MicroRNA miR-181a correlates with morphological sub-class of acute myeloid leukaemia and the expression of its target genes in global genome-wide analysis.

MicroRNAs (miRNAs) are short single-stranded RNAs that have a potentially important role in gene regulation. Using a quantitative real-time polymerase chain reaction assay specific to the mature miRNA, the expression level of a selected group of haematopoietic tissue-specific miRNAs was measured across a set of 30 primary adult acute myeloid leukaemia (AML) with a normal karyotype. The expression levels of each miRNA were correlated with the genome-wide mRNA expression profiles in the same leukaemias. This revealed that miR-181a correlated strongly with the AML morphological sub-type and with the expression of genes previously identified through sequence analysis as potential interaction targets. Three other miRNAs, miR-10a, miR-10b and miR-196a-1, showed a clear correlation with HOX gene expression.

siRNA-mediated AML1/MTG8 depletion affects differentiation and proliferation-associated gene expression in t(8;21)-positive cell lines and primary AML blasts.

The chromosomal translocation t(8;21) is associated with 10-15% of all cases of acute myeloid leukaemia (AML). The resultant fusion protein AML1/MTG8 interferes with haematopoietic gene expression and is an important regulator of leukaemogenesis. We studied the effects of small interfering RNA (siRNA)-mediated AML1/MTG8 depletion on global gene expression in t(8;21)-positive leukaemic cell lines and in primary AML blasts using cDNA arrays, oligonucleotide arrays and real-time reverse transcription-polymerase chain reaction (RT-PCR). Suppression of AML1/MTG8 results in the increased expression of genes associated with myeloid differentiation, such as AZU1, BPI, CTSG, LYZ and RNASE2 as well as of antiproliferative genes such as IGFBP7, MS4A3 and SLA both in blasts and in cell lines. Furthermore, expression levels of several genes affiliated with drug resistance or indicative of poor prognosis AML (BAALC, CD34, PRG2, TSPAN7) are affected by AML1/MTG8 depletion. In conclusion, siRNA-mediated suppression of AML1/MTG8 cause very similar changes in gene expression pattern in t(8;21)-positive cell lines and in primary AML blasts. Furthermore, the results suggest that the specific targeting of AML1/MTG8 function may be a promising approach for complementing existing treatment strategies.

Histone acetylation-mediated regulation of genes in leukaemic cells.

Histone deacetylase (HDAC) and histone acetyltransferase (HAT) functions are associated with various cancers, and the inhibition of HDAC has been found to arrest disease progression. Here, we have investigated the gene expression profiles of leukaemic cells in response to the HDAC inhibitor trichostatin A (TSA) using oligonucleotide microarrays. Nucleosomal histone acetylation was monitored in parallel and the expression profiles of selected genes were confirmed by quantitative polymerase chain reaction (PCR). A large number of genes (9% of the genome) were found to be similarly regulated in CCRF-CEM and HL-60 cells in response to TSA, and genes showing primary and secondary responses could be distinguished by temporal analysis of gene expression. A small fraction of genes were highly sensitive to histone hyper-acetylation, including XRCC1, HOXB6, CDK10, MYC, MYB, NMI and CBFA2T3 and many were trans-acting factors relevant to cancer. The most rapidly repressed gene was MKRN3, an imprinted gene involved in the Prader-Willi syndrome.

The synovial sarcoma associated protein SYT interacts with the acute leukemia associated protein AF10.

As a result of the synovial sarcoma associated t(X;18) translocation, the human SYT gene on chromosome 18 is fused to either the SSX1 or the SSX2 gene on the X chromosome. Although preliminary evidence indicates that the (fusion) proteins encoded by these genes may play a role in transcriptional regulation, little is known about their exact function. We set out to isolate interacting proteins through yeast two hybrid screening of a human cDNA library using SYT as a bait. Of the positive clones isolated, two were found to correspond to the acute leukemia t(10;11) associated AF10 gene, a fusion partner of MLL. Confirmation of these results was obtained via co-immunoprecipitation of endogenous and exogenous, epitope-tagged, SYT and AF10 proteins from cell line extracts and colocalization of epitope-tagged SYT and AF10 proteins in transfected cells. Subsequent sequential mutation analysis revealed a highly specific interaction of N-terminal SYT fragments with C-terminal AF10 fragments. The N-terminal interaction domain of the SYT protein was also found to be present in several SYT orthologs and homologs. The C-terminal interaction domain of AF10 is located outside known functional domains. Based on these results, a model is proposed in which the SYT and AF10 proteins act in concert as bipartite transcription factors. This model has implications for the molecular mechanisms underlying the development of both human synovial sarcomas and acute leukemias.

Identification and molecular characterisation of a CALM-AF10 fusion in acute megakaryoblastic leukaemia.

The t(10;11)(p13;q14-21) is a non-random translocation described in acute lymphoblastic and myeloid leukaemias. It results in the fusion of the gene CALM, which encodes a clathrin assembly protein, on 11q14 to the gene AF10, a putative transcription factor on 10p13. Here we describe for the first time, the occurrence of a CALM-AF10 fusion in a case of acute megakaryoblastic leukaemia. Fluorescence in situ hybridisation and reverse transcriptase polymerase chain reaction were used to confirm the presence of a CALM-AF10 fusion. A novel splice variant of CALM missing nt 1927-2091 was also detected. Though CALM is a cytoplasmic protein, the chimaeric fusion product is able to localise to both the nucleus and cytoplasm. Analysis of the fusion variants suggests, however, that the critical fusion product is likely to be cytoplasmic and contain the interactive leucine zipper of AF10.

Molecular analysis of the genomic inversion and insertion of AF10 into MLL suggests a single-step event.

The interstitial insertion of genetic material from one chromosome into another can achieve the type of gene-gene fusions more usually associated with chromosome translocations. An example of such an interstitial insertion, which has created an MLL-AF10 fusion in an acute myeloid leukaemia, has been analysed at the genomic level. The genomic fusion, which resulted in the juxtaposition of 3' AF10 sequence to 5' MLL sequence, was identified within MLL and AF10 intronic sequences. It was further established that the remaining 3' MLL sequence, from exon 6 onwards, was fused to novel sequence of unknown origin (named FM3 for fused to MLL 3'). The points of fusion of these 5' and 3' portions of MLL matched to adjacent nucleotides and lay between exons 5 and 6. The FM3 sequence was shown to be from chromosome arm 10p and located close to AF10 in a proximal position. It was subsequently demonstrated that in the leukaemia a third fusion existed between 5' AF10 and the FM3 sequence at a point immediately downstream from its fusion to MLL. It was therefore concluded that the MLL-AF10 gene fusion is the result of a simultaneous transposition of genetic material into the MLL gene and the joining of the remaining free ends on chromosome 10. This kind of event, characterised completely here for the first time, is a means to achieve a fusion when the genes involved lie in opposite orientations and results in three genomic junctions.

pRb and Cdk regulation by N-(4-hydroxyphenyl)retinamide.

The cancer chemopreventive synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) can inhibit the growth and induce apoptosis of tumor cells. In this study we analysed the growth suppressive effect of HPR on human breast cancer cell lines in vitro and the role of the retinoblastoma protein (pRb) in this response. Treatment of MCF7, T47D and SKBR3 for 24 - 48 h with 3 microM HPR, a concentration attainable in vivo, resulted in growth inhibition and marked dephosphorylation of pRb involving Ser612, Thr821, Ser795 and Ser780, target residues for cyclin-dependent kinase 2 (Cdk2) the former two, and Cdk4 the latter two. Interestingly, this dephosphorylation of pRb occurred in S-G2-M phase cells, as revealed by experiments on cells fractionated by FACS according to the cell cycle phase, hence suggesting that the retinoid interferes with the regulation of pRb phosphorylation. The in vitro phosphorylation of a GST-pRb recombinant substrate by Cdk2 immunocomplexes from MCF7, T47D and SKBR3 was markedly suppressed after HPR treatment, whereas that by Cdk4 complexes was suppressed in T47D and SKBR3 but not in MCF7. The steady-state levels of Cdk2, Cdk4 and Cyclin A proteins were unaffected by HPR, while those of Cyclin D1 were significantly reduced in all three cell lines. Interestingly, Cyclin D1 downregulation by HPR correlated with transcriptional repression, but not with enhanced proteolysis of Cyclin D1 typically elicited by other retinoids. Collectively, our data suggest that the antiproliferative activity of HPR arises from its capacity to maintain pRb in a de-phosphorylated growth-suppressive status in S-G2/M, possibly through Cyclin D1 downregulation and inhibition of pRb-targeting Cdks. Oncogene (2000) 19, 4035 - 41.

Biochemical analyses of the AF10 protein: the extended LAP/PHD-finger mediates oligomerisation.

Leukaemogenesis correlates with alterations in chromatin structure brought about by the gain or loss of interactive domains from regulatory factors that are disrupted by chromosomal translocations. The gene MLL, a target of such translocation events, forms chimaeric fusion products with a variety of partner genes. While MLL appears to be involved in chromatin-mediated gene regulation, the functions of its partner genes are largely speculative. We report the biochemical analysis of the MLL partner gene AF10 and its possible role in leukaemogenesis. AF10 has been reported to be re-arranged with genes other than MLL leading to the same phenotype, a myeloid leukaemia. We have identified a novel protein-protein interaction motif in the AF10 protein comprising the extended LAP/PHD-finger. This domain mediates homo-oligomerisation of recombinant AF10 and is conserved in several proteins, including MLL itself. AF10 binds cruciform DNA via a specific interaction with an AT-hook motif and is localised to the nucleus by a defined bipartite nuclear localisation signal in the N-terminal region.

Identification of a novel human kinesin-related gene (HK2) by the cDNA differential display technique.

We have used the cDNA differential display technique to isolate genes regulated by the synthetic retinoid N-(4-hydroxyphenyl)-all-trans-retinamide (HPR), a cancer chemopreventive agent in vivo and a powerful inducer of apoptotic cell death in vitro. Here we report the identification of a novel gene, the expression of which is markedly up-regulated in tumor cells after treatment for 30-60 min with HPR. The full-length cDNA of this gene, determined by screening of a human placenta cDNA, is 3.5 kb long and contains an open reading frame of 2037 nt. The gene is > 90% homologous to the mouse KIF2, a gene belonging to the family of kinesin-related motor proteins, and we therefore named it HK2 (human kinesin 2). A shorter form of the HK2 mRNA (HK2s), containing a 57-nt deletion in the open reading frame, has also been detected. Northern analysis revealed that HK2 is widely expressed among hemopoietic and nonhemopoietic cell lines and tissues. By the use of radiation hybrids, HK2 has been localized to chromosome 5q12-q13. Kinesins constitute a superfamily of motor proteins that use energy liberated from ATP hydrolysis to move cargo along microtubules and are implicated in mechanisms of mitosis or meiosis. The role of HK2 in the growth-inhibitory and apoptotic responses elicited by HPR remains to be established.

Multiple G6PD mutations are associated with a clinical and biochemical phenotype similar to that of G6PD Mediterranean.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common red cell abnormalities, is characterized by a wide clinical, biochemical, and molecular heterogeneity. In this study we have determined the molecular basis of G6PD deficiency in a sample of 70 male subjects, originating from different parts of Italy, who all shared a clinical and biochemical phenotype identical or very similar to that of G6PD Mediterranean, the most common variant in Italy. In 59 cases (84%) we found the mutation 563 C --> T, previously known to be underlying the G6PD Mediterranean and the two polymorphic variants G6PD Cagliari and G6PD Sassari. From the remaining 11 we amplified the entire coding region of G6PD in 8 different fragments and subjected them to nonradioactive single-strand conformation analysis. Direct sequencing was then performed on abnormal fragments. By this approach we found six cases (8.5%) with 1360 G --> A mutation (G6PD Union) and two cases (2.8%) with 1376 G --> C (G6PD Cosenza). In the remaining three samples we found two other mutations: 1342 A --> G (two cases, 2.8%) and 1052 G --> T (one case, 1.4%). These two molecular defects have never been described before and were designated by us as G6PD S. Antioco and G6PD Partenope, respectively. Haplotype analysis suggested that all the non-Mediterranean mutations occurred independently on a normal G6PD allele. This study shows that the G6PD Union mutation has a high polymorphic frequency in the Italian population and that the genetic heterogeneity of G6PD Mediterranean-like variants is higher at the molecular level than expected from biochemical characterization.

Solid phase purification and SSCP analysis of amplified genomic DNA by capillary electrophoresis.

Detection and identification of point mutations in genomic DNA has proven increasingly important in biomedical research. A variety of methods for the analysis of single base substitutions have been proposed among which Single Strand Conformational Polymorphism (SSCP) quickly gained success due to its simplicity. In this work we present an analytical on-line tool which combines the ease of solid phase purification of amplified genomic DNA, the simplicity of SSCP and the significant potential advantages offered by capillary electrophoresis (CE).

Anion-exchange HPLC analysis of biotinylated oligonucleotides.

Biotinylated oligonucleotides combined with streptavidin-coated magnetic beads are commonly used in current molecular biology. Their quality and the level of incorporated biotin are essential for yielding good results in either solid-phase DNA sequencing or solid-phase purification procedures. This paper presents a very simple analytical test using anion-exchange HPLC and avidin to ascertain the quality of biotinylated oligonucleotides and to predetermine their ability to bind to avidin, which is a prerequisite for functionality in some solid-phase methods.

Fluorescence-based automated DNA sequencing by limited primer labeling.

The applicability of automated DNA sequencing systems to sequencing strategies that require a large number of primers is limited by the necessity for expensive fluorescence-labeled oligonucleotides. Here we present a simple procedure that allows the use of unlabeled oligonucleotides to perform fluorescence-based DNA sequencing. This method is based on a limited primer extension that incorporates three deoxynucleotides, one of which carries a fluorescent moiety. The elongated fluorescent primer is then used in a standard T7 sequencing reaction. This labeling procedure is both economical and straightforward and offers a valid alternative to current fluorescence-labeling protocols. Results of this method with different DNA templates demonstrate the reliability of the protocol.

Molecular characterisation of an Italian G6PD variant responsible for chronic non-spherocytic haemolytic anaemia.

An Italian deficient G6PD variant associated with chronic non-spherocytic haemolytic anaemia (CNSHA) was biochemically characterised and studied at molecular level. Single-strand conformation polymorphism (SSCP) analysis led to the identification of an abnormal migration pattern of an amplified fragment encompassing exons 10 and 11 of the G6PD gene. Sequence analysis of both strands using an automated fluorescent DNA sequencer revealed a G-->A transition at nt. position 1246 in exon 10. A C-->T substitution at nt. 1311 in exon 11 was also found, which has already been described as a silent mutation common in Caucasians. The 1246 G-->A mutation has been described only in a Japanese subject with CNSHA (G6PD Tokyo) not associated with the 1311T polymorphism, suggesting that this mutation may have arisen independently in Europe and Asia.

Mixed-mode fluorescent DNA sequencing.

Fluorescence-based, automated DNA sequences represent one of the major advances in recent molecular biology. Two main technologies have been developed in this field: the single-label/four-lane system and the four-label/one-lane system. The following present the use of single-label-sequencing chemistry, which resembles traditional radioactive DNA sequencing, using the four-label system ABI 373A that expands its flexibility and obtains data that are immediately interpretable without software manipulation. This method has been named mixed-mode fluorescent DNA sequencing. Here we show one of its possible applications in molecular genetic analysis.

Introduction of magnetic beads in diagnosis: a simple and rapid method to detect mutations of beta globin gene, directly amplified from blood.

In this communication we present the results obtained by the use of magnetic beads in diagnosis, for the identification of genetic variants at the molecular level by sequencing, in comparison with the more laborious method of the production of ssDNA with asymmetric PCR. We compared the two techniques studying variants of beta globin gene: Hb Abruzzo [beta 143 (H21) His -> Arg] and Hb D Los Angeles [beta 121 (GH4) Glu -> Gln].