Christian Johannes

Malignant transformation by cyclin E and Ha-Ras correlates with lower sensitivity towards induction of cell death but requires functional Myc and CDK4

We demonstrate in this paper that the G1 phase specific cell cycle regulator cyclin E is able to provoke focus formation when cotransfected with activated Ha-ras into primary rat embryo fibroblasts (REFs). Cyclin E/Ha-ras transformed cells are highly tumorigenic in synergeneic rats, are able to form colonies in soft agar and show protection towards apoptosis upon serum starvation or DNA damage compared to cells transformed by the combination of Myc, cyclin D1 or SV40 large T-antigen and Ha-ras. Lines that were established after cyclin E/Ha-ras or cyclin D1/Ha-ras transformation contain a large percentage of polyploid cells. This was not observed in cells transformed with other oncoproteins and Ha-ras pointing to an involvement of D- and E type cyclins in genomic instability. The cyclin dependent kinase inhibitors p21 and p27 but also p16 completely abrogate focus formation by cyclin E and Ha-ras suggesting that the oncogenic activity of cyclin E still requires functional G1 specific cyclin/CDK complexes. Moreover, inhibition of Myc function also blocks the oncogenic activity of cyclin E indicating a requirement of Myc for cyclin E function. The findings presented here demonstrate that cyclin E can act as an oncoprotein with a potential involvement in genomic instability and the prevention of cell death. Our data also present more evidence for a strict functional interdependency between G1 cyclin/CDK complexes and c-Myc.

Analysis of X-ray-induced aberrations in human chromosome 5 using high-resolution multicolour banding FISH (mBAND)

Peripheral lymphocytes were exposed to 4 Gy X-rays and aberrations were analysed in human chromosome 5 using high-resolution multicolour banding fluorescence in-situ hybridization (mBAND). This method is suited to detect simple and complex aberrations including peri- and paracentric inversions and exchanges between both chromosomes 5. Additionally, breakpoints can be assigned to specific regions in chromosome 5. Quantitative relationships of induced aberration types are discussed.

The yield of radiation-induced micronuclei in early and late-arising binucleated cells depends on radiation quality

There are conflicting data regarding the effect of culturing time of human peripheral blood lymphocytes on the yield of chromosomal aberrations induced by sparsely ionising radiation in the G0 phase of the cell cycle. While some authors find that the yield of aberrations does not change with time, others find increased frequencies of aberrations with harvesting time. The reasons for the conflicting results are not known, but the majority of studies were performed with lymphocytes of a single donor collected at one time point. We performed a study to verify if individual variability could be a confounding factor. As a positive control, lymphocytes were also exposed to high LET radiation (neutrons and alpha-rays), where an effect of harvesting time on the level of damage is expected to be seen. Blood was drawn from a total of 8 donors at two time points and exposed to X-rays, 6 MeV neutrons or alpha particles generated by an Am-241 source. Whole blood cultures were set up and micronuclei (Mn) were scored in binucleated cells harvested after 72, 96 and 120 h of culture time. The results show that in lymphocytes exposed to X-rays, the frequency of Mn was generally not influenced by the culture time while for both neutrons and alpha particles consistently increased micronucleus frequencies with culture time were detected. Some individual variability was detected and the conflicting results regarding the relationship between the yield of cytogenetic damage and lymphocyte culture time can, at least partly, be due to this variability.

Effect of temperature during irradiation on the level of micronuclei in human peripheral blood lymphocytes exposed to X-rays and neutrons.

Objectives: It has been reported that the level of cytogenetic damage in human peripheral blood lymphocytes (PBL) is higher following irradiation at 37°C than at 0–4°C. The mechanisms of this cytogenetic temperature effect are not fully known. The aim of our study was to check whether the effect was related to the indirect or direct action of radiation. Materials and methods: PBL were kept at 37°C and 0°C for 20 min and exposed to 2 Gy of X-rays. In some experiments PBL were isolated and 0.5 M dimethyl sulfoxide (DMSO) was added for 5 min before exposure. PBL were also irradiated at 37°C and 0°C with 1 Gy of 6 MeV neutrons. Micronuclei were scored as the endpoint. Following exposure to X-rays the level of initial DNA damage was also measured by the alkaline and neutral comet assay. Results: The frequency of micronuclei in cells exposed at 37°C to X-rays or neutrons was higher than that after exposure at 0°C. No effect of temperature was seen when PBL were exposed to X-rays in the presence of DMSO. No effect of temperature was observed on the level of DNA damage measured with the alkaline or neutral comet assay. Conclusions: The results of experiments with DMSO indicate that the temperature effect is due to the indirect action of radiation, i.e., via reactive oxygen species. However, this is not supported by the results with neutrons and the comet assay. Possible reasons for the discrepancies are discussed.

Cross-Contamination of a UROtsa Stock with T24 Cells – Molecular Comparison of Different Cell Lines and Stocks

Background UROtsa is an authentic, immortalized human urothelial cell line that is used to study the effects of metals and other toxic substances, mostly in the context of bladder cancer carcinogenesis. Unusual properties on the molecular level of a provided UROtsa cell line stock prompted us to verify its identity. Methods UROtsa cell line stocks from different sources were tested on several molecular levels and compared with other cell lines. MicroRNA and mRNA expression was determined by Real-Time PCR. Chromosome numbers were checked and PCR of different regions of the large T-antigen was performed. DNA methylation of RARB, PGR, RASSF1, CDH1, FHIT, ESR1, C1QTNF6, PTGS2, SOCS3, MGMT, and LINE1 was analyzed by pyrosequencing and compared with results from the cell lines RT4, T24, HeLa, BEAS-2B, and HepG2. Finally, short tandem repeat (STR) profiling was applied. Results All tested UROtsa cell line stocks lacked large T-antigen. STR analysis unequivocally identified our main UROtsa stock as the bladder cancer cell line T24, which was different from two authentic UROtsa stocks that served as controls. Analysis of DNA methylation patterns and RNA expression confirmed their differences. Methylation pattern and mRNA expression of the contaminating T24 cell line showed moderate changes even after long-term culture of up to 56 weeks, whereas miRNAs and chromosome numbers varied markedly. Conclusions It is important to check the identity of cell lines, especially those that are not distributed by major cell banks. However, for some cell lines STR profiles are not available. Therefore, new cell lines should either be submitted to cell banks or at least their STR profile determined and published as part of their initial characterization. Our results should help to improve the identification of UROtsa and other cells on different molecular levels and provide information on the use of urothelial cells for long-term experiments.

Induction of sister-chromatid exchanges by AluI, DNase I, benzon nuclease and bleomycin in Chinese hamster ovary (CHO) cells

Various endonucleases (AluI, DNase I, benzon nuclease) and bleomycin induce sister-chromatid exchanges (SCE) in Chinese hamster ovary (CHO) cells. The frequencies of SCE are elevated in cells with chromosome-type aberrations, only slightly elevated in cells with chromatid exchanges, and in the control range in cells without chromosomal aberrations. These data indicate that SCE are produced when DNA breaks induced in G1 are either not repaired or misrepaired.

Induction of chromosomal aberrations by DNase I

Chinese hamster ovary (CHO) cells were treated with bovine pancreatic DNase I using the method of electroporation. The enzyme induced chromosomal aberrations in a S-phase independent manner. The frequencies of polycentric chromosomes induced in the G1 phase of the cell cycle are positively correlated with the dose of DNase I. The distributions of DNase I-induced polycentric chromosomes were overdispersed.

Analysis of the action of the restriction endonuclease AluI using three different comet assay protocols.

Background and Purpose:The comet assay offers the opportunity to measure the amount of DNA damage and the effectiveness of DNA repair in single cells. In a first part, experiments are presented comparing three different protocols of the comet assay technique with respect to the analysis of the induction of DNA damage after X-irradiation in isolated human lymphocytes and CHO cells. In a second part, the restriction enzyme AluI, an agent producing DNA double-strand breaks exclusively, was introduced into CHO cells by electroporation and the effects were analyzed using the different comet assay protocols. The experiments were carried out in order to test the assertion that comet assay techniques can measure different types of DNA damages at different pH conditions of lysis and electrophoresis.Material and Methods:Three different comet assay protocols were used for the analysis of DNA damage in lymphocytes and CHO cells.Results:The results clearly indicate that among the three protocols the modified comet assay technique used by the authors showed the highest sensitivity in the radiotherapy-relevant dose range between 0 and 2 Gy. All three protocols were capable of detecting an effect by AluI. This effect, however, was clearly different from radiation effects. Whereas after radiation exposure all cell nuclei show a dose-dependent increase in DNA content in the comet tail, most of the cell nuclei were unaffected by an AluI uptake. Nevertheless, there was an effect by AluI that could be detected in all three assay versions: between 5% and 15% of the nuclei showed clearly abnormal comet morphologies.Conclusion:Neither the strictly alkaline nor the strictly neutral comet assay is applicable in the radiation dose range of about 2 Gy. The restriction enzyme results show that other factors than just DNA strand breaks contribute to DNA migration into the tail of the comets.Hintergrund und Ziel:Der „Kometen-Test“ liefert die Möglichkeit, das Ausmaß von DNA-Schäden und DNA-Reparatur in Einzelzellen zu messen. In einem ersten Teil werden Experimente vorgestellt, in denen drei verschiedene Protokolle des Kometen-Tests auf ihre Fähigkeit überprüft wurden, DNA-Schäden nach Röntgenbestrahlung in isolierten menschlichen Lymphozyten und CHO-Zellen zu erfassen. Im zweiten Teil der Experimente wurde das Restriktionsenzym AluI, ein Agens, das ausschließlich DNA-Doppelstrangbrüche hervorruft, durch Elektroporation in CHO-Zellen eingeführt. Die Effekte wurden mit Hilfe der verschiedenen Kometen-Tests analysiert. Die Experimente wurden durchgeführt, um die Behauptung zu testen, dass durch Veränderung der pH-Bedingungen bei der Lyse und der Elektrophorese unterschiedliche DNA-Schadenstypen erfasst werden können.Material und Methodik:Es wurden drei verschiedene Kometen-Test-Protokolle (Tabelle 1) zur Analyse von DNA-Schäden in Lymphozyten und CHO-Zellen verwendet.Ergebnisse:Die Ergebnisse zeigen eindeutig, dass von den drei Protokollen die von den Autoren verwendete modifizierte Fassung des Kometen-Tests die höchste Empfindlichkeit im für die Strahlentherapie relevanten Dosisbereich von 0–2 Gy aufweist (Abbildungen 1 und 2). Alle drei Protokolle konnten einen Effekt durch AluI erfassen. Allerdings war dieser Effekt deutlich verschieden von Strahleneffekten. Während nach einer Strahlenexposition alle Zellkerne eine dosisabhängige Zunahme an DNA im Kometenschweif aufweisen, blieben die meisten Zellkerne trotz AluI-Aufnahme (Abbildung 5) unbeeinflusst. Dennoch gab es einen AluI-Effekt, der in allen drei Versionen des Tests nachweisbar war: Zwischen 5 und 15% aller Kerne zeigten deutlich anormale Kometenstrukturen (Abbildungen 3 und 4).Schlussfolgerung:Weder der strikt alkalische noch der strikt neutrale Kometen-Test ist in einem Strahlendosisbereich von etwa 2 Gy anwendbar. Die Ergebnisse der Restriktionsenzymexperimente zeigen, dass neben DNA-Strangbrüchen noch andere Faktoren das Ausmaß der DNA-Wanderung in den Kometenschweif bestimmen.

Exposure of CHO cells to AluI : comparison of chromosomal aberrations and cell survival

Chinese hamster ovary (CHO) cells were exposed to various doses of the restriction endonuclease AluI in the presence of 2.2 M glycerol. Some of the cells were cultured for analysis of chromosomal aberrations and some for analysis of colony-forming ability. Cell killing is mainly mediated by chromosomal aberrations.

Chromatid-type aberrations induced by AluI in Chinese hamster ovary cells

Treatment of CHO cells with AluI in the S-phase leads to chromatid-type aberrations whose frequencies are linearly correlated with dose. Treatment in the S-phase leads to fewer aberrations than treatment in the G1-phase, which is comparable to chromosomal aberration induction following X-irradiation in the G1- and S-phases. Treatment in the G1-phase leads to few chromatid-type interchanges, some of these may originate from DNA single-strand gaps induced by AluI in canonical structures of DNA and in DNA.RNA hybrids.