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J. Med. Assoc. Thai, 88, 1- 6

Chromosome analysis of uncultured amniocytes by comparative genomic hybridization in early amniocentesis.

A. Ketupanya, N. Aranyakasemsuk, C. Tocharoentanaphol, C. Vuthiwong

OBJECTIVE: To study chromosome analysis by comparative genomic hybridization (CGH) compared with the conventional technique in early amniocentesis. MATERIAL AND METHOD: Cross-sectional descriptive study design was performed in 32 singleton pregnant women with gestational age between 12-15 weeks. Transabdominal amniocentesis was carried out under ultrasound guidance. The amniotic fluid samples were simultaneously investigated using CGH and the conventional cytogenetics study as a gold standard. RESULTS: Amniocentesis were done for advanced maternal age in all cases. The mean maternal age was 35.8 years (35-42 years). The mean gestational age was 13.7 weeks (12-15 weeks). The chromosome analysis by CGH technique of uncultured amniocyte showed 17 normal female chromosomes (53.1%) and 15 normal male chromosomes (46.9%). This finding was the same as the conventional cytogenetics method. The mean duration of the CGH method was 6 days and that of the conventional cytogenetics method was 13.7 days (10-23 days). CONCLUSION: The CGH technique is a reliable technique for a rapid prenatal diagnosis of chromosome study in early gestation.

Invest Ophthalmol Vis Sci, 46, 2253- 2257

Concurrent loss of chromosome arm 1p and chromosome 3 predicts a decreased disease-free survival in uveal melanoma patients.

E. Kilic, N.C. Naus, van Gils, W., C.C. Klaver, van Til, M.E., M.M. Verbiest, T. Stijnen, C.M. Mooy, D. Paridaens, H.B. Beverloo, G.P. Luyten, de Klein, A.

PURPOSE: Uveal melanoma is a highly malignant disease with a mortality rate of 50% at 10 to 15 years. Previous studies have shown that chromosomal changes are associated with decreased survival of the patient. However, in these studies the small number of tumors analyzed did not allow robust statistical analysis. In the present study, the independent numerical changes in chromosomes 1, 3, 6, and 8 on disease-free survival (DFS) was assessed in a large series of patients with uveal melanoma. METHODS: One hundred twenty tumors from patients with uveal melanoma were analyzed for numerical changes in chromosomes 1, 3, 6, and 8, with cytogenetic analysis, fluorescent in situ hybridization, and/or comparative genomic hybridization. Data were correlated with disease outcome in univariate and multivariate analyses, by Kaplan-Meier and Cox regression analyses. RESULTS: At a mean follow-up time of 45 months, 42 patients had died or had metastatic disease. In the univariate analysis, loss of chromosome 3, gain of 8q, largest tumor diameter, or the presence of epithelioid cells was associated with a decreased DFS. In the multivariate analysis, the effect of monosomy 3 on survival was largely modified by changes in 1p36. Regarding all chromosomal changes, only the concurrent loss of the short arm of chromosome 1 and all of chromosome 3 was an independent prognostic parameter for disease-free survival (P < 0.001). CONCLUSIONS: In uveal melanoma, concurrent loss of the short arm of chromosome 1 and all of chromosome 3 is an independent predictor of decreased DFS.

British Journal of Cancer, 92, 382- 388

Multicolour-banding fluorescence in situ hybridization (mbanding-FISH) to identify recurrent chromosomal alterations in breast tumour cell lines.

A. Letessier, M.-J. Mozziconacci, A. Murati, J. Juriens, J. Adelaide, D. Birnbaum, M. Chaffanet

<p>Recurrent chromosome breakpoints in tumour cells may point to cancer genes, but not many have been molecularly characterised. We have used multicolour-banding fluorescence in situ hybridisation (mbanding-FISH) on breast tumour cell lines to identify regions of chromosome break created by inversions, duplications, insertions and translocations on chromosomes 1, 5, 8, 12 and 17. We delineate a total of 136 regions of break, some of them occurring with high frequency. We further describe two examples of dual-colour FISH characterisation of breakpoints, which target the 1p36 and 5p11-12 regions. Both breaks involve genes whose function is unknown to date. The mbanding-FISH strategy constitutes an efficient first step in the search for potential cancer genes.</p>

Cancer Genet Cytogenet, 163, 44- 56

Chromosomal alterations cause the high rates and wide ranges of drug resistance in cancer cells.

R. Li, R. Hehlman, R. Sachs, P. Duesberg

Conventional mutation-selection theories have failed to explain (i) how cancer cells become spontaneously resistant against cytotoxic drugs at rates of up to 10(-3) per cell generation, orders higher than gene mutation, even in cancer cells; (ii) why resistance far exceeds a challenging drug-a state termed multidrug resistance; (iii) why resistance is associated with chromosomal alterations and proportional to their numbers; and (iv) why resistance is totally dependent on aneuploidy. We propose here that cancer-specific aneuploidy generates drug resistance via chromosomal alterations. According to this mechanism, aneuploidy varies the numbers and structures of chromosomes automatically, because it corrupts the many teams of proteins that segregate, synthesize, and repair chromosomes. Aneuploidy is thus a steady source of chromosomal variation from which, in classical Darwinian terms, resistance-specific aneusomies are selected in the presence of chemotherapeutic drugs. Some of the thousands of unselected genes that hitchhike with resistance-specific aneusomies can thus generate multidrug resistance. To test this hypothesis, we determined the rates of chromosomal alterations in clonal cultures of human breast and colon cancer lines by dividing the fraction of nonclonal karyotypes by the number of generations of the clone. These rates were about 10(-2) per cell generation, orders higher than mutation. Chromosome numbers and structures were determined in metaphases hybridized with color-coded chromosome-specific DNA probes. Further, we tested puromycin-resistant subclones of these lines for resistance-specific aneusomies. Resistant subclones differed from parental lines in four to seven specific aneusomies, of which different subclones shared some. The degree of resistance was roughly proportional to the number of these aneusomies. Thus, aneuploidy is the primary cause of the high rates and wide ranges of drug resistance in cancer cells.

Cytometry, 68, 113- 120

Automatic telomere length measurements in interphase nuclei by IQ-FISH.

R. Narath, T. Lörch, K.M. Greulich-Bode, P. Boukamp, P.F. Ambros

<p>To benefit from the fluorescence-based automatic microscope (FLAME), we have adapted a PNA FISH technique to automatically determine telomere length in interphase nuclei. The method relies on the simultaneous acquisition of pan-telomeric signals and reference probe signals. We compared the quantitative figures to those for existing methods, i.e. Southern blot analysis and quantitative FISH (Q-FISH). Quantitative-FISH on interphase nuclei (IQ-FISH) allows the exact quantification of telomere length in interphase nuclei. Thus, this enables us to obtain not only exact information on the telomere length, but also morphological and topological details. The automatic measurement of large cell numbers allows the measurement of statistically relevant cell populations.</p>

Int. J. Radiat. Biol., 81, 741- 749

The radiation sensitivity of human chromosomes 2, 8, and 14 in peripheral blood lymphocytes of seven donors.

S. Sommer, I. Buraczewska, M. Wojewodzka, E. Bouzyk, I. Szumiel, A. Wojcik

PURPOSE: To investigate if deviations from DNA-proportional distribution of radiation-induced chromosomal aberrations are individually variable. MATERIALS AND METHODS: Peripheral blood lymphocytes were collected from seven healthy donors and exposed to different doses of gamma rays. Chromosomes 2, 8 and 14 were painted in different colors and aberrations scored with the help of an image-analysis system. RESULTS: Chromosome 2 was generally less sensitive than expected on the basis of DNA-proportional distribution and the extent of inter-donor variability was minimal. A higher than expected frequency of aberrations was found in chromosome 14 of five donors, while a higher than expected frequency of aberrations was found in chromosome 8 of two donors. CONCLUSIONS: Inter-donor variability may explain some of the controversies regarding the inter-chromosomal distribution of radiation-induced aberrations.

Leuk Res, 29(9), 987-93

Adequate cytogenetic examination in myelodysplastic syndromes: analysis of 529 patients

C Steidl, R Steffens, W Gassmann, B Hildebrandt, R Hilgers, U Germing, L Trümper, D Haase

In myelodysplastic syndromes (MDS), the karyotype is one of the most significant prognostic markers with profound impact on differential diagnosis and therapeutic decisions. In a retrospective study, we examined karyotypes of bone marrow specimens of an oligocentric cohort comprising 529 patients with MDS to address the question how many metaphases need to be analyzed to detect even small cell clones with an appropriate expenditure. We found a statistically significant difference of the frequency of normal karyotypes in the patient group with 19 or less analyzed metaphases compared to the group with 20 or more metaphases analyzed (56% versus 47%, p=0.041). Furthermore, we demonstrate that the analysis of 25 or more metaphases can further improve the sensitivity of karyotype analysis and leads to the identification of additional clinically relevant abnormal clones or subclones in a substantial proportion of patients. In summary, our data suggest the examination of at least 20 metaphases in MDS.

J. Histochem. Cytochem., 53, 1433- 1440

Standardization of the immunocytochemical detection of neuroblastoma cells in bone marrow.

K. Swerts, P.F. Ambros, C. Brouzes, J.M.F. Navarro, N. Gross, D. Rampling, R. Schumacher-K., A.R. Sementa, R. Ladenstein, K. Beiske

<p>Standard cytomorphological examination of bone marrow (BM) aspirates does not appear to be sensitive enough to detect single neuroblastoma cells. The SIOPEN Neuroblastoma Bone Marrow Committee developed a sensitive and reproducible anti-GD2 immunocytochemical assay and introduced morphological and immunocytological criteria for the interpretation of results. Fixed cytospins were incubated with a commercially available anti-GD2 monoclonal antibody and an APAAP kit. Cells fulfilling all morphological and immunocytological criteria were called criteria-positive cells (CPCs). Not convincingly interpretable cells fulfilled some, but not all, criteria, and negative cells displayed only exclusion criteria. The genetic profile of doubtful cells was checked by fluorescence in situ hybridization. Ideally, 3 x 10(6) cells were analyzed to reach a 95% probability of detecting one tumor cell in 1 x 10(6) mononuclear cells. Four quality control rounds were organized to validate the method. A total of 111 quality control samples were analyzed. Two main improvements were achieved: in discordant cases, the range between the lowest and highest reported result was reduced by half, and discordant results were only found in samples with less than 10 CPCs per 1 x 10(6). This article describes the first internationally standardized protocol to detect and quantify rare neuroblastoma cells by immunocytochemistry. This method is an indispensable tool for multicenter studies evaluating the clinical significance of minimal residual disease in neuroblastoma.</p>

Biochem J, 387, 703- 710

Role of compartmentalized redox-active iron in hydrogen peroxide-induced DNA damage and apoptosis.

M. Tenopoulou, P.-T. Doulias, A. Barbouti, U. Brunk, D. Galaris

<p>Jurkat cells in culture were exposed to oxidative stress in the form of continuously generated hydrogen peroxide, obtained by the addition of glucose oxidase to the medium. This treatment induced a rapid, dose-dependent increase in the ICIP (intracellular calcein-chelatable iron pool). Early destabilization of lysosomal membranes and subsequent nuclear DNA strand breaks were also observed, as evaluated by the Acridine Orange relocation test and the comet assay respectively. Somewhat later, these effects were followed by a lowered mitochondrial membrane potential, with release of cytochrome c and apoptosis-inducing factor. These events were all prevented if cells were pretreated with the potent iron chelator DFO (desferrioxamine) for a period of time (2-3 h) long enough to allow the drug to reach the lysosomal compartment following fluid-phase endocytosis. The hydrophilic calcein, a cleavage product of calcein acetoxymethyl ester following the action of cytosolic esterases, obviously does not penetrate intact lysosomal membranes, thus explaining why ICIP increased dramatically following lysosomal rupture. The rapid decrease in ICIP after addition of DFO to the medium suggests draining of cytosolic iron to the medium, rather than penetration of DFO through the plasma membrane. Most importantly, these observations directly connect oxidative stress and resultant DNA damage with lysosomal rupture and the release of redox-active iron into the cytosol and, apparently, the nucleus.</p>

Diagn Mol Pathol, 14, 77- 82

Fluorescence in situ hybridization for the detection of t(X;18)(p11.2;q11.2) in a Synovial Sarcoma tissuemicroarray using a breakapart-style probe.

J. Terry, T.S. Barry, D.E. Horsman, F.D. Hsu, A.M. Gown, D.G. Huntsman, T.O. Nielsen

Synovial sarcomas (SSs) account for 5% of soft tissue tumors and carry a balanced translocation t(X;18)(p11.2;q11.2), detectable in over 90% of cases. This translocation brings together portions of two genes: SYT and SSX. Detecting interruption of the SYT gene on chromosome 18 would be useful as a diagnostic tool. We describe a scoring method to detect disruption of SYT with breakapart probe fluorescence in situ hybridization (FISH) and the application of this method for identification of SS within a sarcoma tissue microarray. After optimization, SYT disruption was identified in 22 of 23 (96%) of known SS tumor samples but was not in 23 of 23 (100%) of non-SS sarcoma samples. Ten of 11 (91%) blinded test SS tumor samples were also correctly identified. For comparison, commercially available FISH and chromogenic in situ hybridization (CISH) probes were tested. The commercial FISH probes identified SYT disruption in 81% of the SS tumor samples but in none of the non-SS samples. The CISH probes produced signals too weak to interpret. The use of breakapart FISH probes is a relatively quick procedure for detection of synovial sarcoma translocations and can be applied to archival specimens in tissue microarrays.

Cytogenet. Genome Res., 111, 41- 45

Radiosensitivity detected by the micronucleus test is not generally increased in sporadic prostate cancer patients.

D. Varga, I. Michel, B. Patino-Garcia, T. Paiss, W. Vogel, C. Maier

The micronucleus test (MNT) has shown increased micronuclei (MN) frequencies in BRCA associated and sporadic breast cancer patients, Ataxia telangiectasia and Nijmegen Breakage Syndrome patients, demonstrating a common cellular phenotype of increased radiosensitivity. Some genes, causative of these diseases, have also recently been associated with prostate cancer. In order to investigate if prostate cancer exhibits the cellular phenotype of increased radiosensitivity, we performed MNT analysis on 22 sporadic prostate cancer patients and 43 male controls. We determined the baseline MN frequency, in order to see in vivo chromosomal damage without radiation, and induced (after irradiation with 2 Gy) frequency of MN, both in binucleated cells (BNC) obtained from cultured peripheral blood lymphocytes. An automated image analysis system was used to score the MN employing two different classifiers (Classifier A and B) for detection of BNC. The mean baseline frequencies were 48/43 MN/1000 BNC (A/B) for the controls and 42/50 (A/B) for prostate cancer patients. The induced MN frequencies amounted to 107/111 MN/1000 BNC (A/B) for controls and 111/114 MN/1000 BNC (A/B) for prostate cancer patients. The obtained MN frequencies did not result in a statistically significant difference between unselected cases and controls. However, restricting the analysis to young patients (50-60 years, N = 7) and age-matched controls (N = 7) revealed marginally significant higher MN frequencies in patients. We conclude that increased radiosensitivity is not a property of prostate cancer patients in general.

Plant J, 43, 662- 674

Telomerase-independent cell survival in Arabidopsis thaliana.

J.M. Watson, P. Bulankova, K. Riha, D.E. Shippen, B. Vyskot

Telomerase is the reverse transcriptase responsible for the maintenance of telomeric repeat sequences in most species that have been studied. Inactivation of telomerase causes telomere shortening and results in the loss of the telomere's protective function, which in mammals leads to cell-cycle arrest and apoptosis. Experiments performed on Arabidopsis thaliana mutants lacking telomerase activity revealed their unusually high tolerance for genome instability. Here we present molecular and cytogenetic analysis of two cell lines (A and B) derived from seeds of late-generation telomerase-deficient A. thaliana. These cultures have survived for about 3 years and are still viable. However, neither culture has adapted mechanisms to maintain terminal telomeric repeats. One culture (B) suffers from severe growth irregularities and a high degree of mortality. Karyological analysis revealed dramatic genomic rearrangements, a large variation in ploidy, and an extremely high percentage of anaphase bridges. The second cell line (A) survived an apparent crisis and phenotypically appears wild-type with respect to growth and morphology. Despite these indications of genome stabilization, a high percentage of anaphase bridges was observed in the A line. We conclude that the restructured chromosome termini provide the A line with partial protection from end-joining repair activities, thus allowing normal growth.

Cytogenetic and Genomic Research, 108, 217- 222

New insights into the evolution of chromosome 1.

A. Weise, H. Starke, K. Mrasek, U. Claussen, T. Liehr

A complex low-repetitive human DNA probe (BAC RP11-35B4) together with two microdissection-derived region-specific probes of the multicolor banding (MCB) probe-set for chromosome 1 were used to re-analyze the evolution of human chromosome 1 in comparison to four ape species. BAC RP11-35B4 derives from 1q21 and contains 143 kb of non-repetitive DNA; however, it produces three specific FISH signals in 1q21, 1p12 and 1p36.1 of Homo sapiens (HSA). Human chromosome 1 was studied in comparison to its homologues in Hylobates lar (HLA), Pongo pygmaeus (PPY), Gorilla gorilla (GGO) and Pan troglodytes (PTR). A duplication of sequences homologous to human 1p36.1 could be detected in PPY plus an additional signal on PPY 16q. The region homologous to HSA 1p36.1 is also duplicated in HLA, and split onto chromosomes 7q and 9p; the region homologous to HSA 1q21/1p12 is present as one region on 5q. Additionally, the breakpoint of a small pericentric inversion in the evolution of human chromosome 1 compared to other great ape species could be refined. In summary, the results obtained here are in concordance with previous reports; however, there is evidence for a deletion of regions homologous to human 1p34.2-->p34.1 during evolution in the Pongidae branch after separation of PPY.

Int J Mol Med, 16, 463- 469

Studies on the action of mitomycin C and bleomycin on telomere lengths of human lymphocyte chromosomes.

U. Wick, E. Gebhart

In order to address the problem of the action of cytostatics on chromosome ends, telomere length was measured in human lymphocyte cultures exposed to mitomycin C (MMC) and bleomycin (BLM). Telomere-specific PNA probes were used for the quantitative estimation of the relative telomere length of each individual chromosome by fluorescence in situ hybridization. A high inter-cellular and inter-individual variability of relative telomere lengths was found throughout all experiments. Different responses could be observed with respect to the action of the examined mutagens: The total average fluorescence intensity of labeled telomere repeats was decreased under the action of MMC in two of the experiments, while two revealed no significant alteration. BLM caused no significant change of total average telomeric signal intensity in four, a clear decrease in one of the six experiments, and an increase in another. Although all chromosome ends contributed to the observed trends, single telomeres were affected in a very distinct way. The highest concentration of MMC (1 microg/ml) induced significant shortening of telomeres of the chromosome arms; 2q, 3p, 5q, 7p, 10q, 11p, 13q, 17p, 18p&q, and 21q in two independent experiments. In one BLM experiment with 8 microg/ml, the most distinct decrease (p< or =0.005) of telomeric fluorescence was found at the ends of chromosome arms; 1q, 6p, 17p, 20p&q, and 22q. The increase of telomeric signal intensity affected the telomeres of some individual chromosome arms more than others, e.g. 4q, 6p, 7p, 8p, 13p, and 18q. Although the telomere length of the individual chromosome arms varied widely, clear trends could be observed with respect to the rank which was occupied by telomeric length of the various chromosome arms. The telomeres of the 1p, 3p, 4q, 5p, 12q, and 13q chromosome arms throughout all experiments were among the longest; and those of 13p, 15p, 21p, and 22p were among the shortest telomeres of the karyotype. From these data, it can be concluded that MMC affects the telomeric repeat area of chromosomes more than BLM, which mostly had no significant effect on telomere length in the performed experiments.

Int J Radiat Biol, 26, 1707- 1713

Are telomeres a specific target for mutagenic attack by cytostatics in neoplastic cells?

U. Wick, E. Gebhart

Damage to telomeres induced by cytostatic therapy theoretically could generate telomere shortening and, subsequently, induce an additional genomic instability in neoplastic cells. Model experiments were carried out to examine this hypothesis. Cells of the T-ALL derived cell line CCRF-CEM were exposed to various different concentrations of Bleomycin (BLM) or Mitomycin C (MMC) for various times. Telomere lengths of metaphase chromosomes of the exposed cells were compared with those without this exposure (controls). In addition, telomerase activity was determined with a TRAP assay under the given conditions using the BLM experiments as a model. Although slight changes of total telomere length could be found in single experiments, the differences between exposed and non-exposed cells were not significant. Also, a considerable telomerase activity was shown which, however, did not substantially differ between exposed and non-exposed cells. From these data it may be concluded that, at least in the examined cell line, telomeres are not a preferential target for this kind of mutagenic attack.

Leukemia Research, 29, 273- 281

Prognostic value of structural chromosomal rearrangements and small cell clones with high hyperdiploidy in children with acute lymphoblastic leukemia.

Z. Zemanova, K. Michalova, L. Sindelarova, P. Smisek, J. Brezinova, S. Ransdorfova, V. Vavra, A. Dohnalova, J. Stary

In this study, 107 children with acute lymphoblastic leukemia (ALL) were analysed for the presence of hyperdiploidy by cytogenetics and interphase fluorescence in situ hybridisation (I-FISH). Structural aberrations in hyperdiploid cells were investigated by multiple colour FISH (mFISH). Clones with high hyperdiploidy (>50 chromosomes) (HeH) were found in 46 patients (43%). In nine of these (20%), the abnormal clone was present in <20% of the total cell population. There was no significant difference in EFS between those patients with HeH in 2.5-20% or >20% of cells. Structural rearrangements in the HeH clone were found in 10 patients (22%). In this study, HeH karyotypes containing structural aberrations were an indication of a poor prognosis in childhood ALL.

Oncogene, 23(45), 7507–7516
September, 2004

Tumor necrosis factor alpha induces senescence and chromosomal instabilityin human leukemic cells.

Odile Beyne-Rauzy, Christian Recher, Nicole Dastugue, Cécile Demur, Géraldine Pottier, Guy Laurent, Laure Sabatier, Véronique Mansat-De Mas

Previous studies have documented that Tumor necrosis factor alpha (TNFalpha) is a potent negative regulator of normal hematopoiesis. However, the mechanism by which TNFalpha acts at the cellular level is not totally understood. Although apoptotic cell killing appears to be the most common cellular effect of TNFalpha, other studies suggest that this cytokine may elicit other cellular responses such as prolonged growth inhibition. In this context, we have investigated whether TNFalpha may induce senescence in hematopoietic cells, which display intrinsic defect in the apoptotic machinery. The present study described that, in the leukemic KG1 cells, TNFalpha induced no apoptosis but a senescence state characterized by prolonged growth arrest, increased beta-galactosidase activity, p21WAF-1 induction, decreased telomerase activity, telomeric disturbances (shortening, losses, fusions), and additional chromosomal aberrations. Telomerase inhibition correlated with reduced levels of hTERT transcripts. GM-CSF prevented TNFalpha effects and allowed leukemic cells to recover growth capacity. Finally, our study shows for the first time that, at least in some hematopoietic cells, TNFalpha may induce senescence with important functional consequences, including sustained growth inhibition and genetic instability, and that this cellular response is efficiently regulated by hematopoietic growth factors.

Clin Cancer Res, 10(9), 3020–3028
May, 2004

Reliable and sensitive identification of occult tumor cells usingthe improved rare event imaging system.

Stine-Kathrein Kraeft, Andras Ladanyi, Kevin Galiger, Anna Herlitz, Andrew C. Sher, Danielle E. Bergsrud, Gaelle Even, Stephanie Brunelle, Lyndsay Harris, Ravi Salgia, Tom Dahl, John Kesterson, Lan Bo Chen

The purpose of this study was to assess the feasibility of using rare event imaging system (REIS)-assisted analysis to detect occult tumor cells (OTCs) in peripheral blood (PB). The study also sought to determine whether REIS-assisted OTC detection presents a clinically viable alternative to manual microscopic detection to establish the true significance of OTC from solid epithelial tumors.We recently demonstrated proof of concept using a fluorescence-based automated microscope system, REIS, for OTC detection from the PB. For this study, the prototype of the system was adopted for high-throughput and high-content cellular analysis.The performance of the improved REIS was examined using normal blood (n = 10), normal blood added to cancer cells (n = 20), and blood samples obtained from cancer patients (n = 80). Data from the screening of 80 clinical slides from breast and lung cancer patients, by manual microscopy and by the REIS, revealed that as many as 14 of 35 positive slides (40\%) were missed by manual screening but positively identified by REIS. In addition, REIS-assisted scanning reliably and reproducibly quantified the total number of cells analyzed in the assay and categorized positive cells based on their marker expression profile.REIS-assisted analysis provides excellent sensitivity and reproducibility for OTC detection. This approach may enable an improved method for screening of PB samples and for obtaining novel information about disease staging and about risk evaluation in cancer patients.

Int J Oncol, 24, 1279- 1288

Molecular characterizations of derivatives of HCT116 colorectal cancer cells that are resistant to the chemotherapeutic agent 5-fluorouracil.

P.M. De Angelis, B. Fjell, K.L. Kravik, T. Haug, S.H. Tunheim, W. Reichelt, M. Beigi, O.P. Clausen, E. Galteland, T. Stokke

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.

Hum Reprod, 19, 685- 693

Fluorescence in situ hybridization analysis of two blastomeres from day 3 frozen-thawed embryos followed by analysis of the remaining embryo on day 5.

E.B. Baart, D. Van Opstal, F.J. Los, B.C.J.M. Fauser, E.Martini

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.