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K J Hutter

Publications and source records attributed to K J Hutter.

At least 19 recordsLinked to original sources
PubMed2000

Genetic complementation to non-tumorigenicity in cervical-carcinoma cells correlates with alterations in AP-1 composition.

The transcription factor AP-1 represents a central key element in the expression of human pathogenic papillomaviruses (HPV). We here propose a novel role for AP-1 as an essential component of an intracellular surveillance mechanism negatively controlling the proliferation of HPV-positive cells under in vivo conditions. The dissection of AP-1 composition in cervical-carcinoma cells revealed an inverse relationship between the Fos-related antigen Fra-1 and the tumorigenic phenotype. Cervical-carcinoma cell lines were either negative or expressed only low amounts of Fra-1 (jointly with c-Fos) within their AP-1 complexes. Somatic-cell hybridization technique was used to fuse different HPV-positive malignant cell lines. This resulted either in tumorigenic hybrids or in cells in which the malignant phenotype of the parental fusion partners was completely suppressed. The monitoring of AP-1 composition in electrophoretic mobility super-shift assays showed that the amount of Fra-1 was substantially increased within the AP-1 complex of non-malignant cells. In contrast, Fra-1 was even diminished in malignant hybrids, while c-Fos remained expressed. This correlation suggests that the concentration of Fra-1 within the AP-1 transcription complex might be an important marker for predicting the in vivo growth properties of HPV-positive cells.

Female↗
PubMed1998

Selective down-regulation of human papillomavirus transcription by 2-deoxyglucose.

The glycolytic pathway inhibitor 2-deoxyglucose (2-DG) is capable of suppressing the transcription of the human pathogenic papillomavirus type 18 (HPV 18) in cervical carcinoma cells and derived non-tumorigenic somatic cell hybrids at the level of transcription initiation. HPV down-regulation is selective, since other reference genes are not affected or even up-regulated under the same experimental conditions. Moreover, 2-DG appears to restore the normal half-life of the tumor suppressor gene product p53, because the protein is strongly up-regulated after HPV 18 E6/E7 suppression. The observed 2-DG-effect is not cytotoxic and is reversible after refeeding with fresh medium. HPV 18 suppression by 2-DG can be completely abrogated by simultaneous treatment with the intracellular Ca2+ antagonist TMB-8, indicating that Ca2+, a known intracellular "second messenger", is involved in this process. Elevated c-myc and p53 expression appears to be responsible for the time-dependent accumulation of apoptotic cells after prolonged 2-DG treatment. The finding that 2-DG acts selectively against the expression of a human pathogenic papillomavirus strongly suggests that an appropriate level of glycolysis is not only a peculiarity of growing tumors, but even may be an essential prerequisite for the maintenance of virus-specific E6/E7 gene expression. Our results may have substantial implications for the potential therapeutic application of 2-DG or other glucose derivatives in the treatment of precancerous and malignant HPV-associated lesions.

Adenosine Triphosphate↗
PubMed1996

Cell cycle effects of thaliblastine.

The non-myelotoxic antitumor drug thaliblastine (thalicarpine, NSC-68075, CAS-5373-42-21) has a novel chemical structure; it is a complex dimeric-type aporphine benzylisoquinoline alkaloid possessing antiproliferative and antitumor activities in experimental and clinical studies. In this study the effect of this drug on the cell cycle progression of ovarian tumor line O-342 and its cisplatin-resistant subline O-342/DDP was evaluated. As assessed by flow cytometric analysis, thaliblastine affected the cell cycle progression. In both lines, a comparable pattern of cell cycle arrest was found. Within the first 5 h of thaliblastine exposure, a G2/M block was observed; thereafter cell-cycle arrest in G1 became prominent, while S-phase cells finished DNA replication.

Animals↗
PubMed1995

Positive correlation between cellular glutathione and acquired cisplatin resistance in human ovarian cancer cells.

While multiple changes are frequently found to be associated with cisplatin resistance in a variety of tumor cell lines, a cause-effect relationship of these alterations with the resistant phenotype has not been established. In order to identify the resistance-relevant determinants, a series of cisplatin-resistant sublines with different degrees of resistance to cisplatin was developed in a human ovarian carcinoma cell line (O-129). Three derived resistant cell lines displayed 2.1-fold (O-129/DDP4, low), 4.1-fold (O-129/DDP8, moderate) and 6.3-fold (O-129/DDP16, high) resistance, respectively, to cisplatin, compared with the sensitive parental line O-129. While the activity of poly(ADP-ribose) polymerase, an enzyme proposed to be involved in DNA repair, was elevated in all three resistant lines, a significant karyotypic change was observed only in the high-resistance line with the karyotype alteration from near diploidy to heteroploidy. The moderate (4.1-fold) and high (6.3-fold) DDP resistance was associated with a slow proliferation rate in drug-free medium, but cellular glutathione level was highly correlated with DDP sensitivity in all four cell lines. Taken together, the present studies establish that while many changes at cellular level can occur with development of cisplatin resistance, only elevation of intracellular glutathione concentration appears to be related to the resistance phenotype in these human ovarian cancer cells.

Cell Division↗
PubMed1993

Heidelberg Cytometry Symposium, 22-24 October 1992.

Lectures and presentations at the 1992 Heidelberg Cytometry Symposium reflected a rapidly growing research field with widespread activities that cover daily routine diagnosis as well as investigations at the molecular level and the diagnosis of genetic alterations. Both flow and image cytometry and their impact on quantitative cytology were backed and combined with new approaches (i.e. magnetic cell sorting) that enable the isolation of rare cells with high purity for cell biological analyses and thus pave the way for new research fields (i.e. arteriosclerosis). Their combination with the multicolor painting of gene sequences (fluorescent in situ hybridization) represents a further improvement of chromosome quantification and thus the analysis of the topology of cell nuclei. This year's meeting will be held from 21 to 23 October. Its topics will comprise chromosome painting, apoptosis, automated cytology, bio-imaging and proliferation. As usual, space is given to the presentation of new techniques and concepts. The deadline for abstracts is 30 June.

DNA, Neoplasm↗
PubMed1991

Application of laser optical tweezers in immunology and molecular genetics.

Optical tweezers, based on a compact diode pumped Nd:YAG laser providing 350 mW at 1,064 nm coupled into a Zeiss IM 35 microscope, were used to sort CD4+ T cells into a capillary for further mechanical handling and to establish contact between single human natural killer (NK) cells and human erythroleukemia cells (K562) as targets. After contact and a lag phase of a few tens of seconds, the target cell starts to change its morphology and membrane blebbing occurs. The kinetics of the attack of the NK cell on K562 cells is not straightforward but governed by temporal oscillations in the shape of the target cell (zeosis). In a second application, the optical tweezers are combined with a UV laser microbeam based on a pulsed UV laser and with flow cytometry and sorting. With the pulsed laser, segments of sorted chromosome 1 of the chinese hamster karyotype (CHV 79) can be easily micro-dissected and subsequently collected using the optical tweezers. This allows preparation of a few hundred chromosome segments per day without mechanical contact and in an absolutely sterile way and thus may provide an interesting basic technique in any type of genome sequencing project.

Animals↗
PubMed1991

Extinction of the HPV18 upstream regulatory region in cervical carcinoma cells after fusion with non-tumorigenic human keratinocytes under non-selective conditions.

'Universal fuser' clones of a human papillomavirus type 16 positive cervical carcinoma cell line (SiHa) were established to study the effect of a non-tumorigenic fusion partner on the regulation of a stably integrated chloramphenicol acetyltransferase (CAT) gene controlled by the HPV18 upstream regulatory region under non-selective conditions. The CAT expressing cells were fused with both non-tumorigenic, spontaneously immortalized human keratinocytes (HaCaT) and non-modified SiHa cells. The resulting hybrids were characterized by restriction enzyme fragment length polymorphism analysis and flow cytometry. While the non-selectable, HPV18-driven indicator gene is constitutively expressed in SiHa cells, the CAT activity is extinguished in SiHa x HaCaT cells, but still present in SiHa x SiHa hybrids. Examination of the cytokeratin expression pattern reveals that the keratinocyte phenotype seems not only to be dominant in terms of the extinction of the HPV18 regulatory region but also by the conservation of most of the differentiation markers of the non-tumorigenic fusion partner. Cycloheximide treatment and intracellular competition experiments using the transient COS7 fusion-amplification technique are accompanied by the reactivation of the marker gene in previously CAT- SiHa x HaCaT hybrids. These data strongly suggest that trans-acting negative regulatory factors derived from the non-malignant human keratinocytes are responsible for the extinction phenomenon.

Animals↗
PubMed1991

Karyotypic change from heteroploidy to near diploidy associated with development of cisplatin resistance in a rat ovarian tumour cell line.

In a rat ovarian tumour cell line a 33-fold resistance to cisplatin (O-342/DDP) was developed in vitro by continuous exposure of the parental cell line (O-342) to stepwise increase cisplatin concentration in the culture medium. Both cell lines had a similar growth rate in vitro. Development of resistance was accompanied by a change of the karyotype from heteroploidy in chemosensitive O-342 cells to near diploidy in resistant O-342/DDP cells as shown by chromosome number distribution. This finding was confirmed by measuring cellular DNA content using flow-cytometry analysis. Flow karyotyping showed significant differences in chromosomal DNA contents between both cell lines. Our results suggest that the parent line O-342 consists of at least two subpopulations, a cisplatin-sensitive and a cisplatin-resistant one, corresponding to hyperploidy and near diploidy, respectively. Continuous cisplatin exposure of O-342 cells selectively killed the sensitive fraction, resulting in the karyotypic change observed.

Animals↗
PubMed1990

Detection and chromosomal assignment of SV40-DNA integration in Chinese hamster cell lines by chromosome sorting and dot blot hybridization.

A combination of cytometric (chromosome sorting), molecular (dot blot hybridization using radio-active and/or biotinylated DNA probes) and cytogenetic (G-banding) evaluation is described which allows the rapid identification of single copy and repetitive viral integrates and their assignment to chromosome groups or even individual chromosomes. In the case of Chinese hamster cell line CO 631 it could be demonstrated that SV40 DNA was solely integrated into a submetacentric marker chromosome. Such a cytometric/molecular/cytogenetic "identogram" may prove to be a useful tool in many areas of cell and tumor biology. Furthermore, amounts of chromosomes sufficient for analysis as well as subsequent cloning experiments can be accumulated.

Animals↗
PubMed1990

Cytogenetic investigations on a cell line derived from a carcinoma arising in a salivary gland pleomorphic adenoma.

This article discusses the results of cytogenetic investigations of a cell line derived from a malignant tumor arising in a benign salivary gland pleomorphic adenoma. Initial karyotypic studies became possible with cells of the second in vitro passage and revealed the existence of hypertetraploidy. Furthermore, a marked polysomy 7 and a translocation involving 12q13-15, a breakpoint also frequently seen in benign pleomorphic adenomas, were noteworthy. During long term culture, the number of chromosomes per cell decreased as well as the number of copies of chromosome 7. Cell tumorigenicity was proved by heterotransplantation to nude mice. The resulting tumors were karyotyped again. No significant changes of the chromosome number or of the degree of polysomy 7 were found compared to the cells before heterotransplantation. In contrast, the cells from the nude mice tumors showed a remarkable number of different isochromosomes probably indicating an unknown factor supporting the generation of isochromosomes. The consistent presence of the t(12;?)(q13-15;?) makes the cell line well suited for molecular studies of this breakpoint region.

Adenoma, Pleomorphic↗
PubMed1989

Establishment and characterization of human glioblastoma cell lines in vitro and their xenografts in nude mice.

Human gliomas were grown as permanent tissue cultures and xenografts in nude mice. Growth kinetics, immunohistology and karyotypes were established. One tumor showed a distinct change in its karyotype after only two passages in nude mice; by contrast, no change of immunophenotype occurred during and after in vivo passaging. Treatment of this glioma as xenograft in nude mice revealed a high sensitivity towards single agent treatment with BCNU. In view of the cytologic aberrations, however, this result should be interpreted cautiously with regard to the clinical situation.

Animals↗
PubMed1988

Identification of flow-sorted chromosomes by G-banding and in situ hybridization.

The identification of flow-sorted chromosomes is a very important tool for checking the purity of the fractions obtained. An easy and reproducible method for obtaining G-banded chromosomes with good resolution of bands is described. Also, we are able to show that the percentage of chromosomes which can be clearly distinguished by this procedure depends to a large extent on the duration of mitotic arrest. In particular when sorting chromosomes from human-rodent hybrid cell lines, the possibility of using in situ hybridization in addition to conventional staining techniques to characterize the chromosomes can help overcome the problem of highly condensed chromosomes and chromosomal fragments of unknown origin, which cannot be identified otherwise. Thus, we have developed an in situ hybridization technique, based on biotin-labelled human genomic DNA, which allows a clear distinction between human and rodent chromosomal material to be made.

Animals↗
PubMed1985

Detection and separation of the submetacentric marker chromosome of the WALKER (W-256) carcinoma using flow cytometry and sorting.

The chromosomes of WALKER (W-256) carcinoma cells have been separated into different DNA subclasses using DAPI for quantitative DNA staining and laser flow cytometry. The submetacentric marker chromosome could be isolated and its DNA content was determined to be 1.3 pg. One microgram marker DNA was obtained after separation of about 750 000 marker chromosomes by means of electronic flow sorting. The chromosomal composition of sorted fractions was analyzed by microscopy following banding of sorted chromosomes. The average morphological purity obtained was about 83%.

Animals↗
PubMed1982

Assignment of snRNA gene sequences to the large chromosomes of rat kangaroo and chinese hamster isolated by flow cytometric sorting.

Chromosomes from a rat kangaroo (Potorous tridactylus) cell line (PtK2) and from a Chinese hamster (Cricetulus griseus) cell line (CHV79) were isolated by means of fluorescence activated flow cytometric sorting. DAPI (4'-6-diamino-2-phenylindole) was used as the DNA specific fluorescent dye. The karyotype of the PtK2 cells which exhibits 13 chromosomes was separated into 6, and the 22 chromosomes of the CHV79 cells were resolved into 11 fractions. DNA extracted from these chromosomal fractions was used for restriction enzyme digestion and blotting on nitrocellulose filters. The blots were challenged with gene probes corresponding to ribosomal RNA (18S and 28S) and small nuclear RNA (U1-snRNA) genes. The rRNA genes were exclusively assigned to chromosomes containing the nucleolus organizing region (in PtK2: X chromosome; in CHV79: chromosomes 4, 5, 6, and 11). - Solely the largest chromosomes in both cell lines hybridized with U1-snRNA indicating that these gene sequences are located on those chromosomes only. Further possible genetic and biochemical applications of this experimental system are discussed.

Animals↗
PubMed1982

A reliable preparation of mon-dispersed chromosome suspensions for flow cytometry.

A reproducible, simple and rapid protocol was developed to prepare mono-dispersed chromosome suspensions for flow cytometric analysis. The procedure basically employes: 1. twice rinsing of monolayer cultures to eliminate dead cells and cellular debris, 2. mild fixation of mitotic cells with 1% acetic acid, and 3. ultra sonic treatment to release single metaphase chromosomes. The procedure takes less than 30 min. Isolated chromosomes are storable over months at 4 degrees C. Despite mild fixation many biochemical studies and experiments applied on such fixed chromosomes purified and enriched using electronic sorting are feasible: 1. gene and restriction mapping, 2. cloning of specific gene sequences, and 3. gene frequency analysis.

Animals↗
PubMed1982

Rapid detection of mutagen induced micronucleated erythrocytes by flow cytometry.

The micronucleus test is a cytogenetic method for the screening of mutagens and carcinogens which exhibit clastogenic mechanisms of action. After application of clastogenic agents chromosomal fragments or even whole chromosomes can remain as conspicuous structures (micronuclei) in a small fraction of anucleated polychromatic erythrocytes which can be visually scored using a microscope following staining with May-Grünwald-Giemsa solutions. These time-consuming, painstaking, and tedious manual evaluations are often sources of unreliability and uncertainty. Here, a fluorescence technique is presented which applies DNA and protein fluorochromes to discriminate normal anucleated erythrocytes from micronucleated erythrocytes using a fluorescence microscope. This method is particularly tailored to be applied to flow cytometric instrumentation. Data obtained manually and automatically in flow show a strong linear correlation with high significance (r = 0.96) as far as the percentage of micronucleated erythrocytes as an indicator for the mutagenicity of a given drug is concerned. These results have been obtained by means of the established clastogens cyclophosphamide and mitomycin C.

Animals↗
PubMed1980

A flow cytometric study of chromosomes from rat kangaroo and Chinese hamster cells.

Chromosomes from rat kangaroo (PTK) and chinese hamster (CHV 79) cells have been prepared for quantitative flow-cytometric analysis. The preparation time was otimized down to 30 (PTK) and 40 min (CHV 79). DAPI was used as a AT-sensitive fluorescent dye to stain for monoparameter DNA measurements. Simultaneous two-parameter DNA-protein analysis was carried out with DAPI and SR 101 (as a general protein fluorochrome) in combination. The karyotype of the PTK cells with 13 (14) chromosomes was separated into 10DNA peaks. The X-chromosome bearing the nucleolus organizer region generates a distinct peak. The karyotype of the CHV 79 cells with 22 chromosomes was separated inot 15 peaks. The DNA profile obtained indicates a geometric grading of the chromosomal amount of AT components in teh karyotype of this particular cell line. The simultaneous DNA-protein analysis performed show enough sensitivity of the instrument utilizing hihg power UV excitation illumination to discriminate the two color emission consisting of blue (DAPI) and red (SR 101) fluorescence. Color overlapping could be completely avoided. Additionally, the quality (number, location, and resolution of peaks) of the DNA distribution was not influences by the simultaneous application of a second fluorescent stain. Fluorescence activated electronic sorting applied on chromosomal fluorescence distributions providing purified fractions of chromosomes for subsequent biochemical and biological determinations is discussed.

Animals↗