RICERCA ITALIANA     

Research program

Myelodysplastic syndromes: pathogenetic models and promise of new therapies

University Co-ordinator

Università Cattolica del Sacro Cuore - Ematologia - MILANO(MI)

Research Unit Leader

Giuseppe LEONE

Description

Object of our study is the evaluation of the methylation status of the promoter region of several genes in myelodysplastic seyndromes and therapy-related AML, also considering the possibility of treatment of these diseases using demethylating agents and hystone-deacetylase inhibitors.

TASK 1
METHYLATION STATUS OF THE PROMOTER REGIONS OF DNA-REPAIR GENES.
Among gene, important for DNA repair, we will study the methylation status of BRCA-1 and MGMT.
Mutations of the BRCA1 gene are known to confer susceptibility to breast and ovarian cancer in high-risk families. Several studies now suggest promoter hypermethylation that results in reduced expression of the gene, may be common to a significant proportion of sporadic breast and ovarian cancers. We will study the methylation status of BRCA-1 in MDS, to evaluate its role as tumor suppressor in these diseases.
MGMT O6-Methylguanine-DNA metiltrasferase (MGMT) is a DNA-reapir enzyme, which transfers O6 methylic and alchilic groups of guanidine to cysteine, reducing the cell damage due to alchilating agents. Promoter CpG island hypermethylation results in the silencing of protein expression, which progress through mytosis and DNA -metiltrasferase. Aberrant DNA methylation may play a significant role in the induction of dysplastic changes in bone marrow progenitors and malignant transformation.

METHYLATION–SPECIFIC PCR. DNA methylation pattern in the CpG island encompassing DAP-kinase promoter will be determined by Methylation Specific PCR (MSP) of bisulfite-modified DNA. Bisulfite treatment induce modification of the unmethylated but not the methylated cytosines to uracil, then primers specific for either methylated or the modified unmethylated DNA will be used in two distinct PCR reactions. Briefly, DNA 2 µg are denatured by NaOH, modified by sodium bisulfite and then purified using a purification column (Amersham Pharmacia Biotech, Uppsala, Sweden), again treated with NaOH, precipitated with ethanol, and resuspended in water.
Methylation data will be correlated to patient characteristics, including age, sex, type of MDS, karyotype and to . The presence of MDS, will be correlated to the evolution of the disease and to treatment response in s-AML.

We will examine bone marrow samples of patients before treatment start. To exclude potential bias in MDS e s-AML subtype distribution and to establish an association with the methylation pattern 150 samples will be collected. The collaboration to this study of the different participating center is necessary to include this number of patients in 2 years. Furthermore we will extend the population study in a retrospective analysis of patients treated in the past, using our DNA bank which contain about 100 MDS and 30 s-AML samples.

BRCA-1 methylation
BRCA1 promoter methylation will be studied by MSP after bisulfite treatment of DNA, as previously described (Esteller et al, 2000). Primer sequences for the unmethylated reaction are 5'-TTG GTT TTT GTG GTA ATG GAA AAG TGT-3' (sense) and 5'-CAA AAA ATC TCA ACA AAC TCA CAC CA-3' (antisense) and for the methylated reaction 5'-TCG TGG TAA CGG AAA AGC GC-3' (sense) and 5'-AAA TCT CAA CGA ACT CAC GCC G-3' (antisense). The unmethylated product is 86 bp long, and the methylated product is 75 bp. BCRA1 mRNA expression will be studied by RT-PCR on the RNA extracted at the time of the diagnosis, as described elsewhere (Kawakami et al, 2003). The following primers, encompassing exons 18 and 23 of BRCA1 will be used: sense 5'-ATG CTG AAT GAG CAT GAT TTT G-3' and antisense 5'-AGA GTG CTA CAC TGT CCA AC-3'. A fragment of 351 bp will be visualized on a ethidium bromide stained 2% agarose gel. Data about BRCA1 expression will be correlated to promoter methylation.
MGMT methylation
MSP for MGMT will be carried out after bisulfite treatment of DNA, according to Esteller et al (2002). Primer sequences for the unmethylated reaction are 5`-TTT GTG TTT TGA TGT TTG TAG GTT TTT GT-3` (forward) and 5`-AAC TCC ACA CTC TTC CAA AAA CAA AAC A-3` (reverse); primer sequences for the methylated reaction are 5`-TTT CGA CGT TCG TAG GTT TTC GC-3` (forward) and 5`-GCA CTC TTC CGA AAA CGA AAC G-3` (reverse).


Task 2
Hupermethylation of angiogenic factors : COX-2 AND THROMBOSPONDIN
It has been shown that angiogenesis plays a pathogenetic role in MDS. Among genes important for angiogenesis, we will study COX-2, which has a pro-angiogenic and anti-apoptotic role, and thrombospondin, which has an anti-angiogenic function.
Cox-2 codes for a ciclo-oxigenase 2 isoform, induced by inflammatory and mitogenic stimula, which determins an increased prostaglandin production in inflammatory and neoplastic tissues. Prostaglandins, PGE2 in particular, may induce angiogenesis stimulating the productoion of pro-angiogenic factors, like endothelial growth factor. Studieremo l'eventuale ruolo della metilazione nella modulazione dell'espressione di COX-2 nelle MDS.
Thrombospondins are a family of inhibitors of angiogenesis, through direct interaction with vascular endothelial growth factor (VEGF), metalloprotease 9 inhibition and endothelial cell migration and apoptosis induction. Several authors have shown that thrombospondin promoter hypermethylation, and reduced protein expression are frequent in several tumors, including neuroblastoma, gastric cancer and carcinoma of the endometrium. Thrombospondin hypermethylation may play a role also in MDS.

COX-2 Hypermethylation
COX-2 promoter hypermethylation will be studied by methylation-specific (MS) PCR, as described (Hoon Kang et al., 2003). Primer sequences for the unmethylated reaction is 5'-ATAGATTAGATATGGTGGTGGTGGT-3' (sense) and 5'-CACAATCTTTACCCAAACACTTCCA-3' (antisense), and for the methylated reaction: 5'-TTAGATACGGCGGCGGCGGC-3' (sense) and 5'-TCTTTACCCGAACGCTTCCG-3' (antisense). The PCR product is 171 bp for unmethylated and 161 bp for methylated.

Thrombospondin Hypermethylation.
Thrombospondin MS-PCR will be performed using the following olifgonucleotides: 5`-GAATGTGAGTGTTTTTTTAAATGTG-3` (forward) and 5`-CCTAAACTCACAAACCAACTCAA-3` (reverse) for the unmethylated reaction; while for the methylated reaction oligos are: 5`-TGCGAGCGTTTTTTTAAATGC-3` (forward) and 5`-TAAACTCGCAAACCAACTCG-3` (reverse).
The unmethylated PCR product is 78 bp, while that of methylated reaction is 74 bp.

FUNCTIONAL STUDIES ON DAP-KINASE HYPERMETHYLATION IN MDS TREATED WITH DEMETHYLATING AGENTS IN-VITRO AND IN VIVO.
Genetic alterations involving regulation of cell-cycle and apoptosis may influence cell survival, cellular response to chemotherapy and ionizing radiations. Dap-kinase is a serin/threonin kinase, regulated by calmodulin, which participates in a wide array of apoptotic systems started by gamma-interferon, TNF-alpha, Fas, and by detachmnet from the cellular matrix. e dal distacco dalla matrice cellulare. We have shown that DAP-kinasi is hypermethylated in almost 50% of MDS and AML, indicating that its functional block could play a pathogenetic role in these diseases. We will study the DAP-kinase methylation status and its functionl effects in primary cells of MDS and s-AML patients , treated by demethylating agents and hystone acetilase inhibitors

EXPERIMENTAL DESIGN
To study the functional role of DAP-kinase promoter methylation on the gene expression, CD34+ cells from AML/MDS patients, that resulted methylated for DAP-Kinase at the MSP and do not express the corresponding mRNA, will be treated with 5-aza-2'deoxycitidine. Cells will be cultured in RPMI1640/10%FBS at different concentration of drug (from 100 nM to 1 microM). RNA will be extracted at different time points and fluorogenic real-time PCR will be performed.
To study the effect of DAP-kinase reinduction on the apoptotic machinery, the percentage of apoptotic cells will be evaluated by Annexin-V-FLUOS Staining Kit (Roche, Mannheim, Germany) in flow cytometry.

Task 3
EVALUATION OF CLONAL EVOLUTION OF DIFFERENT TYPES OF MYELODYSPLASIA AND CORRELATION TO THE METHYLATION PATTERN AND TREATMENT RESPONSE

The purpose of our study will be to evaluate hemopoiesis clonality in patients which will obtain hematological complete remission after chemotherapy. We will study X-CIP of granulocytes from peripheral blood and bone marrow mononuclear cells performing a tecnique that evaluate the polymorphism of the first exon of human androgen receptor gene (HUMARA). To avoid the risk to consider clonal an acquired or constitutive skewing we will study T-lymphocytes as control tissue.

Experimental design
Peripheral blood will be sedimented with Fycoll and T-lymphocytes fraction will be purified from the mononuclear layer using the anti-CD3-fluorescein isothiocyanate-conjugated antibody. Additional red blood cells were removed from the neutrophil fraction by hipotonic lysis. Bone marrow mononuclear cells will be prepared by Fycoll density gradient centrifugation.
High molecular weight genomic DNA will be extracted from each fraction by standard procedure. After extraction DNA will be resuspended in dH2O; half of the sample will be then digested with RsaI e HpaII restriction enzymes. Five microliters of digested DNA will be added to a PCR reaction mix containing 10pmol of each primer (hum1-TCCAGAATCTGTTCCAGAGCGTGC e hum2-GCTGTGAAGGTTGCTGTTCCTCAT) which will amplify the first exon of HUMARA gene. Thereafter the PCR product will be submitted to fragment analysis that will be able to evaluate the clonality of each cell population.