Topological organization of the MYC/IGK locus in Burkitt's lymphoma cells assessed by nuclear halo preparations.

In Burkitt's lymphoma (BL) cells characteristic chromosomal translocations juxtapose the MYC oncogene to one of the three immunoglobulin (IG) gene loci. This results in deregulation of MYC expression through IG gene enhancer elements. As enhancers and MYC promoters can be as much as several hundred kilobases apart, long-distance effects are to be postulated, which affect chromatin organization. Since transcriptionally active and inactive sequences can be distinguished based on their localization in nuclear halo preparations, we used this technique to assess the topology of wild-type and translocated MYC and IGK genes. Following visualization of these genes by fluorescence in situ hybridization, the signal distribution was determined in nuclear halo structures of human monocytes and the BL-derived cell line LY66. MYC signals derived from the non-translocated chromosome 8 were found equally distributed between the residual nucleus and the surrounding DNA halo. In contrast, the activated MYC and IGK genes on the translocated chromosome in LY66 cells were associated with the residual nucleus in 78 and 88% of cases, respectively. In LY66 cells, attachment to the residual nucleus was restricted to a DNA segment 30 to 50 kb downstream of MYC, while in monocytes it was dispersed over 80 kb around the MYC gene. These findings indicate a specific chromatin organization for the activated MYC locus. Distance measurements between MYC and IGK signals revealed shorter values than expected from their linear distance (325 kb), indicating a back folding of the DNA backbone. Thus, there is strong evidence for a specific topological organization, which is functionally related to the MYC activation status with the specific folding of the DNA strand likely reflecting maintenance of a spatial interaction between IGK enhancer and MYC promoter elements.

Overriding of cyclin-dependent kinase inhibitors by high and low risk human papillomavirus types: evidence for an in vivo role in cervical lesions.

High risk types of human papillomavirus (HPV) are agents in the aetiology of cervical carcinoma. The products of two early genes, E6 and E7, appear to be the principal transforming proteins. Studies of various monolayer cell culture systems have shown that the E7 oncoprotein of human papillomavirus type 16 is able to neutralize or bypass the inhibitory effect of the cell cycle-dependent kinase (CDK) inhibitors (CKIs) p21WAF1/CIP1 and p27KIP1. To understand whether the p21WAF1/CIP1 or p27KIP1 neutralization also plays a role in vivo, we performed studies on clinical specimens. Forty-five cervical biopsies, including HPV-negative mucosa, HPV 16-positive preinvasive (low and high grade lesions) and invasive neoplasia as well as HPV 6-positive condyloma acuminatum were analysed by single and double immunohistology. We examined the positive cell cycle regulator cyclin A and the universal cell cycle marker Ki67 as well as the negative cell cycle regulators p21WAF1/CIP1 and p27KIP1. Here, we show that in a significant fraction of cells the G1 block can be overcome despite high levels of CKIs in HPV lesions. This phenomenon, which was more evident for p21WAF1/CIP1 than for p27KIP1 was most marked in low grade lesions and in condylomata acuminata, in which a high viral productivity is expected. These results indicate that the overriding of CKI inactivation by viral oncoproteins appears to be a conserved property between low and high risk HPV types. We conclude that the CKI neutralization by HPVs is likely to be required for viral DNA replication rather than for malignant transformation of the host cell.

Mapping and chromosome analysis: the potential of fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) is a method widely used for the delineation of chromosomal DNA. FISH is applied in many areas of basic research as well as in clinical cytogenetics. In this review important technical improvements as well as the various applications of this method are summarized. In the first part different labeling and detection procedures are described and the potential of various kinds of probes are discussed. Recent developments in optical instrumentation and digital imaging procedures are outlined in the second part. The following important applications of FISH are discussed: (a) new strategies for high resolution mapping of DNA sequences; (b) detection of chromosomal aberrations in clinical material; (c) techniques allowing the simultaneous detection of numerous probes by multiple color FISH; and (d) the new approach of comparative genomic hybridization, allowing a rapid and comprehensive analysis of chromosomal imbalances in cell populations, which is particularly useful for the cytogenetic analysis of tumor samples.