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Multiple Myeloma (MM) is a neoplastic proliferation of bone marrow plasma cells. Plasma cells, even in MM, can be present in relatively low numbers and divide infrequently. Consequently, conventional metaphase cytogenetic studies can be extremely difficult. Chromosome abnormalities are reported in 18-36% of newly diagnosed MM. This figure is very low when compared to 60-80% with abnormal DNA content as shown by flow cytometry and even higher levels of abnormality suggested by recent in situ hybridisation reports. Standard Fluorescence in situ Hybridisation (FISH) techniques do not discriminate between normal cells and plasma cells, and as abnormalities can be present in only a proportion of the plasma cells, detection of the abnormal clone can be problematic. We present a robust technique for detecting plasma cell abnormalities by immuno-FISH suitable for use in a diagnostic laboratory. The technique involves separation of white cells and preparation of cytospin slides. Plasma cells are then identified by using FITC-conjugated antiantibodies (DAKO) directed against human Kappa and Lambda light chains. This is carried out prior to performing standard in situ hybridisation techniques. Analysis can then be targeted to plasma cells only. We demonstrate the use of the Vysis SpectrumOrange Rb1 probe for 13q deletion detection, but the technique could be extended to dual colour probes such as the Vysis IgH breakapart probe. [ABSTRACT FROM AUTHOR]

Fragile sites appear visually as nonstaining gaps on chromosomes that are inducible by specific cell culture conditions. Expansion of CGG/CCG repeats has been shown to be the molecular basis of all five folate-sensitive fragile sites characterized molecularly so far, i.e., FRAXA, FRAXE, FRAXF, FRA11B, and FRA16A. In the present study we have refined the localization of the FRA10A folate-sensitive fragile site by fluorescence in situ hybridization. Sequence analysis of a BAC clone spanning FRA10A identified a single, imperfect, but polymorphic CGG repeat that is part of a CpG island in the 5′UTR of a novel gene named FRA10AC1. The number of CGG repeats varied in the population from 8 to 13. Expansions exceeding 200 repeat units were methylated in all FRA10A fragile site carriers tested. The FRA10AC1 gene consists of 19 exons and is transcribed in the centromeric direction from the FRA10A repeat. The major transcript of ∼1450 nt is ubiquitously expressed and codes for a highly conserved protein, FRA10AC1, of unknown function. Several splice variants leading to alternative 3′ ends were identified (particularly in testis). These give rise to FRA10AC1 proteins with altered COOH-termini. Immunofluorescence analysis of full-length, recombinant EGFP-tagged FRA10AC1 protein showed that it was present exclusively in the nucleoplasm. We show that the expression of FRA10A, in parallel to the other cloned folate-sensitive fragile sites, is caused by an expansion and subsequent methylation of an unstable CGG trinucleotide repeat. Taking advantage of three cSNPs within the FRA10AC1 gene we demonstrate that one allele of the gene is not transcribed in a FRA10A carrier. Our data also suggest that in the heterozygous state FRA10A is likely a benign folate-sensitive fragile site. [Copyright &y& Elsevier]

CCAAT enhancer-binding protein (CEBP) transcription factors play pivotal roles in proliferation and differentiation, including suppression of myeloid leukemogenesis. Mutations of CEBPA are found in a subset of acute myeloid leukemia (AML) and in some cases of familial AML. Here, using cytogenetics, fluorescence in situ hybridization (FISH), and molecular cloning, we show that 5 CEBP gene family members are targeted by recurrent IGH chromosomal translocations in BCP-ALL. Ten patients with t(8;14)(q11;q32) involved CEBPD on chromosome 8, and 9 patients with t(14;19)(q32;q13) involved CEBPA, while a further patient involved CEBPG, located 71 kb telomeric of CEBPA in chromosome band 19q13; 4 patients with inv(14)(q11q32)/t(14;14)(q11;q32) involved CEBPE and 3 patients with t(14;20)(q32;q13) involved CEBPB. In 16 patients the translocation breakpoints were cloned using long-distance inverse-polymerase chain reaction (LDI-PCR). With the exception of CEBPD breakpoints, which were scattered within a 43-kb region centromeric of CEBPD, translocation breakpoints were clustered immediately 5' or 3' of the involved CEBP gene. Except in 1 patient with t(14;14)(q11;q32), the involved CEBP genes retained germ-line sequences. Quantitative reverse transcription (RT)-PCR showed overexpression of the translocated CEBP gene. Our findings implicate the CEBP gene family as novel oncogenes in BCP-ALL, and suggest opposing functions of CEBP dysregulation in myeloid and lymphoid leukemogenesis.

Somatic cell reprogramming holds great promise for the development of novel cellular therapeutics. A number of sources of reprogramming potential have been identified, including oocytes, embryonic germ (EG) cells and embryonic stem (ES) cells. However, each of these sources of reprogramming factors is problematic, since they are either not freely available or have special growth requirements. Embryonal carcinoma (EC) cells are another source of pluripotent cells that, unlike ES and EG cells, do not usually require special growth conditions. Since they share many of the key characteristics of ES cells, such as pluripotency, EC cells may provide a readily amenable alternative source of reprogramming factors and could serve as a model for ES cells in this respect. Here we show that mouse EC cells can also function as donors of reprogramming factors. PEG-mediated fusion between murine EC cells (P19) and the cells of a human T-lymphoma cell line (CEM-GFP) resulted in inter-species hybrid colony formation. Colonies of hybrid cells displayed heterogeneity in cellular morphology as well as in their pattern of human gene expression. Expression of two human transcription factors characteristic of undifferentiated pluripotent stem cells, Oct-4 and Sox-2, was detected in the hybrid cells, demonstrating activation of endogenous human markers of pluripotency. Simultaneously, down-regulation of CD45, a marker present in lymphocytic cells, was observed in some hybrids. The detection of human specific markers of differentiation, such as nestin, lamininbeta1, and collagen IValpha1, indicates that fusion resulted in reprogramming of the human cells to reflect the differentiation potential of the murine EC partner.