N-3 PUFAs modulate global gene expression profile in cultured rat cardiomyocytes. Implications in cardiac hypertrophy and heart failure.

In cardiac cells the effects of n-3 PUFAs on the whole genome are still unknown despite their recognized cardioprotective effects and ability to modulate gene expression. We have evaluated the effects of n-3 PUFAs supplementation on the global gene expression profile in cultured neonatal rat cardiomyocytes, detecting many genes related to lipid transport and metabolism among the upregulated ones. Many of the downregulated genes appeared related to inflammation, cell growth, extracellular and cardiac matrix remodelling, calcium movements and ROS generation. Our data allow to speculate that the cardioprotective effect of n-3 PUFAs is related to a direct modulation of genes in cardiac cells.

Hh/Gli antagonist in acute myeloid leukemia with CBFA2T3-GLIS2 fusion gene.

BACKGROUND: CBFA2T3-GLIS2 is a fusion gene found in 17% of non-Down syndrome acute megakaryoblastic leukemia (non-DS AMKL, FAB M7) and in 8% of pediatric cytogenetically normal acute myeloid leukemia (CN-AML, in association with several French-American-British (FAB) subtypes). Children with AML harboring this aberration have a poor outcome, regardless of the FAB subtype. This fusion gene drives a peculiar expression pattern and leads to overexpression of some of Hedgehog-related genes. GLI-similar protein 2 (GLIS2) is closely related to the GLI family, the final effectors of classic Hedgehog pathway. These observations lend compelling support to the application of GLI inhibitors in the treatment of AML with the aberration CBFA2T3-GLIS2. GANT61 is, nowadays, the most potent inhibitor of GLI family proteins. METHODS: We exposed to GANT61 AML cell lines and primary cells positive and negative for CBFA2T3-GLIS2 and analyzed the effect on cellular viability, induction of apoptosis, cell cycle, and expression profile. RESULTS: As compared to AML cells without GLIS2 fusion, GANT61 exposure resulted in higher sensitivity of both cell lines and primary AML cells carrying CBFA2T3-GLIS2 to undergo apoptosis and G1 cell cycle arrest. Remarkably, gene expression studies demonstrated downregulation of GLIS2-specific signature genes in both treated cell lines and primary cells, in comparison with untreated cells. Moreover, chromatin immunoprecipitation analysis revealed direct regulation by GLIS2 chimeric protein of DNMT1 and DNMT3B, two genes implicated in important epigenetic functions. CONCLUSIONS: Our findings indicate that the GLI inhibitor GANT61 may be used to specifically target the CBFA2T3-GLIS2 fusion gene in pediatric AML.

Down-regulation of MLL-AF9, MLL and MYC expression is not obligatory for monocyte-macrophage maturation in AML-M5 cell lines carrying t(9;11)(p22;q23).

The MLL-AF9 oncogene originates from the translocation t(9;11)(p22;q23), which is mainly associated with monocytic acute myeloid leukaemia (AML-M5; FAB-classification). In AML-M5 THP-1 cells carrying t(9;11) (p22;q23) and expressing MLL-AF9, we previously showed that MLL-AF9 expression is down-regulated during monocyte-macrophage maturation. We have subsequently observed that in a 'rapid-growing' variant of the THP-1 cell line (THP-1-R) MLL-AF9 down-regulation does not occur. MLL fusion proteins (including MLL-AF9) deregulate MYC transactivation activity, and both presence and absence of MYC down-regulation have been reported during monocyte-macrophage maturation in THP-1 cells. In the present study, we analyze the expression patterns of MLL-AF9, MLL wild-type and MYC after induction of monocyte-macrophage terminal differentiation in the AML-M5 cell lines, THP-1, THP-1-R, Mono-Mac-6 (MM6) and MOLM-13, all of which carry t(9;11)(p22;q23) and express MLL-AF9. RT-PCR analysis indicated that down-regulation of MLL-AF9, MLL or MYC is not necessary to abolish malignant phenotypes by induction of terminal monocyte-macrophage differentiation in leukaemic cells carrying t(9;11)(p22;q23).

Anti-gene peptide nucleic acid specifically inhibits MYCN expression in human neuroblastoma cells leading to cell growth inhibition and apoptosis.

We developed an anti-gene peptide nucleic acid (PNA) for selective inhibition of MYCN transcription in neuroblastoma cells, targeted against a unique sequence in the antisense DNA strand of exon 2 of MYCN and linked at its NH(2) terminus to a nuclear localization signal peptide. Fluorescence microscopy showed specific nuclear delivery of the PNA in six human neuroblastoma cell lines: GI-LI-N and IMR-32 (MYCN-amplified/overexpressed); SJ-N-KP and NB-100 (MYCN-unamplified/low-expressed); and GI-CA-N and GI-ME-N (MYCN-unamplified/unexpressed). Antiproliferative effects were observable at 24 hours (GI-LI-N, 60%; IMR-32, 70%) and peaked at 72 hours (GI-LI-N, 80%; IMR-32, 90%; SK-N-KP, 60%; NB-100, 50%); no reduction was recorded for GI-CA-N and GI-ME-N (controls). In MYCN-amplified/overexpressed IMR-32 cells and MYCN-unamplified/low-expressed SJ-N-KP cells, inhibition was recorded of MYCN mRNA (by real-time PCR) and N-Myc (Western blotting); these inhibitory effects increased over 3 days after single treatment in IMR-32. Anti-gene PNA induced G(1)-phase accumulation (39-53%) in IMR-32 and apoptosis (56% annexin V-positive cells at 24 hours in IMR-32 and 22% annexin V-positive cells at 48 hours in SJ-N-KP). Selective activity of the PNA was shown by altering three point mutations, and by the observation that an anti-gene PNA targeted against the noncoding DNA strand did not exert any effect. These findings could encourage research into development of an anti-gene PNA-based tumor-specific agent for neuroblastoma (and other neoplasms) with MYCN expression.

MLL-AF9 oncogene expression affects cell growth but not terminal differentiation and is downregulated during monocyte-macrophage maturation in AML-M5 THP-1 cells.

The MLL-AF9 oncogene - one of the most frequent MLL/HRX/ALL-1 rearrangements found in infantile and therapy-related leukaemias - originates from t(9;11)(p22;q23) and is mainly associated with monocytic acute myeloid leukaemia (AML-M5; FAB-classification). Here, we investigated the MLL-AF9 function by means of an antisense phosphorothioate-oligodeoxyribonucleotide (MLL-AF9-PS-ODNas) using the THP-1 AML-M5 cell line carrying t(9;11). Having confirmed that MLL-AF9-PS-ODNas induces strong inhibition of THP-1 cell growth, but only a moderate increase in apoptosis, we found that MLL-AF9-PS-ODNas did not induce morpho-functional terminal differentiation or restore M-CSF-, G-CSF- or GM-CSF-induced differentiation. Moreover, THP-1 cells showed the same phenotype with/without MLL-AF9-PS-ODNas. In THP-1 cells differentiated to mature macrophage-like cells by PMA/TPA or ATRA, MLL-AF9 expression was downregulated. Thus, in the monocytic lineage, MLL-AF9 may be expressed only in early phases and can induce deregulated amplification in both nonmalignant and malignant cells, maintaining the monocytic phenotype without blocking final maturation. Our findings suggest that: (1) as well as directly promoting cell growth, MLL-AF9 may also indirectly determine phenotype; (2) other leukaemogenic mutations associated with MLL-AF9-related leukaemias should be searched for mainly in processes of resistance to apoptosis (where MLL-AF9 may play only a limited role) and differentiation blockage (where MLL-AF9 may play no role).

Targeted inhibition of NMYC by peptide nucleic acid in N-myc amplified human neuroblastoma cells: cell-cycle inhibition with induction of neuronal cell differentiation and apoptosis.

We developed an antisense peptide nucleic acid (PNA) targeted against a unique sequence in the terminus of the 5'-UTR of N-myc, designed for selective inhibition of NMYC in neuroblastoma cells. Fluorescent microscopy showed carrier-free delivery of the PNA to two human neuro-blastoma cell lines: GI-LI-N (N-myc-amplified) and GI-CA-N (N-myc-unamplified). Only in the former, PNA treatment determined 70% cell-viability reduction (at 48 h). In N-myc-amplified GI-LI-N cells, the PNA determined NMYC-translation inhibition (Western blotting), accumulation of cells in G1, induction of differentiation and apoptosis. Selectivity of the PNA was demonstrated by altering three point mutations. These findings should encourage development of a PNA-based tumor-specific agent for neuroblastoma (or other neoplasms) with N-myc overexpression.

Transient abnormal myelopoiesis in a phenotypically normal newborn with polyclonal trisomy 21.

We report a rare case of transient abnormal myelopoiesis (TAM) in a phenotypically normal neonate. The presence of a palpable hepatomegaly prompted in-depth laboratory tests, which revealed the presence of severe hyperleukocytosis, with blast cells present in a peripheral blood smear. Although no signs of Down syndrome were present, we suspected TAM. Further analysis identified a mutation in GATA1 along with the unique finding of two different trisomic cell lines, detected upon karyotyping; one with trisomy 21 only, and one with trisomies 21 and 22, which was present in a subpopulation of peripheral blood cells. These genetic abnormalities disappeared by the age of 6 months. The presence of two different trisomic clones may be an evidence of the polyclonal nature of TAM in this patient.

MYCN is a novel oncogenic target in pediatric T-cell acute lymphoblastic leukemia.

MYCN is an oncogene frequently overexpressed in pediatric solid tumors whereas few evidences suggest his involvement in the pathogenesis of haematologic malignancies. Here we show that MYCN is overexpressed in a relevant proportion (40 to 50%) of adult and pediatric T-cell acute lymphoblastic leukemias (T-ALL). Focusing on pediatric T-ALL, MYCN-expressing samples were found almost exclusively in the TAL1-positive subgroup. Moreover, TAL1 knockdown in T-ALL cell lines resulted in a reduction of MYCN expression, and TAL1 directly binds to MYCN promoter region, suggesting that TAL1 pathway activation could sustain the up-regulation of MYCN. The role of MYCN in T-ALL was investigated by peptide nucleic acid (PNA-MYCN)-mediated transcriptional silencing of MYCN and by siRNAs. MYCN knockdown in T-ALL cell lines resulted in a reduction of cell viability, up to 50%, while no effect was elicited with a mismatch PNA. The inhibitory effect of PNA-MYCN on cell viability was due to a significant increase in apoptosis. PNA-MYCN treatment in pediatric T-ALL samples reduced cell viability of leukemic cells from patients with high MYCN expression, while no effect was obtained in MYCN-negative blast cells. These results showed that MYCN is frequently overexpressed in pediatric T-ALL and suggested his role as a candidate for molecularly-directed therapies.

Antitumor activity of sustained N-myc reduction in rhabdomyosarcomas and transcriptional block by antigene therapy.

PURPOSE: Rhabdomyosarcomas are a major cause of cancer death in children, described with MYCN amplification and, in the alveolar subtype, transcription driven by the PAX3-FOXO1 fusion protein. Our aim was to determine the prevalence of N-Myc protein expression and the potential therapeutic effects of reducing expression in rhabdomyosarcomas, including use of an antigene strategy that inhibits transcription. EXPERIMENTAL DESIGN: Protein expression was assessed by immunohistochemistry. MYCN expression was reduced in representative cell lines by RNA interference and an antigene peptide nucleic acid (PNA) oligonucleotide conjugated to a nuclear localization signal peptide. Associated gene expression changes, cell viability, and apoptosis were analyzed in vitro. As a paradigm for antigene therapy, the effects of systemic treatment of mice with rhabdomyosarcoma cell line xenografts were determined. RESULTS: High N-Myc levels were significantly associated with genomic amplification, presence of the PAX3/7-FOXO1 fusion genes, and proliferative capacity. Sustained reduction of N-Myc levels in all rhabdomyosarcoma cell lines that express the protein decreased cell proliferation and increased apoptosis. Positive feedback was shown to regulate PAX3-FOXO1 and N-Myc levels in the alveolar subtype that critically decrease PAX3-FOXO1 levels on reducing N-Myc. Pharmacologic systemic administration of the antigene PNA can eliminate alveolar rhabdomyosarcoma xenografts in mice, without relapse or toxicity. CONCLUSION: N-Myc, with its restricted expression in non-fetal tissues, is a therapeutic target to treat rhabdomyosarcomas, and blocking gene transcription using antigene oligonucleotide strategies has therapeutic potential in the treatment of cancer and other diseases that has not been previously realized in vivo.

HLA-mismatched hematopoietic stem cell tranplantation for pediatric solid tumors.

Even if the overall survival of children with cancer is significantly improved over these decades, the cure rate of high-risk pediatric solid tumors such as neuroblastoma, Ewing's sarcoma family tumors or rhabdomiosarcoma remain challenging. Autologous hematopoietic stem cell transplantation (HSCT) allows chemotherapy dose intensification beyond marrow tolerance and has become a fundamental tool in the multimodal therapeutical approach of these patients. Anyway this procedure does not allow to these children an event-free survival approaching more than 50% at 5 years. New concepts of allogeneic HSCT and in particular HLA-mismatched HSCT for high risk solid tumors do not rely on escalation of chemotherapy intensity and tumor load reduction but rather on a graft-versus-tumor effect. We here report an experimental study design of HLA-mismatched HSCT for the treatment of pediatric solid tumors and the inherent preliminary results.