In previous work, we showed that telomeres of normal cells are organized within the 3D space of the interphase nucleus in a nonoverlapping and cell cycle-dependent manner. This order is distorted in tumor cell nuclei where telomeres are found in close association forming aggregates of various numbers and sizes. Here we show that c-Myc overexpression induces telomeric aggregations in the interphase nucleus. Directly proportional to the duration of c-Myc deregulation, we observe three or five cycles of telomeric aggregate formation in interphase nuclei. These cycles reflect the onset and propagation of breakage-bridge-fusion cycles that are initiated by end-to-end telomeric fusions of chromosomes. Subsequent to initial chromosomal breakages, new fusions follow and the breakage-bridge-fusion cycles continue. During this time, nonreciprocal translocations are generated. c-Myc-dependent remodeling of the organization of telomeres thus precedes the onset of genomic instability and subsequently leads to chromosomal rearrangements. Our findings reveal that c-Myc possesses the ability to structurally modify chromosomes through telomeric fusions, thereby reorganizing the genetic information.genomic instability ͉ 3D nucleus ͉ breakage-bridge-fusion

The role of UVA-radiation-the major fraction in sunlight-in human skin carcinogenesis is still elusive. We here report that different UVA exposure regime (4 Â 5 J/cm 2 per week or 1 Â 20 J/cm 2 per week) caused tumorigenic conversion (tumors in nude mice) of the HaCaT skin keratinocytes. While tumorigenicity was not associated with general telomere shortening, we found new chromosomal changes characteristic for each recultivated tumor. Since this suggested a nontelomere-dependent relationship between UVA irradiation and chromosomal aberrations, we investigated for alternate mechanisms of UVA-dependent genomic instability. Using the alkaline and neutral comet assay as well as c-H2AX foci formation on irradiated HaCaT cells (20-60 J/cm 2 ), we show a dosedependent and long lasting induction of DNA single and double (ds) strand breaks. Extending this to normal human skin keratinocytes, we demonstrate a comparable damage response and, additionally, a significant induction and maintenance of micronuclei (MN) with more acentric fragments (indicative of ds breaks) than entire chromosomes particularly 5 days post irradiation. Thus, physiologically relevant UVA doses cause long-lasting DNA strand breaks, a prerequisite for chromosomal aberration that most likely contribute to tumorigenic conversion of the HaCaT cells. Since normal keratinocytes responded similarly, UVA may likewise contribute to the complex karyotype characteristic for human skin carcinomas.

Background: The observation of multiple genetic markers in situ by optical microscopy and their relevance to the study of three-dimensional (3D) chromosomal organization in the nucleus have been greatly developed in the last decade. These methods are important in cancer research because cancer is characterized by multiple alterations that affect the modulation of gene expression and the stability of the genome. It is, therefore, essential to analyze the 3D genome organization of the interphase nucleus in both normal and cancer cells.

Telomerase- and telomere length regulation in normal human tissues is still poorly understood. We show here that telomerase is expressed in the epidermis in situ independent of age but was repressed upon the passaging of keratinocytes in monolayer culture. However, when keratinocytes were grown in organotypic cultures (OTCs), telomerase was re-established, indicating that telomerase activity is not merely proliferation-associated but is regulated in a tissue context-dependent manner in human keratinocytes. While not inducible by growth factors, treatment with the histone deacetylation inhibitor FK228 restored telomerase activity in keratinocytes grown in monolayer cultures. Accordingly, CHIP analyses demonstrated an acetylated, active hTERT promoter in the epidermis in situ and in the epidermis of OTCs but a deacetylated, silenced hTERT promoter with subsequent propagation in monolayer culture suggesting that histone acetylation is part of the regulatory program to guarantee hTERT expression/telomerase activity in the epidermis. In agreement with the loss of telomerase activity, telomeres shortened during continuous propagation in monolayer culture by an average of approximately 70 base pairs (bp) per population doubling (pd). However, telomere erosion varied strongly between different keratinocyte strains and even between individual cells within the same culture, thereby arguing against a defined rate of telomere loss per replication cycle. In the epidermis in situ, as determined from early-passage keratinocytes and tissue sections from different age donors, we calculated a telomere loss of only approximately 25 bp per year. Since we determined the same rate for the non-regenerating melanocytes and dermal fibroblasts, our data suggest that in human epidermis telomerase is a protective mechanism against excessive telomere loss during the life-long regeneration.

Formation of a well structured epidermis strictly depends on a tight balance between proliferation and differentiation. Accordingly, telomerase, which is restricted to proliferating cells, is downregulated with differentiation. It is unclear, however, whether this inhibition is essential to or only a consequence of the differentiation process. By studying different variants of the HaCaT skin keratinocytes we now show that constitutive overexpression of human telomerase reverse transcriptase (hTERT) in HaCaT-TERT cells (lacking its own differentiation-sensitive promoter) and constitutive expression of the c-myc gene in HaCaT-myc cells caused increased proliferation in conventional cultures; however, this proliferative advantage was not maintained in tissue-like organotypic cocultures. Despite reduced stratification, HaCaT-myc cells were still able to develop a fully differentiated epithelium. HaCaT-TERT cultures, on the other hand, expressed all markers of early but not of terminal differentiation. The failure to differentiate terminally was observed in hTERT mass cultures and individual clones and correlated with an intense nuclear hTERT staining of the uppermost cells of the HaCaT-TERT epithelia. Thus, our data suggest that constitutive overexpression of hTERT does not interfere with epidermal differentiation per se but blocks the terminal stage of differentiation and therefore indicates that hTERT/telomerase plays an active part in the regulatory pathway of epidermal differentiation.

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We describe a boy with multiple congenital anomalies including a complex heart defect, club feet, adducted thumbs, and facial dysmorphic features. He died at the age of 2 months following cardiac surgery. G-banding analysis identified an abnormal chromosome 5q suspected to be an interstitial deletion (5)(q33q35). Breakpoints of the deleted segment were confirmed as del(5)(q33.3q35) by multicolor fluorescence in situ hybridization (FISH) using two sets of combinatorially labeled band specific YAC clones. Findings are discussed in view of previously published cases.

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