Improved Detection of preclinical Colorectal Liver Metastases by High Resolution Ultrasound including Molecular Ultrasound Imaging using the targeted Contrast Agent BR55.

PURPOSE: Aim of the present study was to investigate the sensitivity of high resolution ultrasound (HRU), standard contrast-enhanced ultrasound (CEUS) and CEUS using a novel vascular endothelial growth factor receptor 2 (VEGFR2)-targeted contrast agent for the detection of hepatic metastases in a mouse model of colorectal cancer using clinical standard technology. MATERIALS AND METHODS: The human colon cancer cell line HT29, transfected with luciferase cDNA for in vivo bioluminescence monitoring, was injected intrasplenically into CB17.SCID mice. Mice were monitored weekly by bioluminescence and after 2 and 4.5 weeks by HRU and CEUS. Contrast media (untargeted BR1, targeted BR55) was applied and digital cine loops from the arterial phase (15 - 45 sec), portal venous phase (50 - 120 s) and late phases (3 - 5 min, 1hour) of the whole liver were analyzed. Data were correlated with postmortem histopathology. RESULTS: Without contrast enhancement, lesions > 4 mm were reliably detected. After use of untargeted CEUS, lesions > 2 mm were reliably detected and enhanced rim vascularization and late-phase wash-out was shown. With BR55, lesions > 0.8 mm were reliably detected with excellent documentation of vascularization. A persistent contrast enhancement was seen > 30 min after injection. Contrast-enhancement patterns with BR55 significantly correlated with CD31 (R2 = 0.74) and VEGFR2-immunohistochemistry (R2 = 0.66). CONCLUSION: Detection of metastases by HRU and CEUS was earlier and more accurate than monitoring via bioluminescence. In vivo monitoring of hepatic micrometastases can thus be performed without prior modification of cancer cells using standard technology.

Oviposition behaviour of Gryon gallardoi (Hym.; Scelionidae) on eggs of Spartocera dentiventris (Hem.; Coreidae).

The oviposition behaviour of Gryon gallardoi (Hymenoptera; Scelionidae) on Spartocera dentiventris (Hemiptera; Coreidae) host eggs was investigated in the laboratory. Masses of 12 non-parasitized freshly laid (less than 24 h old) eggs were exposed to 2-5 days old mated females with previous oviposition experience (n = 10). Behaviour was observed for 2 h under the stereomicroscope. The eggs were Then kept individually at 25 degrees +/- 1 degree C/12 h photophase till hatching. The mean number of parasitized eggs was 7.8 +/- 0.81 (mean +/- SE). Five distinct kinds of behaviour were observed: drumming with antennae on the eggs, ovipositor insertion, egg marking, walking and resting. On average, ovipositor insertion was not followed by marking 4.3 +/- 0.76 times per female. In nearly all of these events, parasitism was unsuccessful. Walking and resting were observed less frequently than the other behaviours (1.6 +/- 0.56 and 2.1 +/- 0.48 times/female, respectively). Superparasitism occurred on average 3.6 +/- 0.88 times per egg mass, with 2.7 +/- 0.57 eggs being superparasitized. Among these, on average 87.4 +/- 5.37% led to successful development of an adult parasitoid. The average time spent on the each kind of oviposition behaviour was 1.5 +/- 0.57 min for drumming, 3.9 +/- 0.56 min for ovipositor insertion and 0.4 +/- 0.06 min for marking. There was no significant variation on the duration of each behaviour as the parasitoid progressed in parasitizing an egg mass. Ovipositor insertion almost always (87.58%) occurred in the longitudinal extremities of the egg. In average 31.1 +/- 7.21% of the individual emerging per egg mass were males, the larger proportion of males originating from the 2nd oviposition. The results show a range of oviposition behaviours common to the Scelionidae family. Egg marking behaviour was a good indicator of the effective oviposition by females. Superparasitism is only partially avoided, but its occurrence does not imply a failure of parasitoid emergence. The sex ratio is skewed towards females, and most males come from the first ovipositions.

Oviposition behaviour of Gryon gallardoi (Hym. Scelionidae) on eggs of Spartocera dentiventris (Hem. Coreidae)

The oviposition behaviour of Gryon gallardoi (Hymenoptera; Scelionidae) on Spartocera dentiventris (Hemiptera; Coreidae) host eggs was investigated in the laboratory. Masses of 12 non-parasitized freshly laid (less than 24 h old) eggs were exposed to 2-5 days old mated females with previous oviposition experience (n = 10). Behaviour was observed for 2 h under the stereomicroscope. The eggs were Then kept individually at 25 + or -1C/12 h photophase till hatching. The mean number of parasitized eggs was 7.8 + or - 0.81 (IMG01 + or - SE). Five distinct kinds of behaviour were observed: drumming with antennae on the eggs, ovipositor insertion, egg marking, walking and resting. On average, ovipositor insertion was not followed by marking 4.3 + or -imes per female. In nearly all of these events, parasitism was unsuccessful. Walking and resting were observed less frequently than the other behaviours (1.6 + or - 0.56 and 2.1 + or 0.48 times/female, respectively). Superparasitism occurred on average 3.6 + or - 0.88 times per egg mass, with 2.7 + or - 0.57 eggs being superparasitized. Among these, on average 87.4 + or - 5.37 percent led to successful development of an adult parasitoid. The average time spent on the each kind of oviposition behaviour was 1.5 + or - 0.57 min for drumming, 3.9 + or - 0.56 min for ovipositor insertion and 0.4 + or - 0.06 min for marking. There was no significant variation on the duration of each behaviour as the parasitoid progressed in parasitizing an egg mass. Ovipositor insertion almost always (87.58 percent) occurred in the longitudinal extremities of the egg. In average 31.1 + or - 7.21 percent of the individual emerging per egg mass were males, the larger proportion of males originating from the 2nd oviposition. The results show a range of oviposition behaviours common to the Scelionidae family. Egg marking behaviour was a good indicator of the effective oviposition by females. Superparasitism is only partially avoided, but its occurrence does not imply a failure of parasitoid emergence. The sex ratio is skewed towards females, and most males come from the first ovipositions

Recruitment of the nuclear receptor corepressor N-CoR by the TEL moiety of the childhood leukemia-associated TEL-AML1 oncoprotein.

The t(12;21)(p13;q22) chromosomal translocation is the most frequent illegitimate gene recombination in a pediatric cancer and occurs in approximately 25% of common acute lymphoblastic leukemia (cALL) cases. This rearrangement results in the in frame fusion of the 5'-region of the ETS-related gene, TEL (ETV6), to almost the entire acute myeloid leukemia 1 (AML1) (also called CBFA2 or PEBP2AB1) locus and expression of the TEL-AML1 chimeric protein. Although AML1 stimulates transcription, TEL-AML1 functions as a repressor of some AML1 target genes. In contrast to the wild type AML1 protein, both TEL and TEL-AML1 interact with N-CoR, a component of the nuclear receptor corepressor complex with histone deacetylase activity. The interaction between TEL and N-CoR requires the central region of TEL, which is retained in TEL-AML1, and TEL lacking this domain is impaired in transcriptional repression. Taken together, our results suggest that TEL-AML1 may contribute to leukemogenesis by recruiting N-CoR to AML1 target genes and thus imposing an altered pattern of their expression.

Expression and protein-binding studies of the EEN gene family, new interacting partners for dynamin, synaptojanin and huntingtin proteins.

EEN, identified initially as a fusion partner to the mixed-lineage leukaemia gene in human leukaemia, and its related members, EEN-B1 and EEN-B2, have recently been shown to interact with two endocytic molecules, dynamin and synaptojanin, as well as with the huntingtin protein. In the present study, we show that the expression of the EEN gene-family members is differentially regulated. Multiple-spliced variants were identified for EEN-B2. In the brain, EEN-B1 and EEN-B2 mRNA are preferentially expressed in the cerebellar Purkinje and granule cells, dentate gyrus cells, hippocampal pyramidal neurons and cerebral granule cells. The expression patterns of EEN-B1 and EEN-B2 mRNA in the brain overlap with those of dynamin-I/III, synaptojanin-I and huntingtin, whereas the ubiquitous expression of EEN is consistent with that of dynamin-II. In testes, members of the EEN family are co-expressed with testis-type dynamin and huntingtin in Sertoli cells and germ cells respectively. Our results on the overlapping expression patterns are consistent with the proposed interaction of EEN family members with dynamin, synaptojanin and huntingtin protein in vivo. Although all three EEN family members bind to dynamin and synaptojanin, EEN-B1 has the highest affinity for binding, followed by EEN and EEN-B2. We also demonstrate that amphiphysin, a major synaptojanin-binding protein in brain, can compete with the EEN family for binding to synaptojanin and dynamin. We propose that recruitment of the EEN family by dynamin/synaptojanin to clathrin-coated pits can be regulated by amphiphysin.

The leukaemic oncoproteins Bcr-Abl and Tel-Abl (ETV6/Abl) have altered substrate preferences and activate similar intracellular signalling pathways.

Inappropriate activation of Abl family kinases plays a crucial role in different human leukaemias. In addition to the well known oncoproteins p190Bcr-Abl and p210Bcr-Abl, Tel-Abl, a novel fusion protein resulting from a different chromosomal translocation, has recently been described. In this study, the kinase specificities of the Bcr-Abl and Tel-Abl proteins were compared to the physiological Abl family kinases c-Abl and Arg (abl related gene). Using short peptides which correspond to the target epitopes in known substrate proteins of Abl family kinases, we found a higher catalytic promiscuity of Bcr-Abl and Tel-Abl. Similar to Bcr-Abl, Tel-Abl was found in complexes with the adapter protein CRKL. In addition, c-Crk II and CRKL are tyrosine phosphorylated and complexed with numerous other tyrosine phosphorylated proteins in Tel-Abl expressing Ba/F3 cells. GTPase analysis with a Ras-GTP-specific precipitation assay showed constitutive elevation of GTP-loaded Ras in cells expressing the leukaemic Abl proteins. The mitogenic MAPK/Erk kinases as well as Akt/PKB, a kinase implicated to negatively regulate apoptosis, were also constitutively activated by both Bcr-Abl and Tel-Abl. The results indicate that the leukaemic Abl-fusion proteins have catalytic specificities different from the normal kinases c-Abl and Arg and that Tel-Abl is capable to activate at least some pathways which are also upregulated by Bcr-Abl.

Analysis of the region of the 5' end of the MLL gene involved in genomic duplication events.

Rearrangements of the MLL gene are associated with both myeloid and lymphoid acute leukaemia. The gene is commonly involved in reciprocal translocations leading to the creation of chimaeric genes encoding novel protein products. An alternative mechanism of MLL gene rearrangement is due to intragenic duplication, leading to partial duplication of the amino-terminal portion of the protein. This occurs in leukaemia, but it has recently been shown that partial duplications of the MLL gene are detectable in peripheral blood and bone marrow of healthy donors and in normal non-haemopoietic tissues. Sequence analysis of the 45 kb of the 5' end of the MLL locus encompassing the breakpoints of these genomic duplications has failed to show a definitive reason as to why this region is such a frequent target of rearrangement. Indeed, although the majority of the breakpoint joins are the result of apparent Alu-mediated homologous recombination, several joins do not involve Alu elements in the region, despite a high density of these repetitive elements in the sequence.

MLL2, the second human homolog of the Drosophila trithorax gene, maps to 19q13.1 and is amplified in solid tumor cell lines.

The Mixed Lineage Leukemia (MLL) gene is commonly involved in translocations in infantile leukemia and is amplified in some cases of adult myeloid leukemia. A homolog of MLL denoted MLL2, which represents the second human homolog of the Drosophila trithorax gene, was characterized by assembling ESTs, the KIAA0304 cDNA clone, RT - PCR fragments and a new clone isolated from a cDNA phage library and compared to the available genomic sequence. The MLL2 gene maps to 19q13.1, a region of frequent rearrangement or amplification in solid tumors. MLL2 consists of an 8.5 - 9 kb transcript and spans 20 kb of genomic DNA. The predicted MLL2 protein possesses all of the major domains defined in MLL and the two genes have a similar genomic structure. We find that MLL2 is amplified in two of 14 pancreatic carcinoma cell lines and one of five glioblastoma cell lines and is a likely critical gene in 19q13.1 amplifications. It is also a candidate for chromosomal rearrangements involving this chromosome locus. MLL2 is one additional mammalian trithorax-group gene with involvement in human cancer.

Haemopoietic transformation by the TEL/ABL oncogene.

Rare, novel forms of activated ABL kinase, the result of a fusion between TEL (or ETV6, a member of the ETS transcription factor family), and the non-receptor tyrosine kinase ABL, have been identified. We have analysed the TEL/ABL fusion protein (type A) cloned from an acute lymphoblastic leukaemia patient. In contrast to a second TEL/ABL fusion (type B) identified in two cases of myeloid leukaemia, the portion of TEL contained in the type A TEL/ABL fusion was smaller and did not contain a potential Grb2 binding site. The type A TEL/ABL cDNA we used in this study encoded a 155 kD protein with elevated tyrosine kinase activity and was responsible for the phosphorylation of a number of proteins in vivo. Its expression in factor-dependent murine haemopoietic precursor cells efficiently converted these cells to factor independence for both survival and growth. These cells continued to express high levels of myc mRNA after growth factor depletion. We also demonstrated that type A TEL/ABL self-associated in stably expressing haemopoietic cells. Although the TEL portion of the TEL/ABL fusion protein has no sequence similarity to that of BCR in the BCR/ABL protein, all forms of these fusion proteins contain a structure implicated in oligomerization. Our results support the conclusion that the protein interaction domain of BCR and TEL, but not the Grb2 binding site, are the important functional components in the activation of ABL kinase in haemopoietic discase.

Isolation and characterization of a pufferfish MLL (mixed lineage leukemia)-like gene (fMll) reveals evolutionary conservation in vertebrate genes related to Drosophila trithorax.

The MLL gene is interrupted and fused to a number of partner genes as a result of chromosomal translocations in human leukemias. MLL is a very large protein with a unique domain structure and large regions of homology to Drosophila trx. To define the key structural and functional domains of the MLL protein in vertebrates, we have cloned the genomic region encoding an MLL-like gene in the compact model vertebrate genome of Fugu rubripes. While the similarity between the mouse and human MLL proteins is very high, a lower overall similarity is present between the Fugu and mammalian proteins. Several new highly conserved regions were identified in the portion of the protein included in the MLL leukemia-associated fusion proteins. The conserved nature of regions of similarity between vertebrate forms of MLL and the Drosophila TRX proteins, as well as other domains previously suggested to have a functional role in MLL (including the AT hooks and the DNA methyltransferase domain), was also observed. Therefore, strong evolutionary constraints limited sequence divergence within these domains. The information derived from this comparative analysis will form the basis for the functional study of the MLL protein, particularly as it relates to human leukemogenesis.

The second ETV6 allele is not necessarily deleted in acute leukemias with a ETV6/ABL fusion.

The ETV6 (TEL) locus at chromosome band 12p 13 is a major site of translocations in acute leukemia, particularly in childhood acute lymphoblastic leukemia (ALL). In cases with translocations involving ETV6, the normal ETV6 allele is often deleted. In addition, loss of heterozygosity of ETV6 is frequently observed in childhood'ALL. Thus, it has been suggested that ETV6 may have an anti-oncogenic role to play, in addition to its oncogenic role. We have described an unusual case of ALL in which ETV6 is found fused to the ABL gene; ABL is normally activated by fusion to the BCR gene in the 9:22 translocation. We expanded the primary cells from this ETV6/ABL rearranged case of ALL in SCID animals and analyzed them for expression of both ETV6/ABL and the normal ETV6 mRNA. We found that both the rearranged and normal ETV6 mRNAs are expressed in the expanded cell population. Furthermore, sequence analysis of the ETV6 PCR product revealed no point mutations which would influence the amino acid sequence. Thus, deletion of the second ETV6 allele is not necessary for the transformation to leukemia by ETV6/ABL.

Exon scrambling of MLL transcripts occur commonly and mimic partial genomic duplication of the gene.

The MLL gene is frequently rearranged in acute human leukemia of both the myeloid and lymphoid lineages. Using a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we identified several abnormally spliced transcripts in which MLL exons were joined in an order different from the genomic orientation (scrambled exons). Mis-splicing of MLL was present in both normal and malignant tissues. Although the majority of these scrambled transcripts were joined accurately at consensus splice sites, there were several examples in which the junctions of exons spliced in aberrant order were at non-consensus sites. A number of features differentiate mis-splicing of MLL from the previously described cases of scrambled exons and circular RNAs. Some scrambled transcripts appear to be present in the polyadenylated fraction of RNA. No correlation of exon scrambling with exon skipping was found, and there was no particular tendency for the exons involved to be near large introns. Our data show that splicing of MLL is extremely complex. The presence of scrambled transcripts in both normal and leukemic cells, indistinguishable from transcripts resulting from genomic MLL rearrangements, precludes the use of nested RT-PCR as a screening method for detection of tandem duplication of tandem duplication of MLL.

EEN encodes for a member of a new family of proteins containing an Src homology 3 domain and is the third gene located on chromosome 19p13 that fuses to MLL in human leukemia.

The MLL gene, the closest human homologue to the Drosophila trithorax gene, undergoes chromosomal translocation with a large number of different partner genes in both acute lymphoid and acute myeloid leukemias. We have identified a new partner gene, EEN, fused to MLL in a case of acute myeloid leukemia. The gene is located on chromosome 19p13, where two other MLL partner genes, ENL and ELL/MEN have also been identified. The deduced protein of 368 aa contains a central alpha-helical region and a C-terminal Src homology 3 (SH3) domain most similar to the C-terminal SH3 domain found in the Grb2/Sem-5/Drk family of genes. Sequence analysis of the fusion MLL/EEN transcript in our patient reveals that exon 6 of MLL is fused to the N-terminal end of EEN, a fusion that would create a chimeric protein that includes the major functional domain of EEN. EEN is expressed in a variety of tissue types and encodes a protein of approximately 46 kDa. The EEN protein is the human homologue of a member of a recently described murine SH3 domain-containing protein family. It is also highly related to a putative gene identified in Caenorhabditis elegans, and a number of similar sequences are present in the EST databases of several species.

MLL self fusion mediated by Alu repeat homologous recombination and prognosis of AML-M4/M5 subtypes.

Fifty-six patients with de novo acute myeloid leukemia M4/M5 subtypes were studied for rearrangements of the mixed lineage leukemia gene, MLL (also called HRX, Htrx-1, or ALL-1). Ten patients (18%) showed rearrangements of the MLL gene, 9 in a major breakpoint cluster region within a centromeric 8.3-kb BamHI fragment, whereas rearrangement in one patient was the result of a direct tandem duplication of exons 2-6 of MLL. Analysis of sequences at the duplication junction revealed that the points of MLL fusion within introns 6 and 1 both lie within Alu elements. This suggests the involvement of Alu repeat mediated homologous recombination in MLL self fusion. For the 10 rearranged samples, cytogenetics analysis revealed a normal karyotype in 3, and 3 had abnormalities other than 11q23. Survival analysis of patients revealed no difference between those with rearrangement of MLL and those showing the germ-line configuration.

ts BCR-ABL kinase activation confers increased resistance to genotoxic damage via cell cycle block.

Using a temperature-sensitive mutant of the p210 BCR-ABL gene, transfected into a growth factor-dependent cell line (BaF3), we show that transient BCR-ABL kinase expression increases single cell and clonogenic resistance to apoptosis arising from genotoxic damage induced by ionizing radiation and VP-16/etoposide. This effect is achieved in the absence of any detectable changes in the levels of BCL-2, BAX or BCL-x proteins and is independent of proliferative, MAP kinase-dependent effects of BCR-ABL kinase. In contrast to parental cells that transiently arrest in G2 and then apoptose, p210 BaF3 cells show a pronounced and sustained G2 arrest following radiation coupled with enhanced phosphorylation of cdc2. A cell cycle block in early M phase induced by the mitotic spindle poison, nocodazole, does not provide protection from apoptosis. Reversal of G2 arrest by caffeine abolishes the protective effect of BCR-ABL kinase. These data provide further insight into the transforming properties of BCR-ABL and are relevant to the clinical intransigence of Ph-positive leukaemias.

Frequent somatic deletion of the 13q12.3 locus encompassing BRCA2 in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has consistent 13q chromosomal abnormalities detected by conventional cytogenetics. Using interphase cytogenetics we show deletion of a 1-megabase 13q12.3 locus, encompassing the BRCA2 gene, in 80% of 35 CLL cases studied. Homozygous deletion of BRCA2, located within the minimal deletion consensus, was detected in a significant population of cells in 60% of the cases. Deletion of the previously described 13q14 locus (analyzed with RB1 and D13S25 probes) was seen in 63% of the cases. Homozygous deletion of RB1 was seen in one case. Seven of the cases (32%) with D13S25 deletion had a population of cells with homozygous deletion. Deletions at the 13q12 and 13q14 loci result from distinct events because they were not contiguous. These data provide evidence for the existence of a new tumor suppressor locus in B-cell CLL located at 13q12.3. BRCA2, located within the minimal deletion consensus, is a candidate for the gene whose somatic inactivation could play a role in the initiation and or progression of B-cell CLL.

An activating mutation in the ATP binding site of the ABL kinase domain.

A number of structural alterations have been shown to activate the leukemogenic potential of the ABL oncogene, but there is little understanding of the regulatory mechanisms that are subverted by such changes. We have used directed mutagenesis to examine a potential regulatory motif in cABL, which could directly influence ABL tyrosine kinase activity. A tyrosine to phenylalanine substitution within the ATP binding fold of the ABL kinase domain is sufficient to activate cABL enzymatic activity, and the mutant protein will alleviate growth factor dependence when expressed in the BA/F3 cell line. This growth promotion is dependent upon the structure of the amino terminus of the protein, and the ABL mutation will cooperate with certain BCR sequences in BCR/ABL fusion proteins to deregulate ABL kinase activity.

The fusion of TEL and ABL in human acute lymphoblastic leukaemia is a rare event.

We have recently identified a common ALL patient which harboured a chromosomal fusion between the TEL gene on chromosome 12 and the ABL gene on chromosome 9. We designed an RT-PCR assay to screen 186 adult ALL and 30 childhood ALL patients for this novel translocation. We were unable to identify any additional cases with a TEL/ABL fusion product.