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To develop a sensitive and inducible system to study intestinal biology, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the 12.4 kb murine Villin promoter. The newly generated Villin-rtTA2-M2 mice were then bred with the previously developed tetO-HIST1H2BJ/GFP model to assess inducibility and tissue-specificity. Expression of the histone H2B-GFP fusion protein was observed exclusively upon doxycycline induction and was uniformly distributed throughout the intestinal epithelium. The Villin-rtTA2-M2 was also found to drive transgene expression in the developing mouse intestine. Furthermore, we could detect transgene expression in the proximal tubules of the kidney and in a population of alleged gastric progenitor cells. By administering different concentrations of doxycycline, we show that the Villin-rtTA2-M2 system drives transgene expression in a dosage-dependent fashion. Thus, we have generated a novel doxycycline-inducible mouse model, providing a valuable tool to study the effect of different gene dosages on intestinal physiology and pathology., (Copyright 2008 Wiley-Liss, Inc.)

Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam-2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q-PCR) and NANOG, and the absence of CD30, SSX2-4, and SOX2, confirms that TCam-2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam-2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam-2 is the first well-characterized seminoma-derived cell line, with an exceptional mutation, rarely found in TGCTs., ((c) 2007 Wiley-Liss, Inc.)

Malignant mesothelioma (MM) is an asbestos-induced tumor that acquires aneuploid DNA content during the tumorigenic process. We used instable MM cell lines as an in vitro model to study the impact of DNA copy-number changes on gene expression profiling, in the course of their chromosomal redistribution process. Two MM cell lines, PMR-MM2 (early passages of in vitro culture) and PMR-MM7 (both early and late passages of in vitro culture), were cytogenetically characterized. Genomic gains and losses were precisely defined using microarray-based comparative genomic hybridization (array-CGH), and minimal overlapping analysis led to the identification of the common unbalanced genomic regions. Using the U133Plus 2.0 Affymetrix gene chip array, we analyzed PMR-MM7 early and late passages for genome-wide gene expression, and correlated the differentially expressed genes with copy-number changes. The presence of a high number of genetic imbalances occurring from early to late culture steps reflected the tendency of MM cells toward genomic instability. The selection of specific chromosomal abnormalities observed during subsequent cultures demonstrated the spontaneous evolution of the cancer cells in an in vitro environment. MM cell lines were characterized by copy-number changes associated with the TP53 apoptotic pathway already present at the first steps of in vitro culture. Prolonged culture led to acquisition of additional chromosomal copy-number changes associated with dysregulation of genes involved in cell adhesion, regulation of mitotic cell cycle, signal transduction, carbohydrate metabolism, motor activity, glycosaminoglycan biosynthesis, protein binding activity, lipid transport, ATP synthesis, and methyltransferase activity., (Copyright (c) 2007 Wiley-Liss, Inc.)

The t(7;12)(q36;p13) is a recurrent translocation involving the ETV6/TEL gene (12p13) and a heterogeneous breakpoint at 7q36. A fusion transcript between HLXB9 and ETV6 in AML with t(7;12) is occasionally found. To study the incidence of t(7;12) in infant and childhood acute leukemia, we screened 320 cases <36 months using FISH. Additionally, 28 pediatric cases >36 months with cytogenetic breakpoints at 12p and 7q were investigated. We studied the presence of an HXLB9-ETV6 fusion transcript and quantified the expression of various genes located in the 7q36 breakpoint region. In total, six AML patients carried the t(7;12) of which five were infants and one child of 18 months. Only one out of 99 infant ALL patients harbored the t(7;12). No t(7;12) was found in older children with AML or ALL. AML patients carrying a t(7;12) had a poor outcome with a 3-year EFS of 0%. A fusion of HLXB9 to ETV6 was found in four AML cases with t(7;12). The 7q36 genes NOM1, LMBR1, RNF32, and SHH were equally expressed among t(7;12)-positive AML versus t(7;12)-negative AML, t(7;12)-negative ALL, or normal bone marrow. However, the HLXB9 expression was highly increased in t(7;12)-positive cases, including those with an HLXB9-ETV6 fusion. We conclude that the t(7;12) is almost exclusively present in infant AML and covers 30% of infant AML, while it is extremely rare in infant ALL and older children. The t(7;12) is associated with a poor outcome and an ectopic expression of HLXB9 is commonly involved in this genetic subtype of leukemia.

Chromosome rearrangements are found in many acute leukemias. As a result, genes at the breakpoints can be disrupted, forming fusion genes. One of the genes involved in several chromosome aberrations in hematological malignancies is NUP98 (11p15). As NUP98 is close to the 11p telomere, small translocations might easily be missed. Using a NUP98-specific split-signal fluorescence in situ hybridization (FISH) probe combination, we analyzed 84 patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia, or myelodysplastic syndrome with either normal karyotypes or 11p abnormalities to investigate whether there are unidentified 11p15 rearrangements. Neither NUP98 translocations nor deletions were identified in cases with normal karyotypes, indicating these aberrations may be very rare in this group. However, NUP98 deletions were observed in four cases with unbalanced 11p aberrations, indicating that the breakpoint is centromeric of NUP98. Rearrangements of NUP98 were identified in two patients, both showing 11p abnormalities in the diagnostic karyotype: a t(4;11)(q1?3;p15) with expression of the NUP98-RAP1GDS1 fusion product detected in a 60-year-old woman with AML-M0, and an add(11)(p15) with a der(21)t(11;21)(p15;p13) observed cytogenetically in a 1-year-old boy with AML-M7. JARID1A was identified as the fusion partner of NUP98 using 3' RACE, RT-PCR, and FISH. JARID1A, at 12p13, codes for retinoblastoma binding protein 2, a protein implicated in transcriptional regulation. This is the first report of JARID1A as a partner gene in leukemia., (2006 Wiley-Liss, Inc)

The BCL-2 family has been implicated in the pathogenesis of various hematopoietic malignancies, including follicular non-Hodgkin lymphoma and B-cell chronic lymphocytic leukemia. To identify genes that act synergistically in BCL2-enforced leukemogenesis, we developed a murine B-cell lymphoma/leukemia model based on the IL-3-dependent Balb/C pro-B line (FL5.12). FL5.12 cells were stably transfected with antiapoptotic BCL-2 alone or in combination with proapoptotic BAX or nonfunctional mutant BAX, thereby creating various levels of imbalance within the BCL-2 family. Transfectants were intravenously injected into normal Balb/C mice. Whereas FL5.12 cells did not provoke leukemia, mice injected with stable transfectants died of leukemia over time. Disease incidence and latency time depended on the degree of imbalance in the BCL-2 family, supporting a model whereby BCL2 drives tumorigenesis. All mice presented with hepatosplenomegaly and leukemic FL5.12 cells in peripheral blood and bone marrow compartments. Leukemic conversion was accompanied by secondary genetic aberrations leading to clonal IL-3-responsive leukemia. Cellular transformation was independent of alterations in c-Myc or downstream apoptotic pathway. Leukemic clones retained a normal DNA damage response leading to elevated P53 and P21 levels and cell cycle arrest upon irradiation. In conclusion, our mouse model may prove a valuable tool to identify genes that cooperate in BCL2-enforced lymphoma/leukemogenesis.

We report on a patient with Williams syndrome and a complex de novo chromosome rearrangement, including microdeletions at 7q11.23 and 7q36 and additional chromosomal material at 7q36. The nature of this additional material was elucidated by spectral karyotyping and first assigned to chromosome 22. Subsequent fluorescence in situ hybridization (FISH) experiments showed that it consisted of satellite material only. Refinement of the 7q36 breakpoint was performed with several FISH probes, showing a deletion distal to the triphalangeal thumb (TPT) region. The phenotype of the patient principally results from the microdeletion of the 7q11.23; the small deletion at 7qter and the extra satellite material may not be of clinical significance., (Copyright 2001 Wiley-Liss, Inc.)

Several nonrandom recurrent chromosomal changes are observed in uveal melanoma. Some of these abnormalities, e.g., loss of chromosome 3, gain of the q arm of chromosome 8, and chromosome 6 abnormalities, are of prognostic value. Cytogenetic analysis and/or fluorescence in situ hybridization (FISH) are used to detect these changes. In some cases, however, detailed cytogenetic analysis is not possible due to the presence of complex abnormalities. To define more accurately these cytogenetic changes, we have applied comparative genomic hybridization (CGH) and/or spectral karyotyping (SKY) to two uveal melanoma cell lines and five primary uveal melanomas, with partially defined and/or complex abnormalities. SKY provided additional information on 34/39 partially defined aberrant chromosomes and revealed a new abnormality, a der(17)t(7;17)(?;q?), that had not been recognized by conventional cytogenetics. Additionally, using SKY, abnormalities involving chromosome 6 or 8 were found to be twice as common as observed with cytogenetic analysis. CGH was especially useful in assigning the abnormalities identified by SKY to specific chromosomal regions and, in addition, resulted in the detection of a small deletion of chromosome region 3q13 approximately 21. We conclude that SKY and CGH, as methods complementary to cytogenetic and FISH analysis, provide more complete information on the chromosomal abnormalities occurring in uveal melanoma., (Copyright 2001 Wiley-Liss, Inc.)

Rearrangements of 12p, resulting from deletions or translocations, are common findings in hematologic malignancies. In many cases, these rearrangements target the ETV6 gene (previously called TEL) located at 12p13. Various partner genes have been implicated in the formation of fusion genes with ETV6. These include PDGFRB, JAK2, NTRK3, ABL2, and ABL1, each of which encodes for proteins with tyrosine kinase activity. To date, ETV6/ABL1 transcripts have been detected in only four patients with a leukemic disorder. Here, we describe one adult with chronic myeloid leukemia and a child with T-cell acute lymphocytic leukemia with ETV6/ABL1. Molecular cytogenetic analysis confirmed that formation of an ETV6/ABL1 fusion in these patients required at least three chromosomal breaks and showed that each of these translocations is the result of a complex chromosomal rearrangement. Molecular analysis showed the presence of two fusion transcripts in both patients as the result of alternative splicing, questioning the suggested role of these transcripts in the lineage specificity. Clinical findings of these patients were compared to those of previously reported cases, and the possible clinical and biological similarities between ETV6/ABL1 and other fusion genes leading to increased tyrosine kinase activity are discussed., (Copyright 2001 Wiley-Liss, Inc.)

We report on the establishment and characterization of 2 primary (EOM-3, EOM-29) and 3 metastatic uveal melanoma cell lines (OMM-1, OMM-2, OMM-3) and further cytogenetic characterization of a previously described primary uveal melanoma cell line (OCM-1). Only a few long-term growing primary uveal melanoma cell lines have as yet been established, while of metastatic uveal melanoma cell lines we have found no descriptions. The morphology of the in vitro cultured cells varied from spindle to epithelioid. The cell lines were characterized by immunocytochemistry, electron microscopy and cytogenetical analysis. The relative growth rate was determined by bromodeoxyuridine (BUdR) incorporation. The melanocytic origin of the cell lines was determined by positive staining with antibodies identifying melanoma-associated antigens. Melanosomes and pre-melanosomes were indeed observed by electron microscopy in all cell lines. The stem-cell karyotype was found to be normal in 3 cell lines (EOM-29, OMM-2, OMM-3) and abnormal in 3 others (EOM-3, OCM-1, OMM-1) showing a net loss of chromosome 6. The OCM-1 and the OMM-1 cell lines even demonstrated a large amount of structural chromosomal aberrations, the former being near-tetraploid and the latter triploid. The EOM-29 cell line, cultured from a ciliary body melanoma, did not show the previously described chromosome 3 and 8 abnormalities.

We describe a patient who developed multiple meningiomas but had no clear evidence of neurofibromatosis type 2. Four of the tumors, derived from three different sites, were analyzed cytogenetically and/or at the DNA level using chromosome 22 specific probes. All four tumors showed loss of the same copy of chromosome 22. On the chromosome that was retained in the tumors, we found two constitutional aberrations, a 1.5 kb deletion and a point mutation. The patient had inherited both alterations from her father. The father has not developed any meningiomas so far but he has been treated for a well-differentiated adenocarcinoma of the lung and a brain metastasis from this tumor. The mother and 75 unrelated individuals did not show any of the chromosome 22 alterations. The multiple tumors found in the patient suggest that she has a predisposing gene for the development of meningiomas. The finding that all investigated tumors lost the same, constitutionally normal copy of chromosome 22 could indicate that the predisposing gene resides on chromosome 22 and was affected by the constitutional mutations.