UVA-induced DNA double-strand breaks result from the repair of clustered oxidative DNA damages.

UVA (320-400 nm) represents the main spectral component of solar UV radiation, induces pre-mutagenic DNA lesions and is classified as Class I carcinogen. Recently, discussion arose whether UVA induces DNA double-strand breaks (dsbs). Only few reports link the induction of dsbs to UVA exposure and the underlying mechanisms are poorly understood. Using the Comet-assay and γH2AX as markers for dsb formation, we demonstrate the dose-dependent dsb induction by UVA in G(1)-synchronized human keratinocytes (HaCaT) and primary human skin fibroblasts. The number of γH2AX foci increases when a UVA dose is applied in fractions (split dose), with a 2-h recovery period between fractions. The presence of the anti-oxidant Naringin reduces dsb formation significantly. Using an FPG-modified Comet-assay as well as warm and cold repair incubation, we show that dsbs arise partially during repair of bi-stranded, oxidative, clustered DNA lesions. We also demonstrate that on stretched chromatin fibres, 8-oxo-G and abasic sites occur in clusters. This suggests a replication-independent formation of UVA-induced dsbs through clustered single-strand breaks via locally generated reactive oxygen species. Since UVA is the main component of solar UV exposure and is used for artificial UV exposure, our results shine new light on the aetiology of skin cancer.

Culturing explanted colon crypts highly improves viability of primary non-transformed human colon epithelial cells.

Chemoprotective effects of nutritional compounds are usually studied in cell lines. Studies using primary human colon cells have been limited due to the lack of established methods regarding their culture. We therefore optimized isolation and culture of non-transformed human epithelial cells from individual donors to enrich viable cells and sufficient amounts of intact RNA. Isolated epithelial cells were seeded in different coated cell culture dishes combined with several media (2-24h). To avoid cells from anoikis, also intact colon crypts were isolated to maintain cell interactions. These crypts were incubated with gut fermentation products (24h) derived from indigestible carbohydrates. In none of the coated (fibronectin, laminin) cell culture dishes isolated epithelial cells did attach. The number of these cells remaining in suspension, decreased already after 2h to 20%. Intact colon crypts cultured as pellets showed a stable viability up to 24h (91±4%) and were suitable to gain a sufficient quantity of RNA. The use of colon crypts with an appropriate cell culture medium could double the lifespan of intestinal epithelial cells from 12 up to 24h and represents a promising approach to study early events in carcinogenesis and chemoprevention as well as other diseases of the colon.

Butyrate suppresses mRNA increase of osteopontin and cyclooxygenase-2 in human colon tumor tissue.

The short chain fatty acid (SCFA) butyrate, a product of fermentation of dietary fiber in the human colon, is found to exert multiple regulatory processes in colon carcinogenesis. The aim of this study was to find out whether butyrate affects the tumor-promoting genes osteopontin (OPN) and cyclooxygenase (COX)-2, their respective proteins and/or their functional activity in matched normal, adenoma and tumor colon tissues obtained from 20 individuals at colon cancer surgery. Quantitative real-time polymerase chain reaction experiments showed increased levels of OPN and COX-2 messenger RNA in tumor tissues when compared with the adjacent normal samples (P < 0.001). The addition of butyrate reduced OPN and COX-2 mRNA expression in all tissue types compared with the related medium controls (tumor: P < 0.05). In tumor samples, a downregulation of up to median 35% (COX-2) and 50% (OPN) was observed, respectively. Thereby, tumors with lower levels of OPN basal expression were more sensitive to inhibition and vice versa for COX-2 in normal tissue. At the protein and enzyme level, which were determined by using western blot and enzyme immunometric assays, the impact of the SCFA was not clearly visible anymore. The active proteins of OPN and COX-2 (determined by prostaglandin E(2)) were found to correlate with their respective mRNA expression only in 50-63% of analyzed donors. For the first time, our data reveal new insights into the chemoprotective potential of butyrate by showing the suppression of OPN and COX-2 mRNA in primary human colon tissue with the strongest effects observed in tumors.

The database dbEST correctly predicts gene expression in colon cancer patients.

This study aims to test the predictive power of gene expression data derived from NIH's database dbEST, which collects gene expression results from a large number and variety of DNA array experiments. The motivation of this study is to make comparable experimental studies, which are usually performed only for one or a few tissues or organs, with a wide variety of other tissues. Confirmation of a good predictive power of dbEST would put a number of interesting and partially surprising recent findings, solely based on data mining, on a more solid basis than available so far. The expression of nine genes (eIF4E, DDX6, HAT1, USP28, HSP90(beta, PKM2, PLK1, COX2 and OPN) plus two calibration genes in paired normal and cancer colon tissues of eight individual patients was investigated by quantitative RT-PCR and compared with the predictions made by the data-base. GUS and beta-actin reveal only little variation among different patients, making them good internal calibration standards. In normal colon tissue, data mining correctly predicts the expression of all nine genes, which covers two orders of magnitude. In cancer, dbEST is somewhat less precise, but still valuable for the comparison with clinical results.

Expression levels of 63 p53-related genes add up to similar values in 24 different tissues and are unified in cancer.

The expression patterns of 62 genes interacting with p53 have been investigated in 24 normal and cancerous tissues using NIH's dbEST library. The expression levels of individual genes, such as the TTP53 gene itself, but also other genes, vary up to 33-fold among the 24 different tissues and no consistent pattern can be recognized. However, when expression levels for all 63 genes are summed, these "cumulated levels" are surprisingly constant over the 24 investigated normal tissues. In cancers, the variation is further reduced. Essentially, the cumulated expression levels in cancer are independent of those in normal tissue. We furthermore constructed a linear statistical classifier, i.e., a weighted sum of gene expression levels, which robustly distinguishes normal from cancer tissue independent of the particular kind of tissue. Thus, despite very large differences for individual genes and considerable changes during carcinogenesis, the cumulated expressions have narrowly defined levels.

UVA-induced apoptosis studied by the new apo/necro-Comet-assay which distinguishes viable, apoptotic and necrotic cells.

An adaptation of the Comet-assay was developed which enables the discrimination of viable, apoptotic and necrotic single cells by use of the common Annexin-V staining and a dye exclusion test on the cells already embedded in agarose gel on glass slides. Membrane integrity (Ethidium-Homodimer exclusion), cellular esterase activity (Calcein blue-AM) as well as translocation of phosphadidyl-serine (Annexin-V) were analysed using these stains. The advantage of the 'apo/necro-Comet-assay' is that the viability status of individual cells can be determined and correlated with the DNA fragmentation pattern (comet) formed by the same cells. Hence, DNA damage can be assessed and correlated with viable cells or cells undergoing early, mid- or late stage apoptosis or necrosis as identified by the staining pattern. The staining was verified using heat and etoposide-induced apoptosis. This technique, among others, was used to study whether apoptotic fragmentation interferes with repair kinetics measured with the comet assay following UVA exposure (doses up to 1,280 kJ/m(2)) in the cultured human keratinocytes (HaCaT). Therefore, a time course of apoptotic events (phosphatidyl translocation and TUNEL fragmentation) was established and correlated to the DNA fragmentation in the comet-assay. Apoptotic cells were detected more than 8 h later. The combined three-colour staining method with the comet assay showed that there was no significant interference of DNA repair by apoptotic fragmentation processes since DNA repair was almost completed before the onset of apoptotic fragmentation. The apo/necro-Comet-assay reduces the general problem of false-positive results in genotoxicity tests using the Comet-assay.

Comet-assay in combination with PNA-FISH detects mutagen-induced DNA damage and specific repeat sequences in the damaged DNA of transformed cells.

The Comet-assay was applied to three transformed cell lines (HT1080, CCRF-CEM line and CHO) which were treated with the cytostatics bleomycin (BLM) or mitomycin C (MMC). In addition, PNA probes for the telomere repeat (TTAGGG)(n) were used for detection of telomeric DNA sequences in the damaged DNA. Data were compared with previously obtained results from peripheral leukocytes. The amount of migrating DNA increased in all cell types in a dose-dependent manner after BLM exposure. CHO cells reacted sensitively at low doses of the mutagen, and leukocytes had the highest dose-related effect up to 25 IU/ml which, however, did not further increase. A rather linear dose response characterized the HT1080 cells, the effect was lowest for the CCRF-CEM cells. While MMC at lower doses increased the percentage of migrating DNA in a dose-dependent manner, the higher doses induced shorter comets, on average, than the lower ones in all cell lines. With PNA-Comet-FISH obvious differences were found between the studied cell lines with respect to quantitative head/tail distribution of telomeric signals after BLM exposure. A large number of signal spots of various sizes were found in CHO cells, very small signals could be detected in the comets of both neoplasia cell lines. Dose-dependence of telomeres in the tail was most pro-nounced in CCRF-CEM and normal leukocytes, less in HT1080. The steepest dose-related increase of telomeric signals in the tail was found in CHO cells. The ratio between the migrated DNA and the telomeric signals in the tail varied distinctly between the examined cell types from 3:1 to 1:1. Taken together, Comet-FISH can detect mutagenic effects on specific DNA sequences. This may be of high practical value if amplified DNA sequences will be addressed by those examinations in future.

Genes of glycolysis are ubiquitously overexpressed in 24 cancer classes.

Using NIH's public database dbEST for expression of genes and ESTs, genes of the glycolysis pathway have been found to be overexpressed in a set of 24 cancers representing more than 70% of human cancer cases worldwide. Genes can be classified as those that are almost ubiquitously overexpressed, particularly glyceraldehyde-3-phosphate dehydrogenase, enolase 1, and also pyruvate kinase, and those that are overexpressed in less than 50% of the investigated cancers. Cancers can be classified as those with overexpression of the majority of the glycolysis genes, particularly lymph node, prostate, and brain cancer, in which essentially all glycolysis genes are overexpressed, and those with only sporadic overexpression, particularly cancers of the cartilage or bone marrow. This classification may be useful when cancer therapies aimed at the Warburg effect are designed.

Single molecule techniques for biomedicine and pharmacology.

The present review gives a short summary on techniques useful for single molecule research, describes experiments on in vitro single molecule detection and reactions of single molecules and finally reports on the behavior of single molecules and single virus particles in living cells. One experiment on single molecule enzyme kinetics of lactate dehydrogenase, an enzyme used in the diagnosis of heart attacks and one experiment on restriction analysis of individual DNa molecules are described in some detail. Where it is possible, the relevance to pharmacology and biomedicine is emphasized, often as a perspective or suggestion for experiments, since in this young field of science a not too large variety of experiments have indeed already been devoted directly to drug action.

Putative colon cancer risk factors damage global DNA and TP53 in primary human colon cells isolated from surgical samples.

This study describes a novel in vitro method in genetic toxicology that is based on detection of chemical-induced DNA damage connected with altered migration of TP53 in primary human colonocytes. Techniques were developed to isolate high numbers of human epithelial colon cells from surgical tissues. High quantities of viable cells were obtained per donor. The primary cells were treated with the endogenous risk factors trans-2-hexenal, and hydrogen peroxide. Global DNA damage and repair were measured by single-cell gel electrophoresis (Comet assay). We compared responses of primary colon cells to HT29clone19A, a differentiated human colon tumour cell line, for which the karyotype was analysed with 24-colour FISH. Both compounds were genotoxic in both cell types and most of the induced DNA damage was repaired after 30 min. Specific migration of TP53 was determined by fluorescence in situ hybridization (Comet FISH). Using primary colon cells, we quantified the migration of TP53 signals into the comet tails. In these cells TP53 was more sensitive than global DNA for genotoxicity induced by trans-2-hexenal and H(2)O(2). HT29clone19A cells cannot be used for Comet FISH because of their aberrant karyotype. The approach described allows us to obtain more knowledge of putative risk factors in colon carcinogenesis.

Labelling quality and chromosome morphology after low temperature FISH analysed by scanning far-field and near-field optical microscopy.

A non-enzymatic, low temperature fluorescence in situ hybridization (LTFISH) procedure was applied to metaphase spreads and interphase cell nuclei. In this context 'low temperature' means that the denaturation procedure of the chromosomal target DNA usually applied by heat treatment and chaotropic agents such as formamide was completely omitted so that the complete hybridization reaction took place at 37 degrees C. For LTFISH, the DNA probe had to be single-stranded, which was achieved by means of separate thermal denaturation of the DNA probe only. The DNA probe pUC1.77 was used for all LTFISH experiments. The labelling quality (number of binding sites, relative background intensity, relative intensity of major and minor binding sites) was analysed by confocal laser scanning microscopy (CLSM). An optimum in specificity and signal quality was obtained for 15 h hybridization time. For this hybridization condition of LTFISH, the chromosomal morphology was analysed by scanning near-field optical microscopy (SNOM). The results were compared with the morphology of chromosomes after (a) labelling of all centromeres using the same chemical treatment in the FISH procedure but with the application of target denaturation, and (b) labelling of all centromeres using a standard FISH protocol including thermal denaturation of the DNA probe and the chromosomal target. Depending on the FISH-procedure applied, SNOM images show substantial differences in the chromosome morphology. After LTFISH the chromosome morphology appeared to be much better preserved than after standard FISH. In contrast, the application of the LTFISH chemical treatment accompanied by heat denaturation had a very destructive influence on chromosomal morphology. The results indicate that, at least for certain DNA probes, specific chromosome labelling can be obtained without the usually applied heat and chemical denaturation of the DNA target, resulting in an apparently well preserved chromatin morphology as visualized by SNOM. LTFISH may be therefore a useful labelling technique whenever the chromosomal morphology had to be preserved after specific labelling of DNA regions. Binding mechanisms of single-stranded DNA probes to double-stranded DNA targets are discussed.

Impairment of cytoskeleton-dependent vesicle and organelle translocation in green algae: combined use of a microfocused infrared laser as microbeam and optical tweezers.

A Nd-YAG laser at 1064 nm is used as optical tweezers to move intracellular objects and a laser microbeam to cause impairment of cytoskeleton tracks and influence intracellular motions in desmidiaceaen green algae. Naturally occurring migrations of large nuclei are inhibited in Micrasterias denticulata and Pleurenterium tumidum when the responsible microtubules are targeted with a laser microbeam generating 180 mW power in the focal plane. Impairment of the microtubule tracks appears to be irreversible, as the nucleus cannot pass the former irradiated area in Pleurenterium or remains abnormally dislocated in Micrasterias. The actin filament-dependent movement of secretory vesicles and smaller particles can be manipulated by the same IR-laser at 90 mW when functioning as optical tweezers. In Closterium lunula particles are displaced from their cytoplasmic tracks for up to 10 micro m but return to their tracks immediately after removing the light pressure gained by the optical tweezers. The cytoplasmic tracks consist of actin filament cables running parallel to the longitudinal axis of Closterium cells as depicted by Alexa phalloidin staining and confocal laser scanning microscopy. Dynamics and extensibility of the cytoplasmic strands connecting particles to the tracks are also demonstrated in the area of large vacuoles which are surrounded by actin filament bundles. In Micrasterias trapping of secretory vesicles by the optical tweezers causes irreversible malformations of the cell shape. The vesicle accumulation itself dissipates within 30 s after removing the optical tweezers, also indicating reversibility of the effects induced, in the case of actin filament-mediated processes.

Variations in cell surfaces of estrogen treated breast cancer cells detected by a combined instrument for far-field and near-field microscopy.

The response of single breast cancer cells (cell line T-47D) to 17beta-estradiol (E(2)) under different concentrations was studied by using an instrument that allows to combine far-field light microscopy with high resolution scanning near-field (AFM/SNOM) microscopy on the same cell. Different concentrations of E(2) induce clearly different effects as well on cellular shape (in classical bright-field imaging) as on surface topography (atomic force imaging) and absorbance (near-field light transmission imaging). The differences range from a polygonal shape at zero via a roughly spherical shape at physiological up to a spindle-like shape at un-physiologically high concentrations. The surface topography of untreated control cells was found to be regular and smooth with small overall height modulations. At physiological E(2) concentrations the surfaces became increasingly jagged as detected by an increase in membrane height. After application of the un-physiological high E(2) concentration the cell surface structures appeared to be smoother again with an irregular fine structure. The general behaviour of dose dependent differences was also found in the near-field light transmission images. In order to quantify the treatment effects, line scans through the normalised topography images were drawn and a rate of co-localisation between high topography and high transmission areas was calculated. The cell biological aspects of these observations are, so far, not studied in detail but measurements on single cells offer new perspectives to be empirically used in diagnosis and therapy control of breast cancers.

Comet assay measurements of DNA damage in cells by laser microbeams and trapping beams with wavelengths spanning a range of 308 nm to 1064 nm.

DNA damage induced in NC37 lymphoblasts by optical tweezers with a continuous-wave Ti:sapphire laser and a continuous-wave Nd:YAG laser (60-240 mW; 10-50 TJ/m2; 30-120 s irradiation) was studied with the comet assay, a single-cell technique used to detect DNA fragmentation in genomes. Over the wavelength range of 750-1064 nm, the amount of damage in DNA peaks at around 760 nm, with the fraction of DNA damage within the range of 750-780 nm being a factor of two larger than the fraction of DNA damage within the range of 800-1064 nm. The variation in DNA damage was not significant over the range of 800-1064 nm. When the logarithm of damage thresholds measured in the present work, as well as values reported previously in the UV range, was plotted as a function of wavelength, a dramatic wavelength dependence became apparent. The damage threshold values can be fitted on two straight lines, one for continuous-wave sources and the other for pulsed sources, irrespective of the type of source used (e.g. classical lamp or laser). The damage threshold around 760 nm falls on the line extrapolated from values for UV-radiation-induced damage, while the data for 800-1064 nm fall on a line that has a different slope. The change in the slope between 320 and 340 nm observed earlier is consistent with a well-known change in DNA-damaging mechanisms. The change observed around 780 nm is therefore suggestive of a further change in the mechanism(s). The data from this work together with our previous measurements provide, to the best of our knowledge, the most comprehensive view available of the DNA damage produced by microfocused light.

He-Ne laser irradiation protects B-lymphoblasts from UVA-induced DNA damage.

The effect of He-Ne laser (632.8 nm) pre-irradiation on UVA (343 nm)-induced DNA damage in the human B-lymphoblast cell line NC37 was investigated using the comet assay. He-Ne laser pre-irradiation was observed to result in a dose-dependent decrease in UVA-induced DNA damage. This effect was also found to be dependent on the incubation period between He-Ne laser pre-irradiation and the UVA exposure. Whereas the control cells with a higher DNA damage point to an initial ability of faster repair, both the control and the He-Ne laser pre-irradiated cells subsequently show the same rate of DNA repair. The results suggest that He-Ne laser irradiation protect the cells from UVA-induced DNA damage primarily through an influence on processes that prevent an initial DNA damage.

Cell viability of retinal photoreceptor evaluated by polar distribution of Ca(2+) and electrical charge.

The polar organisation is characteristic to the living cell and disappears with the cell functional decay. Here we report experimental evidence that frog retinal photoreceptor rod cell shows a polar distribution of the electrical charge and of free cytosolic Ca(2+) along its length. Retinal rod cells were loaded with Calcium sensitive dye (Green1) and examined under fluorescence microscopy coupled with an image analysis system. In addition, suspension of rod cells was placed in direct current electric field for electrical polarity assessment. Both polar Ca(2+) and electrical charge distribution can be objectively measured and quantified providing thus a fine test for cell viability. Such a test is required in checking the functional integrity of photoreceptors used in retinal transplant.

UV-A breakage sensitivity of human chromosomes as measured by COMET-FISH depends on gene density and not on the chromosome size.

COMET-FISH, a single cell-based combination of COMET-assay (also known as single cell gel electrophoresis (SCGE)) with fluorescence in situ hybridization (FISH) allows region specific studies on DNA stability and damage. COMET-FISH can be used to investigate UV-A-induced DNA damage of selected whole chromosomes. In the present work, a modified COMET-FISH protocol with whole chromosome painting probes was used to study whether UV-A-induced DNA damage is distributed randomly over the whole genome or occurs at preferred sites. The study was performed with 12 different chromosome painting probes (for chromosomes 1, 2, 3, 8, 9, 11, 14, 18, 19, 21, X and Y). The results on human lymphocytes irradiated with 500 kJ/m2 at a wavelength of 365 nm indicate that the induced number of chromatin strand breaks does not correlate with the chromosome size. They therefore are distributed in a non-random manner. For example, fragments of the gene-rich chromosome chromosome 1 were found in the comet tail in only 3% of the examined cells, and thus chromosome 1 is rather stable, whereas fragmentation of the gene-poor chromosome 8 was observed in 25% of all comets. On the basis of all 12 chromosomes analyzed, an inverse correlation between the density of active genes and the sensitivity toward UV-A radiation is found.

Micromanipulation by laser microbeam and optical tweezers: from plant cells to single molecules.

Complete manipulation by laser light allows precise and gentle treatment of plant cells, subcellular structures, and even individual DNA molecules. Recently, affordable lasers have become available for the construction of microbeams as well as for optical tweezers. This may generate new interest in these tools for plant biologists. Early experiments, reviewed in this journal, showed that laser supported microinjection of material into plant cells or tissues circumvents mechanical problems encountered in microinjection by fragile glass capillaries. Plant protoplasts could be fused with each other when under microscopical observation, and it was no major problem to generate a triple or quadruple fusion product. In the present paper we review experiments where membrane material was prepared from root hair tips and microgravity was simulated in algae. As many plant cells are transparent, it is possible to work inside living, intact cells. New experiments show that it is possible to release by optical micromanipulation, with high spatial resolutions, intracellular calcium from caged compounds and to study calcium oscillations. An example for avian cardiac tissue is given, but the technique is also suitable for plant cell research. As a more technical tool, optical tweezers can be used to spatially fix subcellular structures otherwise moving inside a cell and thus make them available for investigation with a confocal microscope even when the time for image formation is extended (for example at low fluorescence emission). A molecular biological example is the handling of chromosomes and isolated individual DNA molecules by laser microtools. For example, chromosomes can be cut along complex trajectories, not only perpendicular to their long axis. Single DNA molecules are cut by the laser microbeam and, after coupling such a molecule to a polystrene microbead, are handled in complex geometries. Here, the individual DNA molecules are made visible with a conventional fluorescence microscope by fluorescent dyes such as SYBRGreen. The cutting of a single DNA molecule by molecules of the restriction endonuclease EcoRI can be observed directly, i.e. a type of single molecule restriction analysis is possible. Finally, mechanical properties of individual DNA molecules can be observed directly.

Early phase karyotype analysis of chromosome segregation after formation of mouse-mouse hybridomas with chromosome painting probes.

FISH analysis with chromosome painting probes allows, better than karyotyping after Giemsa banding, the study of chromosome segregation after hybridoma formation. FISH is particularly useful for intraspecies hybrids and allows visualization of small chromosome fragments. Cell hybrids were constructed between P3 x 63Ag8.653 mouse myeloma cells and lymphocytes from BALB/c mice by PEG fusion and by selection in hypoxanthine azaserine medium. Three hybridomas (A4, D8, F10) were selected and, after cloning, the cells were cultivated in vitro over a period of 28 days. During this time in culture, air-dried metaphase spreads were prepared by standard methods. For FISH chromosome painting, digoxigenin- and biotin-labeled mouse chromosome painting probes and rhodamine-antidigoxigenin antibodies and fluorescein-avidin were used for dual color detection. Total chromosome numbers and the numbers of mouse chromosomes 1, X, 6 and 12 were estimated as function of days in culture. Mean chromosome numbers of 78 (D8), 82 (F10) and 150 (A4) were observed. The major rearrangements of chromosome numbers occured in the first 28 days in culture and did not change significantly between day 28 and day 56. Mouse chromosome #12, which had the largest chromosome fragments in the parent myeloma, remained stable while the number of X chromosomes, which were significantly fragmented already in the parent myeloma, decreased by approximately 50%.