This study investigated the effects of exposing human embryonic stem cells (hESC) to 4oC and 25oC for extended durations of 24h and 48h respectively. Cell survivability after low temperature exposure was assessed through the MTT assay. The results showed that hESC survivability after exposure to 25oC and 4oC for 24h was 77.3 ± 4.8 % and 64.4 ± 4.4 % respectively (significantly different, P < 0.05). The corresponding survival rates after 48h exposure to 25oC and 4oC was 71.0 ± 0.5 % and 69.0 ± 2.3 % respectively (not significantly different, P > 0.05). Spontaneous differentiation of hESC after low temperature exposure was assessed by morphological observations under bright-field and phase-contrast microscopy, and by immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. hESC colonies were assigned into 3 grades according to their degree of spontaneous differentiation: (1) Grade A which was completely or mostly undifferentiated, (2) Grade B which was partially differentiated, and (3) Grade C which was mostly differentiated. In all low temperature exposed groups, about 95% of colonies remain undifferentiated (Grade A), which was not significantly different (P > 0.05) from the unexposed control group maintained at 37oC. Additionally, normal karyotype was maintained in all low temperature-exposed groups, as assessed by fluorescence in situ hybridization (FISH) of metaphase spreads with telomere and centromere-specific PNA probes. Further analysis with m-FISH showed that chromosomal translocations were absent in all experimental groups. Hence, hESC possess relatively high-tolerance to extended durations of low temperature exposure, which could have useful implications for the salvage of hESC culture during infrequent occurrences of incubator break-down and power failure.

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.

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.

Conventional genetic theories have failed to explain why cancer (1) is not heritable and thus extremely rare in newborns, (2) is caused by non-mutagenic carcinogens, (3) develops only years to decades after initiation by carcinogens, (4) follows pre-neoplastic aneuploidy, (5) is aneuploid, (6) is chromosomally and phenotypically "unstable", (7) carries specific aneusomies, (8) generates much more complex phenotypes than conventional mutation such as multidrug resistance, (9) generates nonselective phenotypes such as metastasis (no benefit at native site) and "immortality" (not necessary for tumorigenesis), and (10) does not contain carcinogenic mutations. We propose, instead, that cancer is a chromosomal disease. Accordingly carcinogenesis is initiated by random aneuploidies, which are induced by carcinogens or spontaneously. Since aneuploidy unbalances 1000s of genes, it corrupts teams of proteins that segregate, synthesize and repair chromosomes. Aneuploidy is therefore a steady source of chromosomal variations from which, in classical Darwinian terms, selection encourages the evolution and malignant progression of cancer cells. The rates of specific chromosomal variations can exceed conventional mutations by 4-11 orders of magnitude, depending on the degrees of aneuploidy. Based on their chromosomal constitution cancer cells are new cell "species" with specific aneusomies, but unstable karyotypes. The cancer-specific aneusomies generate complex, malignant phenotypes through the abnormal dosages of 1000s of genes, just as trisomy 21 generates Down syndrome. In sum, cancer is caused by chromosomal disorganization, which increases karyotypic entropy. Thus, cancer is a chromosomal rather than a genetic disease. The chromosomal theory explains (1) non-heritable cancer because aneuploidy is not heritable, (2) non-mutagenic carcinogens as aneuploidogens, (3) long neoplastic latencies by the low probability of evolving new species, (4) nonselective phenotypes via genes hitchhiking with selective chromosomes, and (5) immortality because, through their cellular heterogeneity, cancers survive negative mutations and cytotoxic drugs via resistant subspecies.

Long-lived, sensitive, and specific biomarkers of particular mutagenic agents are much sought after and potentially have broad applications in the fields of cancer biology, epidemiology, and prevention. Many clastogens induce a spectrum of chromosome aberrations, and some of them can be exploited as biomarkers of exposure. Densely ionizing radiation, for example, alpha particle radiation (from radon or plutonium) and neutron radiation, preferentially induces complex chromosome aberrations, which can be detected by the 24-color multifluor fluorescence in situ hybridization (mFISH) technique. We report the detection and quantification of stable complex chromosome aberrations in lymphocytes of healthy former nuclear-weapons workers, who were exposed many years ago to plutonium, gamma rays, or both, at the Mayak weapons complex in Russia. We analyzed peripheral-blood lymphocytes from these individuals for the presence of persistent complex chromosome aberrations. A significantly elevated frequency of complex chromosome translocations was detected in the highly exposed plutonium workers but not in the group exposed only to high doses of gamma radiation. No such differences were found for simple chromosomal aberrations. The results suggest that stable complex chromosomal translocations represent a long-lived, quantitative, low-background biomarker of densely ionizing radiation for human populations exposed many years ago.

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.

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.

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.

BACKGROUND AND OBJECTIVES: Nodular lymphocyte-predominant Hodgkin's lymphoma (NLPHL) showed recurrent rearrangement of the BCL6 which is gene detected in 48% of cases analyzed by interphase-fluorescent in situ hybridization (FISH). These findings point to a critical role for BCL6 in the development of this distinct Hodgkin's lymphoma. We present our results of metaphase-FISH analyses aimed at identifying and characterizing BCL6-related chromosomal translocations in NLPHL. DESIGN AND METHODS: Four NLPHL cases with available metaphase spreads obtained either at the time of diagnosis or during progression to diffuse large B-cell lymphoma (DLBCL) were collected. Extensive metaphase-FISH analysis was performed to identify the affected partner chromosomes and reciprocal breakpoints. RESULTS: Each of the analyzed NLPHL cases showed a different type of BCL6 rearrangement that included the t(3;22)(q27;q11) targeting immunoglobulin (IG) alpha chain locus, complex t(3;7;3;1) involving the 7p12/Ikaros gene region, t(3;9)(q27;p13) affecting an unknown gene in vicinity of PAX5, and t(3;4)(q27;q32) showing the alternative 3q27 breakpoint outside BCL6 and possibly, an internal deletion of BCL6. Retrospective interphase-FISH analysis of 2 cases with subsequent DLBCL showed the same type of BCL6 translocation as in NLPHL samples. INTERPRETATION AND CONCLUSIONS: The spectrum of BCL6 aberrations targeting IG as well as non-IG loci in NLPHL is similar to that found in DLBCL. These findings further support the hypothesis of a germinal center B-cell-derived origin of NLPHL and of a relationship between these two lymphoma entities. This latter issue is additionally illustrated in two NLPHL patients who subsequently developed DLBCL and showed the same type of BCL6 rearrangements in both tumors.

A clonal origin of hematopoiesis was studied by investigation of X-chromosome inactivation patterns (XCIP) in isolated granulocyte, CD14(+) and CD3(+) subpopulations obtained from bone marrow and peripheral blood of 36 female patients with primary myelodysplastic syndrome (MDS). Clonality was assessed by PCR amplification of polymorphic short tandem repeats of the human androgen receptor (HUMARA) gene and by investigation of silent polymorphism of iduronate sulphatase (IDS) or p55 genes. On the basis of results in a control group of 20 healthy age related females, a ratio of at least 9:1 between the two alleles was considered a significant marker of monoclonal hematopoiesis. Ten of the 11 patients with advanced forms of MDS (RAEB, RAEB-T, CMML) had clonal granulocytes and CD14(+) cells in peripheral blood. In patients with early disease, only 2 out of 11 patients (18%) with RA or RARS, according to WHO classification, had clonal granulocytes and CD14(+) cells in peripheral blood and bone marrow and 2 other patients with 5q-syndrome exhibited extremely oligoclonal granulocyte subpopulation in bone marrow. In contrast, we found clonal granulocytes in 12 out of 14 patients (86%) with refractory cytopenia with multilineage dysplasia (RCMD) and 8 of them simultanously exhibited clonal CD14(+) cells. Estimated 3 years survival of patients with early disease and clonal cell subpopulations was 61% as compared with 88% in patients without clonal hematopoiesis. Karyotype abnormalities were detected in 11 of the 25 females with early disease. Clonal patterns were present in 7 out of 8 patients with abberations diagnosed by routine cytogenetics, nevertheless, FISH revealed 5q deletion in 3 patients without signs of clonality in XCIP assay. No correlation was found between the presence of clonal subpopulations and the degree of telomere shortening in early MDS. Despite some limitations, the measurement of XCIP remains a sensitive tool for diagnosis of the first transforming mutation in the clonal development of MDS especially when combined with FISH and when an age related group is used to establish an appropriate allele ratio to exclude constitutional or acquired skewing. The occurrence of clonal cell subpopulations in most of the RCMD patients in contrast to RA may reflect a proposed multistep pathogenesis of MDS with dysplastic changes limited to erythropoiesis in early step and with subsequent development of multilineage dysplasia. The results also support the usefulness of separation of RCMD from 'pure' RA; however, a more complex insight combining different molecular techniques performed in a large number of patients is needed for refined classification of MDS on the basis of new molecular prognostic factors and for indication of more effective targeted therapy.

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.

<p>Genomic fingerprints of mutagenic agents would have wide applications in the field of cancer biology, epidemiology and prevention. The differential spectra of chromosomal aberrations induced by different clastogens suggest that ratios of specific aberrations can be exploited as biomarkers of carcinogen exposure. We have tested this hypothesis using the novel technique of multicolor banding in situ hybridization (mBAND) in human peripheral blood lymphocytes exposed in vitro to X rays, neutrons, heavy ions, or the restriction endonuclease AluI. In the heavy-ion-irradiated cells, we further analyzed aberrations in chromosome 5 using multicolor FISH (mFISH). Contrary to the expectations of biophysical models, our results do not support the use of the ratios of inter-/intrachromosomal exchanges or intra-/interarm intrachanges as fingerprints of exposure to densely ionizing radiation. However, our data point to measurable differences in the ratio of complex/simple interchanges after exposure to different clastogens. These data should be considered in current biophysical models of radiation action in living cells.</p>

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.

Small supernumerary marker chromosomes (SMCs) in human can be defined as additional centric chromosome fragments smaller than chromosome 20. For most small or minute SMCs a correlation with clinical symptoms is lacking, mostly due to problems in visualizing their euchromatic content. Recently we described two new molecular cytogenetic approaches for the comprehensive characterization of small SMCs, excluding those few cases with neo-centromeres. Minute SMCs, consisting preferentially of alpha-satellite DNA, are characterizable in one step by the centromere-specific multicolor FISH (cenM-FISH) approach. For further characterization of minute SMCs and eventually present euchromatic content, the recently developed centromere-near-specific multicolor FISH (subcenM-FISH) technique can be applied. These two approaches are highly informative and easy to perform, as demonstrated in the present report on the example of a prenatal case with a minute SMC derived from chromosome 3 cytogenetically described as min(3)(:p12.1→q11.2:).

Diagnostic possibilities of CGH and M-FISH techniques for detection of submicroscopic chromosomal imbalancies were compared on the basis of two cases of t(X;Y) and one case of marker chromosome. In cases with t(X;Y), the sequences specific for chromosome Y were detected by PCR and CGH, but the localisation of these sequences on the short arm of chromosome X was confirmed by the FISH technique, employing two Yp-specific probes for SRY and TSPY genes. Significant differences between above cases were revealed in the size of Yp chromosome fragments translocated on chromosome X. An extra material of chromosome marker could not be identified by classical banding and FISH techniques and it was only CGH and M-FISH techniques that enabled detecting the chromosomal origin of the marker. The applied CGH technique enabled finding subtle chromosomal imbalancies in the presented cases with a resolution of approximately 3 Mbp.

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.

We report on a rare additional numerical chromosomal aberration in a child with Down syndrome due to free trisomy 21. The karyotype showed 48,XY,+21,+mar after GTG banding, with the marker present in 80% of cells. The supernumerary marker chromosome (SMC) was as small as approximately one-third of 18p, and with the recently developed centromere-specific multi-color fluorescence in situ hybridization (cenM-FISH) technique, it was shown that the SMC was a derivative chromosome 4. The SMC was not specifically stained by arm-specific probes for chromosome 4; thus, it has been described as der(4)(:p11 --> q11:). Microsatellite analysis resulted in a partial maternal uniparental isodisomy (UPD) for chromosome 4p15-16 and a maternal origin for two chromosomes 21. Until now only two similar cases have been described in the literature, but without clarifying the origin of the SMC and without looking for an additional UPD. This is the only reported case of a UPD 4p in a liveborn child.

A comprehensive and detailed comparative chromosome map of the white-handed gibbon (Hylobates lar = HLA) has been established by hybridizing the recently developed complete human multicolor banding (MCB) probe set on metaphase chromosomes of a male HLA lymphoblastoid cell line. Thus, it was possible to precisely determine the breakpoints and distribution plus orientation of specific DNA-regions in this cytogenetically highly rearranged species compared to Homo sapiens (HSA). In general, the obtained results are in concordance with previous molecular-cytogenetic studies. In this study all 71 breakpoints present in HLA compared to HSA could be determined exactly. This study is a valuable complement to our knowledge on the phylogeny of huminoid chromosomes.

<p>The purpose of this work was to adapt the recently described centromere-specific multicolour (cenM-) FISH technique to human meiotic cells, and evaluate the usefulness of this multiplex fluorescence method for karyotyping human synaptonemal complex (SC), previously analysed by immunocytogenetic approaches. The results obtained demonstrate that cenM-FISH is a reliable one-single-step method, which allows for the identification of all SC present in pachytene spreads. Moreover, when cenM-FISH is applied after immunocytogenetic analysis, the number and distribution of MLH1 foci per chromosome can be established and recombination analysis for each chromosome can be performed easily.</p>