DNA FISH Research Articles

Abstract A40: The incorporation of PTEN/EGFR (DNA) FISH and quantitative biomarker immunofluorescence identifies a phenotype associated with survival in patients with malignant glioma

Abstract Introduction: Glioblastoma multiforme (GBM) is the most common and malignant of adult glial tumors. Despite best standard of care including surgical resection, radiation and chemotherapy (e.g. temozolomide), the median survival remains approximately 12 – 15 months, with fewer than 25% of patients surviving up to 2 years and less than 10% of patients surviving up to 5 years. We sought to identify a preliminary set of baseline tissue-based DNA and protein features which may reflect growth mechanisms responsible for disease progression, and overall survival (OS). Methods: Formalin fixed paraffin embedded specimens from 54 patients (44 Astrocytoma (including 14 GBM), 6 oligodendroglioma and 4 mixed) with outcome data and a median follow-up of 3 years were evaluated using dual-color DNA FISH for PTEN and EGFR. A multispectral immunofluorescent (IF) assay was used as previously described (Cordon-Cardo et al., 2007) to evaluate Ki67, PTEN, and P-AKT. Feature performance was univariately assessed using Kaplan-Meier survival curves. Results: A >10% Loss of heterozygosity (LOH) for PTEN (p-value <0.05) but not EGFR amplification / polysomy was associated with decreased OS in the total cohort. By comparison, the combination of PTEN LOH and EGFR amplification/polysomy in the GBM-only group (n=12, complete data files) was associated with improved survival (p-value 0.03). By quantitative IF, increasing levels of KI67 were associated with shortened OS (p-value 0.004) in the total cohort. Conclusions: The incorporation of DNA FISH and quantitative IF is useful for developing a biologic baseline for understanding disease course in malignant gliomas. PTEN LOH and EGFR amplification / polysomy in GBM may suggest a pathway mechanism for favorable response. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A40

Chromosomal imbalances are uncommon in chagasic megaesophagus.

BackgroundChagas' disease is a human tropical parasitic illness and a subset of the chronic patients develop megaesophagus or megacolon. The esophagus dilation is known as chagasic megaesophagus (CM) and one of the severe late consequences of CM is the increased risk for esophageal carcinoma (ESCC). Based on the association between CM and ESCC, we investigated whether genes frequently showing unbalanced copy numbers in ESCC were altered in CM by fluorescence in situ (FISH) technology.MethodsA total of 50 formalin-fixed, paraffin-embedded esophageal mucosa specimens (40 from Chagas megaesophagus-CM, and 10 normal esophageal mucosa-NM) were analyzed. DNA FISH probes were tested for FHIT, TP63, PIK3CA, EGFR, FGFR1, MYC, CDKN2A, YES1 and NCOA3 genes, and centromeric sequences from chromosomes 3, 7 and 9.ResultsNo differences between superficial and basal layers of the epithelial mucosa were found, except for loss of copy number of EGFR in the esophageal basal layer of CM group. Mean copy number of CDKN2A and CEP9 and frequency of nuclei with loss of PIK3CA were significantly different in the CM group compared with normal mucosa and marginal levels of deletions in TP63, FHIT, PIK3CA, EGFR, CDKN2A, YES and gains at PIK3CA, TP63, FGFR1, MYC, CDNK2A and NCOA3 were detected in few CM cases, mainly with dilation grades III and IV. All changes occurred at very low levels.ConclusionsGenomic imbalances common in esophageal carcinomas are not present in chagasic megaesophagus suggesting that these features will not be effective markers for risk assessment of ESCC in patients with chagasic megaesophagus.

Long nuclear-retained non-coding RNAs and allele-specific higher-order chromatin organization at imprinted snoRNA gene arrays

The imprinted Snurf-Snrpn domain, also referred to as the Prader-Willi syndrome region, contains two approximately 100-200 kb arrays of repeated small nucleolar (sno)RNAs processed from introns of long, paternally expressed non-protein-coding RNAs whose biogenesis and functions are poorly understood. We provide evidence that C/D snoRNAs do not derive from a single transcript as previously envisaged, but rather from (at least) two independent transcription units. We show that spliced snoRNA host-gene transcripts accumulate near their transcription sites as structurally constrained RNA species that are prevented from diffusing, as well as multiple stable nucleoplasmic RNA foci dispersed in the entire nucleus but not in the nucleolus. Chromatin structure at these repeated arrays displays an outstanding parent-of-origin-specific higher-order organization: the transcriptionally active allele is revealed as extended DNA FISH signals whereas the genetically identical, silent allele is visualized as singlet DNA FISH signals. A similar allele-specific chromatin organization is documented for snoRNA gene arrays at the imprinted Dlk1-Dio3 domain. Our findings have repercussions for understanding the spatial organization of gene expression and the intra-nuclear fate of non-coding RNAs in the context of nuclear architecture.

Long nuclear-retained non-coding RNAs and allele-specific higher-order chromatin organization at imprinted snoRNA gene arrays

The imprinted Snurf-Snrpn domain, also referred to as the Prader-Willi syndrome region, contains two approximately 100-200 kb arrays of repeated small nucleolar (sno)RNAs processed from introns of long, paternally expressed non-protein-coding RNAs whose biogenesis and functions are poorly understood. We provide evidence that C/D snoRNAs do not derive from a single transcript as previously envisaged, but rather from (at least) two independent transcription units. We show that spliced snoRNA host-gene transcripts accumulate near their transcription sites as structurally constrained RNA species that are prevented from diffusing, as well as multiple stable nucleoplasmic RNA foci dispersed in the entire nucleus but not in the nucleolus. Chromatin structure at these repeated arrays displays an outstanding parent-of-origin-specific higher-order organization: the transcriptionally active allele is revealed as extended DNA FISH signals whereas the genetically identical, silent allele is visualized as singlet DNA FISH signals. A similar allele-specific chromatin organization is documented for snoRNA gene arrays at the imprinted Dlk1-Dio3 domain. Our findings have repercussions for understanding the spatial organization of gene expression and the intra-nuclear fate of non-coding RNAs in the context of nuclear architecture.

Analysis of allele-specific RNA transcription in FSHD by RNA-DNA FISH in single myonuclei

Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is likely caused by epigenetic alterations in chromatin involving contraction of the D4Z4 repeat array near the telomere of chromosome 4q. The precise mechanism by which deletions of D4Z4 influence gene expression in FSHD is not yet resolved. Regulatory models include a cis effect on proximal gene transcription (position effect), DNA looping, non-coding RNA, nuclear localization and trans-effects. To directly test whether deletions of D4Z4 affect gene expression in cis, nascent RNA was examined in single myonuclei so that transcription from each allele could be measured independently. FSHD and control myotubes (differentiated myoblasts) were subjected to sequential RNA-DNA FISH. A total of 16 genes in the FSHD region (FRG2, TUBB4Q, FRG1, FAT1, F11, KLKB1, CYP4V2, TLR3, SORBS2, PDLIM3 (ALP), LRP2BP, ING2, SNX25, SLC25A4 (ANT1), HELT and IRF2) were examined for interallelic variation in RNA expression within individual myonuclei. Sequential DNA hybridization with a unique 4q35 chromosome probe was then applied to confirm the localization of nascent RNA to 4q. A D4Z4 probe, labeled with a third fluorochrome, distinguished between the deleted and normal allele in FSHD nuclei. Our data do not support an FSHD model in which contracted D4Z4 arrays induce altered transcription in cis from 4q35 genes, even for those genes (FRG1, FRG2 and SLC25A4 (ANT1)) for which such an effect has been proposed.

Spatial juxtaposition of HIV-1 provirus with PML and KAKA bodies as revealed by 3D Immuno DNA FISH

Various members of tripartite motif (TRIM) protein family display antiviral properties, targeting retroviruses in particular. The potential activity of TRIM19, better known as promyelocytic leukemia protein (PML) against several viruses has been well documented, yet it's role in HIV-1 infection remains elusive. One of the most important cellular partners of HIV-1, P-TEFb kinase complex, composed of CDK9 and CyclinT1 plays a crucial role in regulation of HIV-1 transcription. We have previously demonstrated that both members of P-TEFb interact with PML protein and localize inside the PML Nuclear Bodies (PML NB) [1,2]. In particular, we found that the acetylated and enzymaticaly inactive form of CDK9 binds PML and can be separated with PML in the insoluble chromatin fraction. Our ChIP analysis revealed that acetylated form of CDK9 localizes to the integrated and transcriptionally silent viral genome, thus indicating that PML might have yet unidentified role in regulation of HIV-1 infection and latency [2]. We indeed identified PML protein bound to the silent viral genome in Jurkat cells harboring single integrations of HIV-1 provirus. The distribution of PML protein along the viral genome could be correlated with bimethylated lysine 9 (K9) on Histone 3 (2MetK9 H3), a mark of facultative heterochromatin. Transcriptional activation of the provirus with TNF-α led to a displacement of PML protein from the genome and to a substantial loss of 2MetK9H3 mark. By three-dimensional immuno fluorescent in situ hybridization (3D Immuno DNA FISH) that we developed we visualized the viral genome in close proximity to PML NB and we characterized this position as adjacent. Within the population of cells tested in the silenced state, 40% of the cells showed proximity at the distance of less that 0.6μm. PML NB were recently described to have a close spatial relationship with the so called KAKA foci, characterized by the presence of KAP-1 (or Trim28) and KRAB-Zinc Finger proteins [3] thus prompting us to analyse the relationship of the silent HIV-1 provirus with these nuclear structures. We detected numerous PML and KAKA foci in both Jurkat cells (J-LAT model of latency) and primary human CD4+T lymphocytes, with HIV-1 residing either in close proximity to PML or to KAKA foci (~40% showing less than 0.6μm distance) in J-LAT latency model. Whether this spatial localization of HIV-1 provirus is correlated to silencing of the virus genome or is a general feature of HIV-1 integration and replication remains still to be determined.

A new enrichment model for high sensitivity detection and downstream analyses of circulating tumor cells in breast cancer patients.

Abstract Abstract #4162 The detection of circulating tumor cells (CTCs) in breast cancer patients have the potential to improve prognostication and the monitoring of response to treatment. Most CTC enrichment technologies are based on binding to anti-EpCAM antibodies. The sensitivity of such assays is limited by tumors that express no or undetectable levels of EpCAM. Improvements in CTC detection coupled with the development of systems to interrogate CTCs for therapeutic target expression could lead to novel applications for patient monitoring, clinical diagnosis and treatment. In this study, we describe a sensitive and reproducible enrichment method for CTCs. We defined cells as circulating tumor cells with three criteria: Positive for cytokeratin (CK+) and DAPI (nuclear) (DAPI+) and negative staining for CD45 (CD45-). We have previously reported that this system has a higher sensitivity for circulating tumor cell detection and provides a better platform for CTC downstream analyses compare to the methods currently available in the market. Herein, we describe the use of this platform for the evaluation of breast cancer biomarkers in CTCs. Blood samples from patients with metastatic breast cancer were used for CellSearch™ assay (Veridex , LLC ) and our CTC assay (A1000 CTC enrichment and detection kit, Genetix). We performed the CTC enrichment assay using the combination of anti-CK and anti-EpCAM antibodies. CTCs were identified with brightfield and fluorescence labeled anti-CK, anti-CD45 and DAPI (nuclear stain) images. The Ariol® system (Applied Imaging Corporation) was employed for automated cell image capture and analysis of CTCs on glass slides. CTCs enriched on the glass slides were used for CTC downstream analysis. Our CTC enrichment model is designed to have the capability to enrich all the three types of CTCs including CK+ & EpCAM+, CK+ & EpCAM-/low and CK-/low & EpCAM+ cells. Compared to the enrichment methods using anti-EpCAM or anti-cytokeratin antibody alone, a higher CTC detection rate and a larger dynamic CTC detected range were obtained with our new enrichment model. Interestingly there were clear CTC number differences with enrichment methods in the higher CTC count patient samples which indicate that the different enrichment methods may enrich different types of CTCs from patient blood samples.
 Results of DNA and RNA FISH analyses on enriched CTCs indicate that the CTCs on glass slides can be used for its downstream analyses directly or indirectly. Our method may have better performance on enrichment of heterogeneous CTCs and provide a better platform for CTCs profiling for biomarker evaluations and CTC downstream analyses. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4162.

In situ analysis of the bacterial community associated with the reindeer lichen Cladonia arbuscula reveals predominance of Alphaproteobacteria

The diversity and spatial pattern of the bacterial community hosted by the shrub-like reindeer lichen Cladonia arbuscula were investigated by general DNA staining and FISH, coupled with confocal laser scanning microscopy (CLSM). Using an optimized protocol for FISH using cryosections of small lichen fragments, we found about 6 x 10(7) bacteria g(-1) of C. arbuscula. Approximately 86% of acridine orange-stained cells were also stained by the universal FISH probe EUB338. Using group-specific FISH probes, we detected a dominance of Alphaproteobacteria (more than 60% of all bacteria), while the abundance of Actinobacteria and Betaproteobacteria was much lower (<10%). Firmicutes were rarely detected, and no Gammaproteobacteria were present. Bacterial cells of different taxonomic groups are embedded in a biofilm-like, continuous layer on the internal surface of the C. arbuscula podetia, mainly occurring in small colonies of a few to a few hundred cells. The other parts of the lichen showed a lower bacterial colonization. alpha-proteobacterial 16S rRNA genes were amplified using total DNA extracts from C. arbuscula and separated by single-strand conformation polymorphism (SSCP). Sequencing of excised bands revealed the dominance of Acetobacteraceae.

β-Globin LCR and Intron Elements Cooperate and Direct Spatial Reorganization for Gene Therapy

The Locus Control Region (LCR) requires intronic elements within β-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional β-globin intron 2 elements that rescue LCR activity directed by 5′HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igμ 3′MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igμ 3′MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5′HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI) of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN) lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5′HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate correction of hemoglobinopathies using single copy vectors.

Radiosensitivity of Prostate Cancer and BPH Patients Studied by DNA Repair Capacity, CA and FISH.

An individual’s genetic constitution and lifestyle, e.g., diet and levels of physical activity, can affect the body’s response to various exogenous agents including therapeutic treatment. There is a strong need to combine studies on variability in a cellular response to genotoxic exposure with predisposition of the patient to diseases development and healing. In this study a variation in responses to challenging dose of X rays in lymphocytes from healthy donors and prostate cancer patients were compared on the molecular and mitotic levels. Blood was collected from healthy donors BPH (Benign Prostate Hyperplasia), and prostate cancer patients. Among cancer patients 33% never smoke 46.7% were former smokers. Immediately after collecting the blood a challenging irradiation was performed followed by culturing procedures for classic cytogenetics and FISH techniques with a probe for the whole chromosome 1. In DNA damage investigations, isolated and cryopreserved lymphocytes were thawed and their viability examined before molecular studies. To evaluate individual susceptibility, defrosted lymphocytes were exposed to 4 Gy dose of X-rays and the extent of DNA damage was studied right after irradiation with the alkaline version of the single cell gel electrophoresis (SCGE) assay. To asses variability in the DNA repair competency the residual (unrepaired) DNA damage was detected again after one hour of incubation, during which irradiated cells had the condition allowing to complete the fast DNA repair process. Visible difference between DNA susceptibilities to the challenging dose was observed between investigated groups. However, variation between individuals in repair efficiency of the DNA damage induced by the challenging treatment was significantly higher in lymphocytes of prostate cancer patients than that of BPH and healthy donors (p<.05). That findings correlate with results from cytogenetic studies. Significantly higher amount of chromosomal damage was detected in irradiated lymphocytes of prostate cancer than that in BPH patients by classic cytogenetics. Results obtained by FISH technique have also shown a statistically significantly higher level of translocations frequency (4.3 ± 0.33 vs 2.9 ± 0.31, p<.005) in chromosome 1 of prostate cancer lymphocytes than that of BPH. Our results, clearly suggest that vulnerability of chromosome 1 and DNA repair competence assays have shown a possible potency as predictive assays in preclinical studies. *the MSC foundation’ fellow from Kazakh National University, Almaty, Kazakhstan

Diagnosis of bovine freemartinism by fluorescence in situ hybridization on interphase nuclei using a bovine Y chromosome-specific DNA probe

A heifer co-twin to a bull, in most cases, is a sterile freemartin which needs to be identified and culled from replacement stock. Various methods are available for the diagnosis of freemartinism, but none is ideal in terms of speed, sensitivity, or specificity. The present study was thus conducted to develop and validate a satisfactory fluorescence in situ hybridization procedure on interphase nuclei (I-FISH) for identifying the bovine XX/XY-karyotypic chimerism, the hallmark of freemartinism. A 190-bp DNA FISH probe containing the bovine male-specific BC1.2 DNA sequence was synthesized and labeled with digoxigenin by PCR. The FISH was performed on metaphase spreads and interphase nuclei of blood lymphocytes. Upon FISH, the probe expectedly bound to the nucleus of the male cell or to a region of the p12 locus of the Y chromosome. Twenty-four young heterosexual twins (Holstein-Friesian and Korean Cattle breeds; 10 pairs and 4 singletons) were analyzed in the present study; all but three exhibited the XX/XY-karyotypic chimerism to varying extents in both I-FISH and karyotyping. One heifer was identified to have 100% XX cells by both analyses, whereas two bulls were judged as 100% XY- and XX/XY-chimeric karyotypes by karyotyping and I-FISH, respectively. Nevertheless, the ratios of the XY to XX cells in these animals were very similar between the two analyses. In conclusion, the present I-FISH was a rapid and reliable procedure that can be used for early-life diagnosis of bovine freemartinism.

A novel algorithm for reliable detection of human papillomavirus in paraffin embedded head and neck cancer specimen

Human papillomavirus type 16 (HPV16) plays a role in the development of a subgroup of head and neck squamous cell carcinomas (HNSCC). However, uncertainty exists about the true impact of HPV in this tumor type as conflicting reports have been published with prevalence rates from 0 to 100%. We aimed to find a detection algorithm of a biologically and thus clinically meaningful infection, applicable for high-throughput screening of frozen and formalin-fixed paraffin embedded (FFPE) specimens. By considering detection of HPV E6 oncogene expression in frozen biopsies as gold standard for a meaningful HPV infection, the value of several assays was evaluated on FFPE tumor specimens and sera of 48 HNSCC patients. The following assays were evaluated on FFPE tissue samples: HPV DNA general primer (GP)5+/6+ PCR, viral load analysis, HPV16 DNA FISH detection, HPV16 E6 mRNA RT-PCR, p16 immunostaining, and on corresponding serum samples detection of antibodies against the HPV16 proteins L1, E6 and E7. Comparing single assays on FFPE tissue samples detection of E6 expression by RT-PCR was superior, but application remains at present limited to HPV16 detection. Most suitable algorithm with 100% sensitivity and specificity appeared p16 immunostaining followed by GP5+/6+ PCR on the p16-positive cases. We show that clinically meaningful viral HPV infections can be more reliably measured in FFPE HNSCC samples in a standard and high throughput manner, paving the way for prognostic and experimental vaccination studies, regarding not only HNSCC, but possibly also cancer types with HPV involvement in subgroups such as penile and anal cancer.

Early S phase DNA replication: A search for targets of carcinogenesis

Our studies have revealed that replicating DNA is more vulnerable to adduction than is non-replicating DNA. Contrary to our expectations that the vulnerability to neoplastic transformation induced by carcinogens in synchronized cells would parallel the rate of DNA replication, we actually found that the vulnerability was notably increased early in the S phase and more closely paralleled the rate of entry cells into the S phase (the very beginning of S phase) rather than the overall rate of DNA synthesis. From these findings we hypothesized that there were targets for the neoplastic transformation of cells that were among the earliest replicated sequences in the genome. To test that this hypothesis was plausible we investigated the temporal order of DNA replication during the S phase and showed that the order of DNA replication was far more precisely defined than had been recognized previously. The cell synchronization techniques that made those findings possible made it feasible to demonstrate that only a relatively few sites of DNA replication are identifiable in chromosomal bands at the earliest times in the S phase. The same synchronization techniques enabled us to label DNA replicated when populations of cells were very early in S phase and to isolate and clone this DNA. The clonal elements of this library of DNA prepared in this manner have been sequenced and mapped to the human genome. Efforts are in progress to characterize the genes and sequence features associated with these regions. We have utilized methods to identify and characterize origins of DNA replication as a means of locating the earliest replicating part of these early replicating regions. We have identified several new origins of DNA replication that are activated early and late in the S phase but the features of the chromatin at the origin that determines its time of activation remain obscure. In an effort to improve our ability to identify more origins, particularly adjacent origins in genomic regions, we have combined the methods of DNA combing and FISH analysis of combed DNA to search for DNA precursor incorporation patterns characteristic of origins of DNA replication. Preliminary nascent strand abundance studies appear to have proven the existence of two origins of DNA replication predicted from the precursor incorporation studies. We have found that the combed DNA techniques can be combined with precursor incorporation studies and antibodies to sites of DNA damage to address questions of mechanisms of DNA damage and repair. For example these studies have shown recently that DNA damage is not randomly distributed in the genome and that both inhibition of replicon initiation and inhibition of strand elongation are separately distinguishable as components of the S checkpoint function. It is our hope and expectation that these results and the opportunities that they provide for future studies will enable us to identify possible targets for malignant transformation that explain our observation that cells at the start of S phase are vulnerable to the initiation of carcinogenesis.

Efficient detection of genetic abnormalities in Barrett??s esophagus patients by automated analysis of DNA FISH on brush cytology specimens

Rygiel, A1; Bergman, JJGHM2; van Baal, JWPM1; Milano, F1; Peppelenbosh, MP3; Krishnadath, KK2 Author Information

Nuclear Dynamics and Gene Activation during Erythroid Maturation.

We have investigated the relationships among nuclear positioning, association with RNA polymerase II (PolII) and expression of the murine β-globin locus during erythroid differentiation, as well as the role of the locus control region (LCR) in these processes. Fetal liver cells from wildtype and LCR-deletion mouse strains were stained with a panel of antibodies, and flow cytometry was used to define and isolate cells from four stages of erythropoiesis spanning pro-erythroblasts (stage 1) to orthochromatic normoblasts and nucleated RBC (stage 4). DNA FISH analyses reveal that with increasing erythroid maturation the β-globin locus is less likely to be located in the nuclear periphery. Immuno-FISH demonstrates that PolII speckles initially are spread diffusely throughout the nucleus, with the exception of extreme periphery. With erythroid maturation the number of speckles decline and become more centrally located. Combined DNA FISH /PolII immuno-staining reveals an increase in co-localization of PolII and β-globin alleles during erythroid maturation. These results are consistent with a model in which the locus is more likely to be in the nuclear periphery and away from PolII foci prior to activation, and with maturation, the locus re-locates more centrally and associates with PolII speckles, leading to β-globin activation. To investigate this model, α and β-globin primary transcript FISH were performed and revealed that while β activation lags behind α, both start at stage 2 and increase with maturation. Notably, activation occurs prior to the increased localization of the locus away from the periphery during maturation, suggesting that localization of the locus away from the periphery may be secondary to the redistribution of PolII during erythroid maturation. Analysis of sorted erythroid cells from mice with a targeted deletion of the LCR reveals less co-localization of the β-globin locus and PolII speckles and loss of re-localization away from the periphery during maturation. Moreover, primary transcript FISH analysis of sorted cells from mice lacking the LCR and those carrying a combined deletion of HS 2 and 3 suggests that the LCR affects the likelihood that an allele is expressed, as well as the amount of transcript generated during periods of expression. Taken together our results suggest that the LCR plays a role in locating to, or stabilization of, interactions between the locus and PolII speckles, increasing the probability of β-globin expression, after which the LCR also affects the rate of transcription.

Multi-target interphase fluorescence in situ hybridization assay increases sensitivity of sputum cytology as a predictor of lung cancer

Survival rates for lung cancer are low because patients have disseminated disease at diagnosis; therefore tests for early diagnosis are highly desirable. This pilot study investigated occurrence of chromosomal aneusomy in sputum from a 33 case-control cohort matched on age, gender, and date of sample collection. Subjects had chronic obstructive pulmonary disease and ≥30 pack-years of tobacco use, and aneusomy was tested using a multi-target DNA FISH assay (LAVysion, Abbott/Vysis). In specimens collected within 12 months of lung cancer diagnosis, abnormality was more frequent among the 18 cases (41%) than the 17 controls (6%; P = 0.04). Aneusomy had no significant association with cytologic atypia, which might indicate that molecular and morphological changes could be independent markers of tumorigenesis. Combining both tests, abnormality was found in 83% of the cases and 20% of the controls ( P = 0.0004) suggesting that FISH may improve the sensitivity of cytologic atypia as a predictor of lung cancer.

Bladder Transitional Cell Carcinomas: A Comparative Study of Washing and Tumor Bioptic Samples by DNA Flow Cytometry and FISH Analyses

Objective and Methods: We compared information obtained from samples of tumor biopsy and bladder washing to evaluate the representatives of the latter type of sampling. Both types of samples from 44 cases of superficial bladder TCCs and one papilloma were analyzed by FCM, to evaluate cellular DNA content, and FISH, to detect numerical aberration of chromosome 9. Results: The use of both tumor and washing samples by FCM and FISH analyses evidenced alterations in 95% of cases. FCM evidenced higher aneuploid frequency in bladder washing than in bioptic specimens. In bladder washing, FISH analysis showed higher frequency of monosomy and lower frequency of trisomy than in biopsy. No correlation was found between histological grade and centromeric chromosome 9 loss while correlation was evidenced with centromeric 9 gain. Conclusion: Irrigation specimens, analyzed by FCM and FISH, can complement information obtained by biopsy in cytodiagnosis and follow–up of patients with bladder TCC.

Deletion of a region that is a candidate for the difference between the deletion forms of hereditary persistence of fetal hemoglobin and deltabeta-thalassemia affects beta- but not gamma-globin gene expression.

The analysis of a number of cases of beta-globin thalassemia and hereditary persistence of fetal hemoglobin (HPFH) due to large deletions in the beta-globin locus has led to the identification of several DNA elements that have been implicated in the switch from human fetal gamma- to adult beta-globin gene expression. We have tested this hypothesis for an element that covers the minimal distance between the thalassemia and HPFH deletions and is thought to be responsible for the difference between a deletion HPFH and deltabeta-thalassemia, located 5' of the delta-globin gene. This element has been deleted from a yeast artificial chromosome (YAC) containing the complete human beta-globin locus. Analysis of this modified YAC in transgenic mice shows that early embryonic expression is unaffected, but in the fetal liver it is subject to position effects. In addition, the efficiency of transcription of the beta-globin gene is decreased, but the developmental silencing of the gamma-globin genes is unaffected by the deletion. These results show that the deleted element is involved in the activation of the beta-globin gene perhaps through the loss of a structural function required for gene activation by long-range interactions.

Oestradiol, a new hapten for detecting nucleic acid sequences by FISH.

Oestradiol has been conjugated to allylamine-dUTP with an 11-atom spacer to allow enzymatic incorporation of the label into DNA sequences. In a comparative DNA and mRNA FISH study we have used DNA probes that were either labelled with digoxigenin, biotin or oestradiol. Results show that oestradiol-labelled probes can detect DNA and RNA sequences in FISH equally well as digoxigenin- and biotin-labelled probes. Further, no crossreactivity between the various hapten-specific antibodies and the three haptens were observed. Binding of the rabbit anti-oestradiol antibody to endogenous oestrogen in various tissues was not observed under the conditions tested. In view of the increasing demands for multi-colour DNA and mRNA FISH applications, oestradiol is a welcome addition to the collection of haptens employed in FISH.