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.

A physical-biological coupled model for algal dynamics in lakes.

A coupled model is presented for simulating physical and biological dynamics in fresh water lakes. The physical model rests upon the assumption that the turbulent kinetic energy in a water column of the lake is fully contained in a mixed layer of variable depth. Below this layer the mechanical energy content is assumed to vanish. Additionally, the horizontal currents are ignored. This one-dimensional two-layered model describes the internal conversion of the mechanical and thermal energy input from the atmosphere into an evolution of the mixed layer depth by entrainment and detrainment mechanisms. It is supposed to form the physical domain in which the simulation of the biological processes takes place. The biological model describes mathematically the typical properties of phyto- and zooplankton, their interactions and their response to the physical environment. This description then allows the study of the behaviour of Lagrangian clusters of virtual plankton that are subjected to such environments. The essence of the model is the dynamical simulation of an arbitrary number of nutrient limited phytoplankton species and one species of zooplankton. The members of the food web above and below affect the model only statically. The model is able to reproduce the typical progression of a predator-prey interaction between phyto- and zooplankton as well as the exploitative competition for nutrients between two phytoplankton species under grazing pressure of Daphnia. It suggests that the influence of the biological system on the physical system results in a weak increase of the surface temperature for coupled simulations, but a considerably higher seasonal thermocline in spring and a lower one in autumn.

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.

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.

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.

[Acute myocardial infarction mortality in a hospital department during the last decades]. / Az acut myocardialis infarctus halálozásának alakulása osztályunkon az utóbbi évtizedekben.

Authors surveyed the data of 2677 patients with acute myocardial infarction treated in cardiological department between 1970 and 1993. Activity of Coronary Care Unit (has been working from 1974) reduced the earlier hospital mortality in general internal medicine possibilities with 50%. Main cause of this reduction was the temporal electrotherapy of cardiac arrhythmias. Since the middle of last decade the goal of has been generally introduced combined therapy (beta-blockers, intravenous nitrates, acetyl-salicic acid, thrombolytics) is to reduce the mass of damaged myocardium and hereby increasing the ratio of survival. They reduced the hospital mortality around 10% with consistent application of this therapy. According to the authors application of thrombolytic therapy has basic importance in management of myocardial infarction, but they could not treat a lot of patients with streptokinase in consequence of too long prehospital time period and not properly controlled hypertension. The proportion of systemic thrombolytic therapy on their patients is almost a quarter of them. It would be very important to increase this beneficial ratio with reducing the time of patients decision to turn to health care and applying systemic thrombolytic therapy more than six hours after the onset of symptoms of myocardial infarction.

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.

In vivo and in vitro genotoxicity of several N-nitrosamines in extrahepatic tissues of the rat.

Toxicological mechanisms involved in organotropism of tumor induction may include cell-specific metabolic activation of the carcinogen, in vivo distribution of active metabolites and persistance of induced DNA damage. In order to elucidate which factors are involved in the organotropic action of environmentally relevant N-nitrosamines, we have studied their genotoxic and cytotoxic effects within primary intact cells of lung and kidney. The end-points determined were cytotoxicity by trypan blue exclusion and DNA single-strand break (SSB) induction by alkaline filter elution. The assays were performed in vitro to determine organ-specific metabolic activation by incubating the cells with the test compounds. The results obtained with N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodiethanolamine (NDEIA), N-nitrosoethylvinylamine (NEVA), N-nitrosodibutylamine (NDBA), N-nitrosobutylbutanolamine (NBBOH), N-nitrosobutylcarboxypropylamine (BCPN), N-nitrosomethylbenzylamine (NMBzA) and N-nitrosodibenzylamine (NDBzA) indicate that several compounds may be activated to reactive metabolites by cells of kidney and lung, NDBzA revealing the highest degree of cytotoxicity. In contrast, genotoxicity in kidney cells was induced only by NBBOH and BCPN and at relative low levels. Primary lung cells could not be employed as indicators for genotoxic effects in vitro because the cell yield was not sufficient to perform the alkaline elution assay. To assess the distribution of NDMA in the whole rat and the persistence of the induced DNA damage in the two organs, further studies were carried out after oral application of 1, 2, 4, 10, 20, 32 and 40 mg/kg to the animals. Following 1 h exposure of rats to NDMA, the lowest effective genotoxic dose for lung and kidney was 2 mg NDMA/kg body wt. A plateau was achieved after a dose of 20 mg/kg in both organs. Furthermore, the persistence of DNA damage was studied in the lung. After 4 h exposure, DNA damage was still detectable at 32 mg NDMA/kg, but for the lower doses it was reduced nearly to control levels. After 16 h exposure the SSB rate in lung cells was reduced for all dose levels except for the highest dose of 40 mg/kg.

Secondary metabolites by chemical screening. 8. Decarestrictines, a new family of inhibitors of cholesterol biosynthesis from Penicillium. I. Strain description, fermentation, isolation and properties.

A family of new 10-membered lactones was detected by chemical screening. Taxonomic studies and fermentation conditions of the producing organisms, which belong to the species Penicillium simplicissimum and Penicillium corylophilum, are presented. The isolation as well as physico-chemical data of the new compounds named decarestrictines A to D are reported. In vitro testing using the HEP-G2 cell assay showed the decarestrictines to be inhibitors of cholesterol biosynthesis, which could be confirmed in vivo. In addition to the decarestrictines from P. corylophilum epoxyagroclavine-I (1) was isolated.

Secondary metabolites by chemical screening. 9. Decarestrictines, a new family of inhibitors of cholesterol biosynthesis from Penicillium. II. Structure elucidation of the decarestrictines A to D.

The structures of the novel 10-membered lactones, named decarestrictines A1/A2 (1/2), B (3), C1/C2 (5/6) and D (7), are presented. The structures of these secondary metabolites, isolated from different Penicillium species, were established by spectroscopic analysis and confirmed by X-ray analysis of 7 and a derivative of 3 leading to the stereochemical information. The decarestrictines vary in the oxygenation pattern between C-3 and C-7 and show structural similarities to known lactones from other fungi.

Further characterization of acquired-resistance to Cisplatin in a rat ovarian tumor-cell line.

The proliferation of a rat ovarian tumor cell line (O-342) was completely inhibited by a 2 h exposure to 20 muM cisplatin (DDP) in vitro up to 120 h after its removal, while in its DDP resistant subline (O-342/DDP), the same treatment only caused a transient growth inhibition within the first 24 h post the exposure, followed by the recovery of proliferation at a similar rate as the control cells. DNA interstrand cross links (ISCL) were maximally formed 12 h post DDP treatment in either O-342 or O-342/DDP cells, with a 2.8-fold increase in the sensitive cells at this time (262 vs. 95 rad eq.). After further 12 h incubation, however, 75% of DNA-ISCL was removed in O-342/DDP cells, while only 22% of them were repaired in O-342 cells. DNA single strand breaks (SSB) were produced to a similar extent in both lines but reached a maximum at 12 and 24 h in the resistant and the sensitive cells, respectively. ADP-ribosyl transferase (ADPRT) activity, a DNA repair-associated enzyme, was 2.6-fold higher in O-342/DDP cells compared to the sensitive subline. Following DDP treatment, the activity was stimulated in the sensitive cells with a maximum of about 1.5-fold at 24 h, whereas the inhibitory effect was observed in the resistant cells, although at 12 h it recovered almost to the control level. Flow cytometric analysis showed that there were at least two sub-populations (2n and 4n) in O-342 cells, while only 2n population was observed in O-342/DDP cells. Following DDP exposure, O-342/DDP cells progressed through the cell cycle with only a small and transient accumulation of cells in S-phase at 12 h and 24 h, while in the sensitive cells, it was impossible to distinguish cell-cycle distribution at 12 h due to severe damage, at 24 h most of the cells became arrested in G2-phases, which persisted until the end of the observation (48 h). Our results suggest that both reduced interaction of cellular DNA with DDP and increased DNA repair are contributing factors for development of DDP resistance, which might be directly or indirectly subsequent to alterations of poly (ADP-ribose) metabolism in the resistant cells.

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.

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.

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.

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.

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.

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.

[Polychlorinated biphenyls: determination of individual components in raw milk and contaminated fodder]. / Polychlorierte Biphenyle: Bestimmung der Einzelkomponenten in Rohmilch und kontaminierten Futtermitteln.

Since 1980, 2145 samples of raw milk from producers in the district of Freiburg have been analysed for polychlorinated biphenyls (PCB) during a monitoring programme. In 1983, we started to search for the source of PCB, whenever milk was contaminated above the average value (above 0.2 mg/kg fat, calculated as Clophen A60). In 7 cases, previous coats of paint in the silos proved to be the cause of the PCB contamination. Five samples of milk and 4 corresponding silages as well as 1 sample of wood (from a cowshed) were investigated for all of the individual PCB components included in the technical mixtures of Clophen A30 and A60. Two samples of silages contained both low and high chlorinated biphenyls, whereas low chlorinated biphenyls (di-, tri-, and tetrachlorinated biphenyls) were not detected in the corresponding milk samples. Pentachlorinated biphenyls were only detected in the range of 1%-2%. In the samples of wood and corresponding milk, a different PCB pattern appeared. There were remarkably high percentages of hepta- and octachlorinated biphenyls as well as a lower percentage of hexachlorinated biphenyls. The exact PCB content of the milk, determined by the addition of all single components, proved to be approximately half the value obtained by the usual calculation based on the evaluation of the three main peaks of the technical PCB mixture (Clophen A60). During 1984-1987, 607 samples of raw milk were analysed for six single PCB components, for which legal tolerance levels became valid in 1988.(ABSTRACT TRUNCATED AT 250 WORDS)