PML-RARA fusion transcripts in irradiated and normal hematopoietic cells.

It is believed that two important factors in the genesis of reciprocal chromosomal translocations in malignant cells are the physical proximity of the involved regions and local structural features of the chromatin fiber that make them more susceptible to breakage and rearrangement. In this work we sought to investigate whether PML-RARA fusion transcripts, characteristic of acute promyelocytic leukemia (APL), could be induced by a clastogenic agent in cells known to have, a priori, a favorable spatial distribution of these genes. A lymphoid-cell line, lacking the t(15;17) but having the PML and RARA genes in close proximity in specific phases of the cell cycle, was irradiated with 10 Gy of (60)Co, and the incidence of PML-RARA transcripts was analyzed by a highly sensitive PCR assay. Despite gene proximity, typical PML-RARA transcripts were only rarely detected in irradiated cells. The same phenomenon was observed at similar frequency in control non-irradiated cells. These findings made us investigate whether such transcripts could also be detected in peripheral blood cells from normal individuals. PML-RARA transcripts were observed at low frequencies in isolated lymphoid and granulocytic cell populations, with similar incidence in both cell types. The data thus indicate that the PML and RARA genes are not particularly susceptible to the clastogenic effects of gamma-irradiation, and that, similar to what has been reported for other chromosomal translocations, transcriptionally active PML-RARA rearrangements can be generated in normal hematopoietic cells of different lineages without apparent oncogenic consequences.

The spatial distribution of human immunoglobulin genes within the nucleus: evidence for gene topography independent of cell type and transcriptional activity.

The three-dimensional positioning of immunoglobulin (Ig) genes within the nucleus of human cells was investigated using in situ hybridization and confocal microscopy. The visualization of heavy and light chain genes in B-lymphoid cells showed that the three Ig genes are differentially and nonrandomly distributed in different nuclear subvolumes: the kappa genes were found to be preferentially confined to an outer nuclear volume, whereas the gamma and lambda genes consistently occupied more central positions within the nucleus, the lambda genes being more interior when compared with the gamma genes. The data further show that these overall topographical distributions are independent of gene transcriptional activity and are conserved in different cell types. Although subtle gene movements within those defined topographical regions cannot be excluded by this study, the results indicate that tissue specificity of gene expression is not accompanied by drastic changes in gene nuclear topography, rather suggesting that gene organization within the nucleus may be primarily dependent on structural constraints imposed on the respective chromosomes.

Influence of transcription and replication on the in situ resolution of immunoglobulin heavy-chain constant region genes: an interphase cytogenetics analysis.

An interphase cytogenetics analysis was performed to investigate whether replication and transcription could influence in situ resolution of immunoglobulin (Ig) heavy chain constant region genes. A plasmid probe recognizing five C gamma segments separated by known linear DNA distances was hybridized in situ and visualized by digital fluorescence microscopy. In interphase nuclei from phytohemagglutinin (PHA)-stimulated lymphocytes, the gamma genes were resolved as one to three signals per allele in the majority of nuclei, whereas in a minority, complex patterns of several signals per allele could be observed. The latter were restricted to nuclei in an early stage of the S phase, as assessed by hybridization experiments performed in cells grown in the presence of bromodeoxyuridine. To investigate whether the in situ resolution of the C gamma segments could vary as a function of the transcription activity of the locus, the C gamma probe was subsequently hybridized to nuclei from a mature B cell line (JVM-2), which produces gamma transcripts as shown by in situ RNA hybridization experiments. Primary human fibroblasts were further used as representative of a non-lymphoid cell type with transcriptionally inactive Ig genes. When Gl nuclei from the three cell types were compared in terms of the in situ resolution of the C gamma locus, JVM-2 cells were found to include the highest percentage of higher resolution patterns (three to five signals per allele in 28% of nuclei), fibroblasts the lowest (three signals per allele, 2%), while PHA-stimulated lymphocytes occupied an intermediate position between the other two cell types (three or four signals per allele, 15%). The data show that the in situ resolution of Ig C gamma genes varies throughout the cell cycle and is influenced by the transcriptional activity of the locus. The variability of the resolution patterns observed appears to reflect different levels of chromatin packaging, which in turn are likely to influence the probe accessibility to its target. These observations are relevant for the interpretation of data from interphase cytogenetics analysis of independent, but closely spaced, DNA segments.

In situ visualisation of immunoglobulin genes in normal and malignant lymphoid cells.

Aims-To directly visualise immunoglobulin (Ig) heavy (H) and light chain genes (kappa and lambda) in metaphase chromosomes and interphase nuclei of normal and malignant lymphocytes using small genomic probes targeted to intragenic sequences.Methods-Cytogenetic preparations from phytohaemagglutinin stimulated lymphocytes, B-chronic lymphocytic leukaemia (B-CLL) cells, and a B-prolymphocytic leukaemia (B-PLL) cell line, containing a t(11;14), were hybridised in situ using biotin or digoxigenin labelled plasmid probes. The kappa genes were visualised with a combination of probes for the Ckappa, Jkappa, Vkappa1, and Vkappa2 segments, the lambda genes with a probe containing the Jlambda2-Clambda2, Jlambda3-Clambda3 segments and the H genes with a probe for Clambda2. Hybridisation sites were visualised using appropriate fluorochrome conjugates and images were analysed by digital microscopy.Results-In both normal and malignant lymphoid cells, the kappa and lambda genes were visualised as a single dot signal, whereas the H lambda genes were resolved as either two or three separate signals per chromatid in metaphase chromosomes or per allele in interphase nuclei. In the malignant PLL cells, double hybridisation experiments with a painting library specific for the chromosome 11 showed that the lambda region was retained in the translocated chromosome, with an in situ resolution pattern similar to that of the normal allele.Conclusions-This study shows that a high resolution in situ analysis of the three Ig loci can be efficiently performed with small size genomic probes on both normal and malignant lymphoid cells. Such an approach offers a flexible tool for the molecular characterisations of these loci on chromosomes and individual neoplastic cells.

Effect of dexamethasone and phenobarbital on run-on transcription rate and CYP3A mRNA concentration in rat liver: changes during development.

Modulation of CYP3A1 and CYP3A2 mRNA expression by dexamethasone and by phenobarbital has been studied in immature (21-day-old) and adult (90-day-old) rat liver. Positive modulation of these forms by both agents markedly declines with the age of the animals. However, CYP3A2 mRNA, although physiologically extinguished in the adult females, still responds to dexamethasone stimulation. The regulatory mechanisms underlying the differential behavior of CYP3A1 in the immature and adult animals have been further investigated by analyzing the early changes in the run-on transcription rates and the subsequent mRNA accumulation in the liver in response to the inducer agents. CYP3A genomic clones were constructed and characterized for this purpose. The use of a unique cosCYP/3A1 intronic sequence, identified in this work, made possible the selective determination of the transcription rate of this gene by run-on assay, as a function of ontological development and inducer treatment. Parallel determination of the mRNA concentration in the liver by dot blot analysis demonstrated that dexamethasone induces CYP3A1 essentially through transcription regulation in immature animals, while in adults it is suggested to act mainly at a post-transcriptional level.