The proliferative life span of human cells is limited by telomere shortening, but the specific telomeres responsible for determining the onset of senescence have not been adequately determined. We here identify the shortest telomeres by the frequency of signal-free ends after in situ hybridization with telomeric probes and demonstrate that probes adjacent to the shortest ends colocalize with gammaH2AX-positive DNA damage foci in senescent cells. Normal BJ cells growth arrest at senescence before developing significant karyotypic abnormalities. We also identify all of the telomeres involved in end-associations in BJ fibroblasts whose cell-cycle arrest at the time of replicative senescence has been blocked and demonstrate that the 10% of the telomeres with the shortest ends are involved in >90% of all end-associations. The failure to find telomeric end-associations in near-senescent normal BJ metaphases, the presence of signal-free ends in 90% of near-senescent metaphases, and the colocalization of short telomeres with DNA damage foci in senescent interphase cells suggests that end-associations rather than damage signals from short telomeres per se may be the proximate cause of growth arrest. These results demonstrate that a specific group of chromosomes with the shortest telomeres rather than either all or only one or two sentinel telomeres is responsible for the induction of replicative senescence.

5-Fluorouracil (5-FU) is the chemotherapeutic drug of choice for the treatment of metastatic colorectal cancer, but resistance to 5-FU remains a major obstacle to successful therapy. We generated 5-FU-resistant derivatives of the HCT116 human colon cancer cell line by serial passage of these cells in the presence of increasing 5-FU concentrations in an attempt to elucidate the biological mechanisms involved in resistance to 5-FU. Two resultant resistant derivatives, HCT116 ResB and ResD, were characterized for resistance phenotypes, genotypes, and gene expression using cells maintained long-term in 5-FU-free media. Compared to parental HCT116 cells that respond to 5-FU challenge by inducing high levels of apoptosis, ResB and ResD derivatives had significantly reduced apoptotic fractions when transiently challenged with 5-FU. ResB and ResD cells were respectively 27- and 121-fold more resistant to 5-FU, had increased doubling times, and significantly increased plating efficiencies compared to the parental cells. Both resistant derivatives retained the wild-type TP53 genotype, TP53 copy number and CGH profile characteristic of the parental line. Alterations in gene expression in the resistant derivatives compared to the parental line were assessed using oligonucleotide microarrays. Overall, the 5-FU-resistant derivatives were characterized by reduced apoptosis and a more aggressive growth phenotype, consistent with the observed up-regulation of apoptosis-inhibitory genes (e.g., IRAK1, MALT1, BIRC5), positive growth-regulatory genes (e.g., CCND3, CCNE2, CCNF, CYR61), and metastasis genes (e.g., LMNB1, F3, TMSNB), and down-regulation of apoptosis-promoting genes (e.g., BNIP3, BNIP3L, FOXO3A) and negative growth-regulatory genes (e.g., AREG, CCNG2, CDKN1A, CDKN1C, GADD45A). 5-FU metabolism-associated genes (e.g., TYMS, DTYMK, UP) and DNA repair genes (e.g., FEN1, FANCG, RAD23B) were also up-regulated in one or both resistant derivatives, suggesting that the resistant derivatives might be able to overcome both 5-FU inhibition of thymidylate synthase and the DNA damage caused by 5-FU, respectively. Development of 5-FU resistance thus appears to encompass deregulation of apoptosis-, proliferation-, DNA repair-, and metastasis-associated regulatory pathways.

BACKGROUND: Chromosomal mosaicism in human embryos may give rise to false positive or false negative results in preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). Therefore, we have investigated whether the results obtained from a 2-cell biopsy of frozen-thawed embryos and fluorescence in situ hybridization (FISH) analysis are representative for the chromosome constitution of the remaining embryo on day 5. METHODS: Cryopreserved day 3 embryos were thawed and from surviving embryos two blastomeres were biopsied. FISH analysis was performed for chromosomes 1, 7, 13, 15, 16, 18, 21, 22, X and Y. After biopsy, the embryos were cultured until day 5 and further analysed using the same probe panels. RESULTS: In all, 17 embryos were available with a diagnosis based on two blastomeres on day 3 and confirmatory studies on day 5. In 10 of these 17 cases the initial diagnosis could be confirmed. However, in only six cases cytogenetic results were concordant. Besides the 10 cases with a 'correct' diagnosis, there were six false positive results and one false negative, all involving mosaicism. CONCLUSIONS: Investigating the chromosomal constitution of two blastomere nuclei offers a good opportunity to study the incidence of chromosomal mosaicism in early embryo development. The confirmation rate of the results obtained on day 3 depends on the interpretation and is higher when considered from a clinical than from a cytogenetic point of view.

Precise breakpoint definition of chromosomal rearrangements using conventional banding techniques often fails, especially when more than two breakpoints are involved. The classic banding procedure results in a pattern of alternating light and dark bands. Hence, in banded chromosomes a specific chromosomal band is rather identified by the surrounding banding pattern than by its own specific morphology. In chromosomal rearrangements the original pattern is altered and therefore the unequivocal determination of breakpoints is not obvious. The multicolor banding technique (mBAND, see Chudoba et al., 1999) is able to identify breakpoints unambiguously, even in highly complex chromosomal aberrations. The mBAND technique is presented and illustrated in a case of intrachromosomal rearrangement with seven breakpoints all having occurred on one chromosome 16, emphasizing the unique analyzing power of mBAND as compared to conventional banding techniques.

No abstract available.

Routine cytogenetic analysis provides important information of diagnostic and prognostic relevance for hematological malignancies. In spite of this, poorly spread metaphase chromosomes and highly rearranged karyotypes with numerous marker chromosomes, are often difficult to interpret. In order to improve the definition of chromosomal breakpoints multicolor banding (MCB) was applied on 45 bone marrow samples from patients suffering from hematological malignancies like myelodysplastic syndrome (MDS), acute myelocytic leukemia (AML), chronic myelocytic leukemia (CML) or acute lymphoblastic leukemia (ALL). The breakpoints defined by GTG banding were confirmed by MCB in 8 cases, while in the remaining 37 cases the breakpoints had to be redefined. In 20/45 cases the breakpoints could only be characterized after application of MCB. In summary, 73 different breakpoints were characterized, thereof 33 were previously undescribed. Eleven cases showed known acquired aberrations and 21 cases had previously described aberration types such as del(5q-), del(7q-), del(13q-) or t(1;5) as sole rearrangement or in connection with other complex ones. In a total of 11 cases 19 breakpoints as described before were involved in hematological malignancies, while in 14 cases 33 breakpoints were identified which have not been described previously. Thus, MCB has proven to be a powerful and reliable method for screening of chromosomal aberrations, which considerably increased the accuracy of cytogenetic diagnosis.

BACKGROUND AND METHODS: 44 peripheral nerve sheath tumors (PNST) (27 schwannomas, 9 neurofibromas and 8 malignant peripheral nerve sheath tumors (MPNST)) were analyzed to determine DNA ploidy pattern and to clarify the conflicting data in the literature concerning this topic (whether benign PNSTs are aneuploid or not). For further insight we analyzed 6 schwannomas, one atypical neurofibroma and five MPNSTs by fluorescence in situ hybridization (FISH) technique using centromeric chromosome probes (7, 17 and 18) and automatic image analysis station, Metafer 4. RESULTS: Benign schwannomas (including the problematic variants as ancient, cellular, neuroblastoma like and multiplex schwannomas) could be characterized by euploid-polyploidisation and by their 4c peak height value which was usually more than 10% of total cell number measured. These characters were not found among neurofibromas and MPNST-s. FISH analysis revealed and confirmed that the 'normal' euploid-polyploid cells are mainly eusomic-polysomic containing two, four, eight or sixteen signals for each chromosomes examined, but in a small proportion aneusomy was found among tumor cells of benign schwannomas (average: 2.58; range 1.33-3.44). In contrast, the atypical neurofibroma displayed marked aneusomy (18.44%) but it contained normal eusomic and polysomic cells too. Two diploid MPNSTs proved to be clearly aneusomic with trisomy of chromosome 17 and monosomy of chromosome 18. CONCLUSIONS: All these data suggest that ploidy pattern determination combined with FISH analysis may be a very useful supplementary tool for making a right diagnosis (to differentiate benign versus malignant schwannomas in problematic variants) and to understand better the malignant transformation in PNSTs.

Tumor samples of 53 patients with t(11;14)-positive mantle cell lymphomas (MCLs) were analyzed by matrix-based comparative genomic hybridization (matrix-CGH) using a dedicated DNA array. In 49 cases, genomic aberrations were identified. In comparison to chromosomal CGH, a 50% higher number of aberrations was found and the high specificity of matrix-CGH was demonstrated by fluorescence in situ hybridization (FISH) analyses. The 11q gains and 13q34 deletions, which have not been described as frequent genomic aberrations in MCL, were identified by matrix-CGH in 15 and 26 cases, respectively. For several genomic aberrations, novel consensus regions were defined: 8p21 (size of the consensus region, 2.4 megabase pairs [Mbp]; candidate genes: TNFRSF10B, TNFRSF10C, TNFRSF10D); 10p13 (2.7 Mbp; BMI1); 11q13 (1.4 Mbp; RELA); 11q13 (5.2 Mbp; CCND1); 13q14 (0.4 Mbp; RFP2, BCMSUN) and 13q34 (6.9 Mbp). In univariate analyses correlating genomic aberrations and clinical course, 8p- and 13q14- deletions were associated with an inferior overall survival. These data provide a basis for further studies focusing on the identification of pathogenetically or clinically relevant genes in MCL.

Multicolor fluorescence in situ hybridization (FISH) assays are nowadays indispensable for a precise description of complex chromosomal rearrangements. Routine application of such techniques on human chromosomes started in 1996 with the simultaneous use of all 24 human whole chromosome painting probes in multiplex-FISH (M-FISH) and spectral karyotyping (SKY). Since then different approaches for chromosomal differentiation based on multicolor-FISH (mFISH) assays have been described. Predominantly, they have been established to characterize marker chromosomes identified in conventional banding analysis. Their characterization is of high clinical impact and is the requisite condition for further molecular investigations aimed at the identification of disease-related genes. Here we present a review on the available mFISH methods including their advantages, limitations and possible applications.

Intratumor heterogeneity in chromosomal aberrations is believed to represent a major challenge in the treatment of cancer. The aim of our work was to assess the chromosomal heterogeneity of advanced cervical carcinomas and to distinguish aberrations that had occurred at a late stage of the disease from early events. A total of 55 biopsies, sampled from 2-4 different sites within 20 tumors, were analyzed by use of comparative genomic hybridization. Heterogeneous aberrations were identified as those present in at least 1 of the biopsies and which were not seen, nor seen as a tendency, in the others of the same tumor. The homogeneous aberrations were those seen in all biopsies of the tumor. The most frequent homogeneous aberrations were gain of 3q (65%), 20q (65%) and 5p (50%), indicating that these are early events in the development of the disease. Chromosomal heterogeneity was observed in 11 tumors. The most frequent heterogeneous aberrations were loss of 4p14-q25 (60% of 10 cases with this aberration), and gain of 2p22-pter (50% of 6 cases), 11qcen-q13 (33% of 9 cases) and 8q (27% of 11 cases), suggesting that these events promote progression at a later stage. Many of the heterogeneous regions contained genes known to influence the prognosis of cervical cancer, such as 7p (EGFR), 8q (c-MYC), 11qcen-q13 (CCND1) and 17q (ERBB2). Three evolution sequences for the subpopulations in the heterogeneous tumors were identified: a serial, a parallel and a mixed sequence. In 2 tumors with a serial sequence, it was indicated that the aberrations +8 and -X had occurred after the other heterogeneous aberrations and hence were the aberrations most recently formed. Our results suggest pronounced chromosomal instability in advanced cervical carcinomas. Moreover, aggressive and treatment-resistant subpopulations may emerge at a late stage and possibly contribute to a poor prognosis of the advanced stages.

<p>Persistant infection with human papillomavirus (HPV) of the uterine cervix is related with cytological atypia (SIL), the oncogenic potential of which is unclear in a given time point of monitoring. HPV-induced genetic instability result in polyploidization as well as in low frequency random chromosome aberrations in squamous cells. In the present work we analyzed whether highly polyploid/aneuploid cells reflect genomic changes at the chromosomal level. 13 samples with the cytological diagnosis of HSIL were analyzed for HPV type and nuclear DNA content measured by laser scanning cytometry (LSC). Hyperdiploid cells with &gt;5c and with &gt;9c DNA content were further analyzed for numerical aberrations of the chromosomes 3 and 17 by fluorescence in situ hybridization (FISH) following repositioning. Cells with &gt;5c DNA content were found more frequently than cells with &gt;9c DNA content (5-98 and 1-44 cells, respectively). The FISH analysis demonstrated frequent polysomies, however, the rate of aneusomy (other than 2, 4, 8 or 16 chromosome copies) was significantly higher in cells with &gt;9c DNA content than in cells with &gt;5c DNA content or the normal diploid cells. The imbalance of chromosome 3 and 17 copy number was also increased in cells with &gt;9c DNA content. Moreover, in three out of the 13 analyzed HSIL samples, recurrent abnormal chromosome 3/17 ratio was demonstrated in a significant part of the cells, indicating a common origin of these cells. Highly polyploid/aneuploid cells in HSIL accumulate cytogenetic aberrations detectable by FISH analysis. These cells may reflect early changes with tumorigenic potential in a very concentrated fashion.</p>

Chromosome 17 is severely rearranged in breast cancer. Whereas the short arm undergoes frequent losses, the long arm harbors complex combinations of gains and losses. In this work we present a comprehensive study of quantitative anomalies at chromosome 17 by genomic array-comparative genomic hybridization and of associated RNA expression changes by cDNA arrays. We built a genomic array covering the entire chromosome at an average density of 1 clone per 0.5 Mb, and patterns of gains and losses were characterized in 30 breast cancer cell lines and 22 primary tumors. Genomic profiles indicated severe rearrangements. Compiling data from all samples, we subdivided chromosome 17 into 13 consensus segments: 4 regions showing mainly losses, 6 regions showing mainly gains, and 3 regions showing either gains or losses. Within these segments, smallest regions of overlap were defined (17 for gains and 16 for losses). Expression profiles were analyzed by means of cDNA arrays comprising 358 known genes at 17q. Comparison of expression changes with quantitative anomalies revealed that about half of the genes were consistently affected by copy number changes. We identified 85 genes overexpressed when gained (39 of which mapped within the smallest regions of overlap), 67 genes underexpressed when lost (32 of which mapped to minimal intervals of losses), and, interestingly, 32 genes showing reduced expression when gained. Candidate genes identified in this study belong to very diverse functional groups, and a number of them are novel candidates.

Telomere length was evaluated by terminal repeat fragment method (TRF) in 50 patients with myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) arising from MDS and in 21 patients with untreated primary AML to ascertain, whether telomere erosion was associated with progression of MDS towards overt leukemia. Heterogeneity of TRF among MDS FAB subgroups (P=0.004) originated from its shortening in increased number of patients during progression of the disease. Chromosomal aberrations were present in 32% MDS patients with more eroded telomeres (P=0.022), nevertheless a difference between mean TRF in the subgroups with normal and abnormal karyotype diminished during progression of MDS. A negative correlation between individual TRF and IPSS value (P=0.039) showed that telomere dynamics might serve as a useful prognostic factor for assessment of an individual MDS patient’s risk and for decision of an optimal treatment strategy.

The 8p11-21 region is a frequent target of alterations in breast cancer and other carcinomas. We surveyed 34 breast tumor cell lines and 9 pancreatic cancer cell lines for alterations of this region by use of multicolor fluorescence in situ hybridization (M-FISH) and BAC-specific FISH. We describe a recurrent chromosome translocation breakpoint that targets the NRG1 gene on 8p12. NRG1 encodes growth factors of the neuregulin/heregulin-1 family that are ligands for tyrosine kinase receptors of the ERBB family. Breakpoints within the NRG1 gene were found in four of the breast tumor cell lines: ZR-75-1, in a dic(8;11); HCC1937, in a t(8;10)(p12;p12.1); SUM-52, in an hsr(8)(p12); UACC-812, in a t(3;8); and in two of the pancreatic cancer cell lines: PaTu I, in a der(8)t(4;8); and SUIT-2, in a del(8)(p). Mapping by two-color FISH showed that the breaks were scattered over 1.1 Mb within the NRG1 gene. It is already known that the MDA-MB-175 breast tumor cell line has a dic(8;11), with a breakpoint in NRG1 that fuses NRG1 to the DOC4 gene on 11q13. Thus, we have found a total of seven breakpoints, in two types of cancer cell lines, that target the NRG1 gene. This suggests that the NRG1 locus is a recurring target of translocations in carcinomas. PCR analysis of reverse-transcribed cell line RNAs revealed an extensive complexity of the NRG1 transcripts but failed to detect a consistent pattern of mRNA isoforms in the cell lines with NRG1 breakpoint.

The detection of disseminated tumor cells (DTCs) in the hematopoetic system is important for various reasons. It is essential for tumor staging. According to the International Neuroblastoma Staging System (INSS) only the cytomorphological examination of bone marrow smears is accepted despite the fact that an infiltrate below 0.1%, can hardly be detected and even infiltrates of more than 10% are sometimes overlooked. Another important aspect is the monitoring of the disease response to cytotoxic drugs by quantifying DTCs. Moreover, bone marrow aspirates represent an ideal source to determine the genetic and biological make up of DTCs at diagnosis and during follow up. Key issues that can be tested on DTCs are: determination of the proliferation capacity, the apoptotic rate, the drug sensitivity etc. The prerequisite for such a bone-marrow diagnosis, however, is the unequivocal identification of disseminated tumor cells. Thus, in order to avoid false positive and false negative results, which are a risk in bone-marrow diagnostics, a system was developed to distinguish tumor cells from non-neoplastic cells and to facilitate the gain of insights into the biological make-up of tumor cells more easily.