoter methylation or mutation may be involved in the regulation of IRF-4 expression in leukemia cells. Whereas promoter mutations or structural rearrangements could be excluded as a cause of altered IRF-4 expression in hematopoietic cells, the IRF-4 promoter methylation status was found to significantly influence IRF-4 transcription. First, treatment of IRF-4-negative lymphoid, myeloid and monocytic cell lines with the methylation-inhibitor 5-aza-2-deoxycytidine resulted in a time- and concentration-dependent increase of IRF-4 mRNA and protein levels. Second, using a restriction-PCR-assay and bisulfite-sequencing we identified specifically methylated CpG sites in IRF-4-negative but not in IRF-4-positive cells. Third, we clearly determined promoter methylation as a mechanism for IRF-4 down-regulation via reporter gene assays, but did not detect an association of methylational status and mRNA expression of DNA methyltransferases or methyl-CpG-binding proteins. Together, these data suggest CpG site-specific IRF-4 promoter methylation as a putative mechanism of down-regulated IRF-4 expression in leukemia.

Impact of molecular profiling and cytogenetics in acute lymphoblastic leukemia

"Impact of molecular profiling and cytogenetics in acute lymphoblastic leukemia." Hematology, 10(sup1), pp. 176–177

Genetics and molecular biology of chronic lymphocytic leukemia

B cell chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by accumulation of malignant CD5+ B cells. Multiple molecular events likely contribute to malignant transformation; no single genetic abnormality or event has been shown to be responsible for development of the disease. Significant advances have recently been made towards understanding the genetic and molecular basis for the etiology and clinical course of CLL. Our current understanding is only now bringing us to the point where we can use this information in management and in developing new therapies for patients with CLL. Familial clustering of CLL cases is not uncommon and implicates a genetic basis for the development of the disease in some individuals. Potential interventions in this instance could employ strategies of gene transfer or gene therapy to correct genetic defects or strategies of chemoprevention, none of which is currently under investigation. Greater potential for therapeutic intervention rests with targeting molecular aberrations and altered gene expression in leukemia cells, for example, over expression of the anti-apoptotic proteins of the Bcl-2 family. CLL follows a variable clinical course, with some patients not needing treatment for many years and responding to therapy completely and repeatedly. Other patients have rapidly progressive disease that is refractory to currently available agents and they quickly succumb to their disease. One major recent advance has been the identification of molecular and genetic prognostic factors that can be used in early-stage patients to identify those likely to rapidly progress. This affords the opportunity to tailor management for patients based on the predictable aggressiveness of their disease. Molecular and genetic findings are increasingly influencing management decisions in CLL. Bone marrow transplantation may be considered for a patient with unfavorable prognostic features earlier than for a patient with favorable features and same clinical stage of disease. It is likely that these genetic and molecular-based factors will be targets of new treatment modalities that fundamentally change the management of this disease. In this review we detail the current understanding of the genetics and molecular biology of CLL and introduce potentials for therapeutic intervention.

Differential Gene Expression in Leukemic Cells from Bone Marrow (BM) and Peripheral Blood (PB) of Pediatric Patients with Acute Lymphoblastic Leukemia (ALL).

Although the genetic mechanisms underlying the induction of ALL are similar, the molecular events leading to diverse clinical presentation and course are not well characterized. High PB white blood cell (WBC) counts reflect tumor burden in PB and have been used in the past as a clinical risk factor in ALL. We hypothesized that, given differential microenvironment signaling provided by BM and PB, ALLs with high or low WBC counts (WBChigh and WBClow) might differ with regard to their dependence on the microenvironment. To approach this issue, we investigated gene expression changes in PB blasts as compared to their BM counterparts in patients with precursor B-cell ALL (n=15). Blasts were isolated from BM and PB samples by flow sorting and investigated for mRNA levels using Affymetrix HG U133A microarrays. Gene expression data, normalized by variance stabilization on probe level, were analysed in matched PB-BM pairs using a SAM-like analysis of log ratios in a balanced permutation test. By these procedures, a limited number of 38 genes consistently changed in PB vs BM blasts was identified (false discovery rate < 0.01). Leukemic PB cells were characterized by upregulation of genes encoding for cell adhesion- and trafficking-related proteins (CD11b, CD73, S100A4, EMP3). Furthermore, downregulation of the cell cycle- and proliferation-associated genes (thymidylate synthetase TYMS, kinesin KIF11, topoisomerase TOP2A, ribonucleotide reductase RRM2, kinetochore protein ZWINT) indicated decreased proliferative activity of leukemic cells in PB microenvironment. Finally, the gene encoding for Bcl-2 protein, known to be a powerful downstream mediator of survival signaling from microenvironment in various experimental settings, was consistently downregulated in PB as compared to BM blasts. Within the group of WBClow patients (n=8, mean WBC=17x103/μl), most of these genes retained their statistical significance. By contrast, no consistent expression changes in PB vs BM blasts were found in WBChigh patients (n=7, mean WBC=201x103/μl). In summary, investigation of differential gene expression in leukemic cells from PB and BM indicated decreased levels of proliferative and survival signaling in the PB microenvironment and a higher dependence on this signaling of leukemic cells from WBClow as compared to WBChigh patients.

Genetic Polymorphisms in the Promoter Region of the beta-2 Adrenergic Receptor Are Associated with the Early Response of Acute Lymphoblastic Leukemia to Chemotherapy.

Acute lymphoblastic leukemia (ALL) in children is a paradigm of disseminated cancer that is curable with chemotherapy, yet current treatment fails to cure about 20% of patients, for reasons that remain unknown. In a genome-wide assessment of in vivo treatment-induced changes in gene expression in ALL cells using the Affymetrix U95A and U133A oligonucleotide microarray, we found that patients who eventually relapsed did not up-regulate expression of the pro-apoptotic beta-2 adrenergic receptor gene (ADRB2) in their ALL cells after initial treatment with methotrexate and mercaptopurine. After treatment we observed a 5-fold lower level of ADRB2 gene expression in leukemia cells of patients who ultimately relapsed. We found a common genetic polymorphism in the ADRB2 promoter that was significantly linked to high-dose methotrexate induced up-regulation in ADRB2 gene expression in ALL cells. Moreover, the ADRB2 promoter haplotype was significantly linked to poor early treatment response in ALL cells from 242 children (i.e., probability of event-free survival at two years, p=0.0275 stratified by risk groups). These findings have revealed a germline polymorphism that is linked to the early antileukemic effects of ALL chemotherapy and provide new insights into genetic determinants of ALL treatment efficacy.

Detection of an acquired mutation in an HLA-A∗03 allele impairing gene expression in leukemia cells of a patient with all

Detection of an acquired mutation in an HLA-A∗03 allele impairing gene expression in leukemia cells of a patient with all

Genetics of childhood acute lymphoblastic leukemia

Genetic alterations that underlie the pathogenesis of individual leukemia subtypes have important prognostic implications in acute lymphoblastic leukemia (ALL). Heretofore, specific genetic abnormalities with prognostic relevance were identified mainly by cytogenetic analysis or reverse-transcriptase polymerase-chain-reaction, and were largely limited to B-lineage ALL cases. Moreover, there is heterogeneity among specific genetic subtypes of ALL and treatment has over-riding impact on the prognostic information conveyed by the genetic abnormalities. In Philadelphia chromosome-positive (Ph+) ALL, an age of 1 to 9 years and a low initial leukocyte count confer a favorable prognosis. In each category of 11q23/MLL abnormality, infants less than 1 year of age have a worse treatment outcome than do older patients. Allogeneic transplantation with HLA-matched related donor improved outcome for patients with Ph+ ALL but not those with the t(4;11) andMLL-AF4 fusion. Using oligonucleotide microarrays, T-cell ALL can now be classified into four major subtypes with prognostic significance:MLL-ENL andHOX11 clusters with favorable prognosis andTAL1 andLYL1 clusters with inferior outcome. In our recent study of 327 ALL cases, distinct expression profiles could not only accurately identify each of the known prognostically important leukemia subtypes, but also individual cases that would eventually relapse or develop therapy-related acute myeloid leukemia. We have also identified a novel ALL subgroup with unique expression profile. Examination of the genes comprising the different expression signatures provided important insights into the biologic differences between the leukemia subgroups. The importance of host pharmacogenetics has also been recognized. Thus, genetic polymorphisms of certain drug-metabolizing enzymes, drug transporters or drug receptors have been linked with treatment efficacy and toxicity, as well as host susceptibility to the development of de novo leukemia or therapy-related second cancers. The best-characterized genes (e.g. thiopurine methyltransferase, cytochrome P450, glutathione transferase, methylenetetrahydrofolate reductase and NAD(P)H: quinone oxidoreductase) will be discussed. Ultimately, patients will be treated with individualized therapy based on leukemic cell gene expression profile and host pharmacogenetics.

G6PD activity and gene expression in leukemic cells from G6PD-deficient subjects

In the present study we examined gene expression and glucose-6-phosphate dehydrogenase (G6PD) activity in leukemic cells isolated from G6PD normal and deficient subjects. The results have shown that G6PD activity strongly increases in G6PD normal leukemic cells as well as in G6PD deficient leukemic cells when compared to peripheral blood mononuclear cells (PBMC). Higher levels of G6PD gene expression were observed in leukemic cells from G6PD deficient patients compared to G6PD normal. A similar pattern of gene expression was also observed for 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase. These results support the hypothesis that G6PD deficient cell, in order to sustain their growth, must respond to the low activity of their mutant enzyme with an increase in quantity through an induction of gene expression.

Regulation of low Km (ecto-) 5'-nucleotidase gene expression in leukemic cells.

Ecto-5’-NT has been considered a biochemical marker in leukemias and lymphomas (1). The enzyme activity is low in lymphocytes (2) and decreased in B-cells in patients with X-linked agammaglobulinemia (3), decreased or not detectable in lymphocytes from chronic lymphocytic leukemia and infectious mononucleosis (4) and decreased in CD8+ lymphocytes in AIDS (5). In general, low activity has been observed during development and in undifferentiated cells which increases markedly in some cell types at the final stages of differentiation and during senescence and aging. Although there are some data showing correlation of low ecto-5’-NT activity with efficacy of nucleoside analogues of anti-cancer activity, no specific role which would explain the pattern of distribution of this enzyme has been proposed.

Thymosin β4 Gene Expression in Leukemic Cells.

Thymosin β4 (Tβ4) was originally isolated as a thymic hormone from calf thymosin fraction 5 which exhibited both immune and endocrine functions in vivo and in vitro. Tβ4 is a ubiquitous peptide located in various tissues of mammalian species and other vertebrate classes. Recent studies on the molecular cloning and sequence analysis of rat and human Tβ4 cDNA have demonstrated that Tβ4 lacks a signal peptide which makes it unlikely that Tβ4 is a secretory peptide. The real function of Tβ4 is presently unknown, however, we focus this review on the molecular biology of Tβ4 with a special reference to the aspects of the Tβ4 gene expression in leukemic cells and cell lines during growth and differentiation.

The c-sis gene expression in cells from a patient with acute megakaryoblastic leukemia and Down's syndrome

The c-sis gene expression in leukemia cells from a patient with acute megakaryoblastic leukemia and Down's syndrome was studied. The leukemia blasts were identified as megakaryoblasts by the platelet peroxidase reaction and the reactivity against antiplatelet monoclonal antibodies. Leukemia cells obtained from peripheral blood or bone marrow specimens before and after initiation of chemotherapy were analyzed for c-sis gene expression by the RNA-DNA dot blot hybridization. Although the level varied, the mRNA of the c-sis gene was detected in all megakaryoblastic leukemia cells obtained at different clinical stages. The c-sis mRNA level in cells obtained after relapse was higher than that obtained before the initiation of therapy. The 25S c-sis mRNA was detected in a megakaryoblastic leukemia cell line established from this patient. The role of the expression of the c-sis gene in acute megakaryoblastic leukemia cells is discussed.