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E Gebhart

Publications and source records attributed to E Gebhart.

At least 19 recordsLinked to original sources
PubMed2002

Comparative M-FISH and CGH analyses in sensitive and drug-resistant human T-cell acute leukemia cell lines.

Cell lines of human T-cell acute lymphoblastic leukemias (T-ALL) have gained high interest for study of mechanisms of cytostatic drug resistance. However, they should also be suited to examine the validity and reliability of molecular cytogenetic techniques in detecting genomic alterations in neoplastic cells. Therefore, comparative genomic hybridization (CGH) and 24-color-fluorescence-in-situ-hybridization (M-FISH) were applied to eight sublines of CCRF-CEM leukemia cells selected in vitro for drug resistance and to their drug-sensitive parental counterparts. All cell lines were characterized by altered chromosome numbers and by a variety of chromosomal structural aberrations as shown by M-FISH. The great majority of anomalies detected by this technique were confirmed by CGH. Interestingly, a considerable number of the rearrangements found were imbalanced. Amplifications of 5q13 in the six methotrexate-resistant cell lines, a del(9)(p21pter) in all lines examined, and a gain of chromosome 20 in 9 of the 10 lines examined were readily detected by both techniques. The same held true for losses of chromosomes 17 and 18 in the near tetraploid cell lines which could also be confirmed by CGH. Some imbalances of genomic material detected by CGH were, however, not observed by means of M-FISH, possibly due to the limited extension of the corresponding chromosomal segment involved or the small subpopulation of cells affected. On the other hand, reciprocal translocations, balanced isochromosomes, and small deletions remained mainly undetected by CGH. A comparison of chromosomal alterations in drug-resistant and parental cell lines showed not only amplifications of chromosomal segments harboring well-known drug resistance genes, e.g., the dihydrofolate reductase gene, but also chromosomal changes which may involve novel genes associated with drug resistance. Thus, the present study has clearly unveiled the strengths and weaknesses of both techniques which can excellently complement each other. Their combination allowed a distinct improvement of the definition of the complex karyotypes of drug-resistant cell lines.

Chromosome Aberrations↗
PubMed2002

Thin section arrays for I-FISH analysis of chromosome-specific imbalances in squamous cell carcinomas of the head and neck.

Thin section arrays of 20 head and neck squamous cell carcinomas were studied by I-FISH for gains (including amplification) and losses of specific genomic segments. These arrays allow the examination not only of a number of tumor sections but also of the surrounding margins and of inconspicuous control tissue in one experiment. All tumor sections examined significantly differed from the inconspicuous control tissues by containing more or less extensive cell populations with aberrant signal constitutions. In no case, however, did the aberrant population constitute the whole area of the section. Gains of signals were strikingly more frequent than were losses. All tumors showed significant gains of the segments examined, the highest differences between tumor and control sections were found for the segments 9q34 and 8q24, followed by 5p15.3 and 11q13. Amplifications were most frequently found of 11q13: 8 of the 20 tumors showed amplifications in more than 20% of the nuclei, while no nucleus with more than four signals was found in any of the control tissues (control: 0%). Amplifications of the target sequences on chromosomes 8 (14 tumors) and 9 (8 tumors) were observed in low but significant percentages of nuclei, no significant cell population was detected with an amplification of 5p15.3. Fourteen tumors exhibited a significant loss of 13q14, and only 8 tumors a significant loss at any other site. In the tumor margin sections, in most cases, the margins apparently were also affected by the one or the other of the genomic changes of the pertinent primary tumor. Nevertheless, there were, in some cases, also large differences depending on the way of analysis, but also on the specific signal constitution considered. Tumor stages T3 and T4 tended to have higher frequency of nuclei with gains of 5p15.3, 8q24, and 11q13 as compared to T2 tumors and less gains of 9q34 and loss of 13q14. With the exception of 8q24 and 13q14 alterations there was also a trend to higher percentages of aberrant nuclei in the margin of T3-4 tumors vs. T2 tumors.

Aged↗
PubMed2001

Chromosome painting for cytogenetic monitoring of occupationally exposed and non-exposed groups of human individuals.

The suitability of a three-color fluorescence in situ suppression hybridization technique was examined for monitoring five different groups of individuals: 30 occupied in radiology, 26 occupied in nuclear medicine or radiation physics, 32 patients with breast cancer, 26 occupied with military waste disposal, all presumably exposed to low doses of radiation or chemical mutagens and a non-exposed control group (N=29). The average frequency of breaks constituting the various aberrations did not significantly differ between the groups of medical radiation appliers and the control group. However, breast tumor patients and military waste disposers, as groups, showed a higher aberration rate than did healthy controls. Stable rearrangements mainly characterized the groups of controls, tumor patients, and radiation appliers, while a higher proportion of unstable aberrations was found in the chemically exposed individuals. Individuals with an increased frequency of aberrations could be detected within each examined group, which clearly determined the average values of the whole group. With respect to interchromosomal distribution of the breakpoints constituting the found aberrations and the involvement of the labeled chromosomes in rearrangements, the observed values were very close to the expected ones in the controls. A rather similar trend of deviations from expectation was observed in all other groups. Chromosome 4 was slightly over-affected, while chromosome 2 was slightly underrepresented in all analyzed groups (except tumor patients). Rearrangements of the labeled chromosomes with the unlabeled ones exceeded expectation. In conclusion, chromosome painting if included in further attempts of human population monitoring will broaden the basis of argumentation with respect to health risks introduced by mutagen exposure.

Adult↗
PubMed2001

Comparative genomic hybridization-aided unraveling of complex karyotypes in human hematopoietic neoplasias.

The information obtained by conventional cytogenetics (CC) in human leukemias is sometimes limited, in particular by complex karyotypes with many marker chromosomes. While CC is restricted to metaphases with a good quality, interphase fluorescence in situ hybridization (I-FISH) is also capable of analyzing specific anomalies in the interphase nuclei. Comparative genomic hybridization (CGH) gives additional information about the imbalanced karyotype changes in the whole genome. The aim of this study was to assess the contribution of CGH to the unraveling of complex GTG karyotypes, which are difficult to evaluate by banding analysis, and to compare these results with those by CC and FISH. Thirteen bone marrow samples and one sample obtained from peripheral blood of 13 leukemia patients were examined by CC, FISH and CGH. The GTG banding analysis showed complex karyotypes with many marker chromosomes. The most frequent abnormalities were numerical and structural aberrations on chromosomes 5 and 7. In 12 of the 14 samples, the CGH analysis was able to detect chromosomal imbalances with losses of material on chromosome 5 and 7 as the most frequent aberrations. In all 14 samples, additional FISH analyses were performed. For most of the studied neoplasias, a close correlation between CC, FISH and CGH data was observed. CGH was considerably helpful in adding additional information to classical karyotyping, if the low quality or number of metaphases was insufficient for a reliable CC analysis. Even in cases where whole chromosome painting could be applied, it added information on the breakpoints of the observed rearrangements. In only 2 of the studied 14 samples, neither CGH nor I-FISH could improve the result of karyotyping. CGH, nevertheless, can be regarded as a powerful additional technique in leukemias with unsuccessful CC, incomplete, or complex karyotypes with many marker chromosomes. A systematic analysis by three techniques such as CC, FISH and CGH guarantees an optimal genetic characterization of the neoplasias.

Acute Disease↗
PubMed2001

Gp130 and ras mediated signaling in human plasma cell line INA-6: a cytokine-regulated tumor model for plasmacytoma.

INTRODUCTION: Cytokines of the gp130-family, particularly interleukin(IL)-6, play a crucial role in the propagation of malignant plasma cells. MATERIALS AND METHODS: The role of IL-6 and other gp130-cytokines was studied in the human plasma cell line INA-6 in vitro and in INA-6 xenografts. The proliferative response to gp130-cytokines was evaluated and activated components of gp130-signaling pathways were identified by Western blotting and DNA binding studies. Specifically, expression of IL-6 and receptors for IL-6 and leukemia inhibitory factor were analysed by RT-PCR and ELISA. RESULTS: The plasma cell line INA-6 was cultured for several years remaining strictly dependent on exogenous IL-6. Other gp130-cytokines had no significant effect on INA-6 cell proliferation in vitro. Due to an activating mutation in the N-ras gene, mitogen-activated protein kinases (MAPK) were constitutively phosphorylated. In contrast, signal transducer and activator of transcription(STAT)-3 activation was dependent on stimulation with IL-6. Blocking of either one of these pathways resulted in a significant decrease of INA-6 cell proliferation. Remarkably, INA-6 xenografts did not require exogeneous IL-6 for proliferation in vivo. Instead, an autocrine IL-6 loop and, in certain tumor sublines, responsiveness to additional gp130-cytokines was induced during in vivo growth. CONCLUSION: Activation of the gp130 signal transducer is mandatory for INA-6 cell growth in vitro and in vivo. Both the MAPK and the Jak/STAT pathway are operative in malignant plasma cells and either one is essential for plasma cell growth. The INA-6 cell line provides a preclinical model to study growth regulation of human plasmacytoma cells and to evaluate novel therapeutic strategies.

Aged↗
PubMed2001

Increased translocation frequency of chromosomes 7, 11 and 14 in lymphocytes from patients with neurocysticercosis.

Neurocysticercosis (NCC) has been associated with a high frequency of DNA damage in human circulating lymphocytes and more recently with the development of hematological malignancies. Chronic inflammation, a common feature of helminthic infections, has been proposed to play a key role in carcinogenesis induced by parasites. However, this mechanism is more likely to occur during local tumorigenesis rather than in systemic neoplasia such as that reported for patients with NCC. As an alternative, constant antigen stimulation, which is a feature of chronic NCC, may increase the frequency of aberrations in chromosomes that harbor regions constantly rearranged during T and B lymphocyte maturation, e.g. chromosomes 7 and 14. Therefore, in this study we determined the frequencies of aberrations in chromosomes 7, 11 and 14 in lymphocytes from 10 NCC patients and 10 controls and compared them with the frequency observed in chromosomes 1, 2 and 4 in the same cell samples. Chromosome aberrations were analyzed using a chromosome painting technique. Although the genome painted by probes for chromosomes 1, 2 and 4 was almost twice as large as that painted by probes for chromosome 7, 11 and 14, translocations involving the later (median 7.6 per 1000 metaphases) were more frequent than those occurring in chromosomes 1, 2 and 4 (median 2.5 per 1000 metaphases, P = 0.002). These results suggest that persistent antigen stimulation can cause chromosome instability in lymphocytes from patients with NCC and should be considered as an additional mechanism whereby parasites may induce cancer.

Adult↗
PubMed2001

DNA amplification on chromosome 7q in squamous cell carcinoma of the tongue.

Squamous cell carcinoma of the head and neck exhibit a highly variable picture of chromosomal aberrations. In the present study the clearly defined anatomical region of the tongue was analyzed for potentially specific patterns of chromosomal alterations. Fresh tumor samples from 18 patients afflicted by squamous cell carcinoma of the tongue constituted the clinical basis of the present investigation. The tumor samples were analyzed on the basis of comparative genomic hybridization (CGH), a molecular cytogenetic FISH-approach. Gains in DNA copy numbers were detected as the predominant imbalance on chromosomes 7q (9/18), 3q (48/18), 16p (7/18) and 20q (7/18). The regions of minimal overlap on these chromosomes were mapped to 7q11.2q11.3 and 3q26. A conspicuous finding was the frequent detection of amplifications in the 7q11 region. Gains in the 7q region have been rarely reported in CGH studies of tumors derived from different regions of the head and neck. Amplifications on 7q could thus be specifically linked with the tongue region and could correlate with specific clinical factors of this tumor entity.

Adult↗
PubMed2001

FISH monitoring of 100 courses of human leukemias: the cytogenetic viewpoint.

Interphase fluorescence in situ hybridization (I-FISH) analyses were performed from 2 up to 13 times along the course of 100 human leukemias (36 chronic myeloid leukemias, 38 acute myeloblastic leukemias, 17 acute lymphoblastic leukemias, and 9 additional hematopoietic neoplasias) in order to control clonality, evolution, and disappearance of the basic cytogenetic changes. The relevance of these data to confirm clinical remission or to detect minimal residual disease and/or relapse was evaluated. Fifty-four patients were monitored following hematopoietic stem cell transplantation, and 46 cases after chemotherapy. Various chromosome or aberration specific DNA probes were applied for follow-up in the time frame of 1 month up to 13 years. From the cytogenetic point of view, the aim was to determine the power of resolution of the I-FISH technique in the detection of clinically significant changes in the course of disease and its usefulness in daily routine cyto-genetics as compared with classical cytogenetics. In addition, reliability standards of the various DNA probes should be established. In 75 patients with remissions during the entire period of I-FISH monitoring no conspicuous signal constitution was detected. Of 25 relapses or progresses of disease, which were clinically confirmed, 22 were reliably detected by I-FISH, in only 1 case I-FISH monitoring failed to detect the aberrant clone. In 2 patients conventional karyotype analysis confirmed the relapse by detecting complex chromosomal aberrations, but specific probes for I-FISH confirmation were not available. These data suggest that I-FISH analyses in the follow-up of leukemias is a simple and in most cases sufficiently sensitive and highly reliable way of monitoring the outcome of therapy. It may well serve to close the gap between conventional karyotyping and the highly sensitive molecular techniques.

Adolescent↗
PubMed2000

Patterns of genomic imbalances in human solid tumors (Review).

Based on comparative studies on CGH-detected genomic imbalances in head and neck squamous cell carcinomas and in ovarian tumors it was supposed that the patterns of genomic imbalances in human tumors are not only related to the oncogenic progress and tumor progression or specific for the tissue of origin, but also could be influenced by environmental (mutagenic) factors present in the environment of the evolving cancer. To base this hypothesis on a more solid ground, data obtained by use of comparative genomic hybridization (CGH) which were reported up to early 1999 from a large variety of more than 2400 human solid tumors, representing 18 different organs of origin or physical localizations, were collected and comparatively analyzed. Patterns of inter- and intra-chromosomal distribution of DNA sequence copy number changes pointed to high conformity on several chromosomal segments, but also revealed striking differences between the tumor types, the latter suggesting tumor specific, tissue specific and/or environment specific influences on the generation of genomic imbalance in human neoplasia. The clinical relevance of these findings must be examined further on by increasing the studied material.

Chromosome Banding↗
PubMed2000

Delimiting the use of comparative genomic hybridization in human myeloid neoplastic disorders.

Hematopoietic disorders can be used as a suitable tool of additional information on the actual resolving power of comparative genomic hybridization (CGH). Therefore, CGH examination was performed of DNA extracted from 23 acute and 15 chronic myeloproliferative disorders which had just been analyzed using classical cytogenetic techniques. In nearly all cases CGH analysis was repeated with reversely labeled probes. A Zeiss axioplan microscope was equipped with the ISIS 3 system for photometric evaluation of the CGH data. A main group was selected of 34 cases showing karyotypic mosaics when routinely diagnosed by classical cytogenetics. The grade of mosaicism was basically determined from the classical cytogenetic analysis and was additionally defined examining target anomalies by I-FISH analysis in 28 of the cases. The second group included 23 cases with deletions, and in 1 case another informative genomic imbalance could be analyzed. Every target anomaly irrespective of its type could be detected in all cases with an affected cell population equalling or exceeding about 25%, but in none was it below 23%. This value was the lowest and was found in a case, with CGH-detected 20q deletion. The smallest deletions of two bands on 20q which could visually be detected by CGH were estimated in the range of 5-7 Mb. CGH was also suitable to detect imbalances which were not clearly detected by routine cytogenetics. Reverse labelling, performed in nearly all cases, confirmed the result of the original CGH analysis. These data not only document the readiness and reliability of CGH studies on human leukemia, but also further contribute to a clearer definition of the limits of the resolving power of this technique.

Adult↗
PubMed1999

Deletion monitoring in skin tumors by interphase-FISH using band-specific DNA probes.

Thin sections from archival paraffin blocks of various skin tumors (26 melanomas, 15 squamous cell carcinomas, 5 keratoacanthomas, 5 basal cell carcinomas) were subjected to interphase-FISH (I-FISH) with DNA probes which are specific for chromosomal regions often involved in deletions in human cancer. These were probes for chromosome 3p21, the p53 gene on chromosome 17p13, and, in a few selected cases, a probe for chromosome 9p21. It was demonstrated that deletions of these regions could be reliably detected and related to tumor type and histology, i.e. grading. The most common deletion was that of 3p21 which was found in all studied squamous cell carcinomas (SCC) of low differentiation, in 60% of the Bowen carcinomas, in 70% of the metastatic melanomas less than 1.5 mm thick, and in over 55% of those which thickness over 2 mm. In contrast, FISH-detected p53 deletion was a rare finding in the investigated tumors. However, this gene was even found in an increased copy number in 60% of the poorly differentiated SCCs (grade 4) and in 50% of the non-metastatic melanomas less than 1.5 mm thick. Deletion of 9p21 was detected in 13 of the 14 tumors on which pertinent examinations could be performed. I-FISH was shown to be a reliable technique for the rapid detection of chromosome band specific deletions in archival material of human skin tumors.

Adult↗
PubMed1999

Differentiation between Spitz nevi and malignant melanomas by interphase fluorescence in situ hybridization.

Spitz nevi are benign melanocytic neoplasias which have distinct pathological features that make the pathological differential diagnosis from malignant melanomas extremely difficult. The Spitz nevi may be misdiagnosed as malignant melanoma and vice versa. Therefore, interphase fluorescence in situ hybridization (I-FISH) was used for a possible discrimination between Spitz nevi and malignant melanomas on the basis of numerical aberrations of the chromosome complement in interphase nuclei of thin sections. Previous studies had shown changes in malignant melanomas which were not found at the same level in normal tissue or benign tumors. Thin sections of archival paraffin material from 42 Spitz nevi with different histological type and grade of anomaly were subjected to FISH-analyses using commercially available biotinylated and/or digoxigenated alphoid DNA probes of chromosomes 1, 6, 7, 9, 17 and 18, which were applied in combinations in a two- or three-color-FISH. Unaffected epithelial areas from the same sections served as. The obtained data were compared with those collected previously from thin sections of malignant melanomas prepared in the same way. Due to the sometimes limited nevus area investigated, the number of evaluable nuclei was lower than expected from previous experiences with malignant melanomas. Therefore, only 20 nevi could be reliably evaluated. The comparison of the group of Spitz nevi with the group of controls did not show any significant difference regarding chromosomes 1, 6, 7, 9 and 17 (Wilcoxon test). The method used to detect chromosomal loss or gain in the individual Spitz nevi demonstrated only two nevi (one of the spindle cell type with a low to middle grade of anomaly, the other of the epitheloid cell type with a middle grade of anomaly) with a gain of chromosome 7 and chromosome 17, respectively. So, with respect to the histological type and grade of anomaly, no numerical aberrations could be detected in Spitz nevi. The comparison of the group of Spitz nevi with subgroups of malignant melanomas (metastatic, non-metastatic, melanomas with a thickness <1.5 mm and melanomas with a thickness >2. 0 mm) and with the whole group of malignant melanomas showed significant differences concerning chromosome 9 (Mann-Whitney U test), signal indices, which were higher in the melanomas than in the Spitz nevi. Regarding chromosomes 6, 7 and 17 no significant differences could be shown, although a trend of gain in melanomas and of loss in Spitz nevi was observed of these chromosomes.

Adolescent↗
PubMed1998

New eukaryotic semaphorins with close homology to semaphorins of DNA viruses.

Semaphorins were initially described as a family of repulsive guidance molecules in embryonal development. Their basic structure consists of an N-terminal signal sequence, the defining semaphorin domain ofapproximately 500 amino acids, an Ig-like domain,and a variable carboxy-terminus. We recently described a viral semaphorin homologue encoded by the alcelaphine herpesvirus type 1. Less conserved, truncated homologues were also identified in poxviruses. Here we describe new human and murine semaphorin homologues. The respective genes were cloned and sequenced, and they were termed H-Sema-L and M-Sema-L (HGMW-approved symbols SEMAL and Semal, respectively). A multiply spliced mRNA of 3.2 kb is expressed in human placenta, spleen, thymus, and gonadal tissue. H-Sema-L maps to chromosome 15q22.3-q23 and M-Sema-L to the homologous locus 9A3.3-B in the mouse genome. The expression patterns and the presence of related genes in large DNA viruses suggest that this new semaphorin has a relevant function in the immune system.

Adjuvants, Immunologic↗
PubMed1998

Chromosomal in-vitro radiosensitivity of lymphocytes in radiotherapy patients and AT-homozygotes.

BACKGROUND: We investigated the in-vitro radiosensitivity of peripheral blood lymphocytes with a special FISH/CISS-technique. PATIENTS AND METHODS: From October 1993 through April 1996, a total number of 52 cancer patients was enrolled in the study. The tumor sites in these patients were: breast (n = 41), lung (n = 4), head and neck (n = 3) as well as prostate, bladder, rectal cancer and Hodgkin's disease (each n = 1). Twenty-six of them were examined prior to planned radiotherapy (prospective group) and 26 after radiotherapy (retrospective group). Three additional individuals (without cancer or radiotherapy) with proven ataxia telangiectasia (Louis-Bar syndrome, AT-homozygotes) were also investigated and their blood samples served as positive control for radiosensitivity. The clinical radiation response of normal tissue in radiotherapy patients was scored according to the WHO grading system for acute and according to the RTOG grading system for late effects. For to estimate the intrinsic radiosensitivity, blood samples were taken and irradiated in vitro with 0 (control) or 0.7 or 2 Gy with a 6 MV-linear accelerator, standard 48-hour lymphocyte cultures were prepared, chromosomes #1, #2 and #4 were simultaneously labeled with a FISH/CISS-technique and 200 to 1,000 metaphase spreads were scored for chromosomal aberrations. The radiation sensitivity of lymphocytes was expressed as the number of radiation-induced chromosomal breaks per mitosis after 0.7 Gy or 2 Gy corrected for the 0-Gy control value. RESULTS: The frequency of chromosomal breaks/mitosis in the unirradiated control lymphocytes was 0.020 +/- 0.015 in prospective patients who had not yet received radiotherapy. It was significantly higher in retrospective patients (0.264 +/- 0.164 breaks/mitosis) as a result of the previous radiation exposure. The 3 AT-homozygotes showed also an increased number of spontaneous chromosomal breaks (0.084 +/- 0.016 breaks/mitosis), probably resulting from the chromosomal instability in this disease. This figure, however, was significantly lower than in retrospective patients. The number of radiation-induced breaks after in-vitro irradiation was comparable in lymphocytes of patients who showed no normal tissue reaction (n = 11) as compared to those with mild to moderate radiation reaction (n = 32, acute reactions Grade 1 to 2, late reactions Grade 0 to 2). In 9 patients with unexpected severe plus late Grade 3 to 4 reactions, however, a significantly higher number of radiation-induced chromosomal breaks was measured; the highest number was observed in a patient with a radiation myelitis. The 3 AT-homozygotes showed, as expected, an extreme radiosensitivity of their lymphocytes. The number of breaks/mitosis after 0.7 Gy in vitro irradiation of lymphocytes was 0.103 +/- 0.059 in patients with no normal tissue radiation reaction (n = 11), 0.122 +/- 0.146 in the group with mild to moderate radiation reactions Grade 1 to 2 (n = 32), 0.359 + 0.226 in patients with unexpected Grade 3 to 4 normal tissue reactions (n = 9) and 0.550 +/- 0.243 in the 3 AT-homozygotes (p < 0.01, t-test). The difference in lymphocyte radiosensitivity between these 4 groups was also detected after in-vitro irradiation with 2 Gy (0.484 +/- 0.132 vs. 0.535 +/- 0.228 vs. 0.926 +/- 0.349 vs. 1.423 +/- 0.072). CONCLUSIONS: We found a significantly higher number of chromosomal breaks in lymphocytes of patients with severe or extreme radiation reaction of normal tissues as compared to patients with no or only mild to moderate radiation reactions. The radiosensitivity of lymphocytes in these radiosensitive patients was in the range between normal radiosensitivity and the radiosensitivity of AT-homozygotes. Detection of patients with severely enhanced intrinsic radiosensitivity might be possible with this method.

Adult↗
PubMed1998

Estimation of DNA single-strand breaks by single cells gel electrophoresis in tumor cells.

To estimate the frequency of single-strand breaks on a single cell level the so-called comet-assay (single cell gel electrophoresis) is a well-established technique. We present a modified protocol suitable for testing primary tumors, a kind of tissue very uneasy to be analysed in former single cell gel electrophoresis assays. Tumor cells of 12 studied cases showed a typical dose-rate effect on in vitro irradiation with different X-ray doses, as observed in peripheral blood leukocytes. Interestingly, the repair capability of primary tumor cells was lower than that of peripheral blood leukocytes of the same patients.

Adenocarcinoma↗