A laser scanning confocal microscopy method. Simultaneous detection of intracellular Ca2+ and apoptosis using Fluo-3 and Hoechst 33342.

OBJECTIVE: To develop a simple and direct method to simultaneously determine apoptotic cells from a treated population of cells and detect the changes of intracellular Ca2+ in these apoptotic cells, in particular single ones, by confocal microscopy. STUDY DESIGN: MGC-803 cells treated with As2O3 were used as the double-staining cell model with Hoechst 33342 as a DNA probe and Fluo-3AM as a Ca2+ indicator. MGC-803 cell apoptosis induced by As2O3 was first demonstrated by DNA ladder in gel electrophoresis. Based on the difference in DNA stainability with Hoechst 33342 and corresponding fluorescence intensity between live and apoptotic cells, apoptotic cells and the changes in intracellular Ca2+ were detected at the same time by confocal microscopy. No necrotic cells in the group treated with As2O3 were found by the trypan blue exclusion test. RESULTS: The results from confocal microscope detection showed that intact and apoptotic cells were successfully recognized and the changes of intracellular Ca2+ in apoptotic and intact cells were simultaneously detected in the same sample. CONCLUSION: We provided a useful method to exactly detect changes in intracellular Ca2+ in apoptotic cells, especially in single ones, by confocal microscopy and to exclude the artifact effect of necrotic and intact cells.

Spectroscopic comparison of different DNA structures formed by oligonucleotides.

Six different nucleic acid structures including duplex, triplex and quadruplex are formed by oligonucleotides. Their structural properties are studied in detail by four spectroscopic techniques, i.e. CD, UV, NMR and fluorescence. Results are: CD Spectra: The common characteristics is a negative band at 240 nm, and the spectra are different from each other in the range 260-300 nm. Many factors such as chain direction, sugar puckering, orientation of the glycosyl bond, base stacking and sequence can effect their conformation and then show diversity and complexity in the spectra. UV Spectra: The UV spectra of all forms are quite similar, all of them exhibit a sharp positive peak around 210 nm and a broad positive band in the region of 240-280 nm. Although the bands are different in absorbance, the spectra are not characteristic enough to distinguish these forms. In addition, their thermal denaturation is also observed by UV spectrum, different melting curves and points are shown and some thermodynamic information is provided. NMR Spectra: Since the G residues in the six samples all participate in hydrogen bond, the imino proton can not exchange with the solvent freely so as to allow an observable resonance to arise. The resonance number and chemical shift will vary with the change in base-pairing number and mode as well as the whole geometry of its molecule. Fluorescence Spectra: The interaction mechanisms between EB and these structures are different. B type duplex and triplex adopt an intercalative mode in which the efficiency of energy transfer is relatively high and the fluorescence of EB can not be quenched easily. While for the parallel duplex, outside binding is predominant in which energy transfer can hardly happen and most of its fluorescence can be quenched. As for the quadruplex, groove binding is possible, so the efficiency of energy transfer is higher than that in outside binding, but lower than that in intercalative binding, and fluorescence is quenched partly.

Induction of apoptosis and inhibition of human gastric cancer MGC-803 cell growth by arsenic trioxide.

Arsenic trioxide (As2O3), used to treat human diseases for centuries in traditional Chinese medicine, has been identified as a very effective antileukaemic agent, but its effect on solid tumours which could be more suitable for clinical treatment with arsenic compounds is still unknown. In this study, we investigated the in vitro effect of As2O3 at concentrations of 0.01-1 microM against six human malignant cell lines, MGC-803, HIC, MCF-7, HeLa, BEL-7402 and A549 cells. As2O3 inhibited growth and induced apoptosis in these malignant cells at varying degrees, in a time dose-dependent manner. The most marked effects were seen in the gastric cancer cell line, MGC-803. In contrast, minimal growth inhibition and induction of apoptosis occurred in human embryonic pulmonary cells following treatment with As2O3 found at the same concentrations. Changes in intracellular Ca2+, following As2O3 treatment were measured by Ca2+ sensitive fluorescent probe Indo-1/AM in flow cytometric assays. The increase in intracellular Ca2+ correlated with the sensitivity of these cells to As2O3, possibly indicating that a critical intracellular Ca2+ signal transduction pathway could be involved in As2O3-mediated cell-death and its selectivity. The marked sensitivity of MGC-803 cells in vitro suggests that As2O3 may be a potential antigastric cancer agent.

The involvement of singlet oxygen in copper-phenanthroline/H2O2-induced DNA base damage: a chemiluminescent study.

Copper in the presence of excess 1,10-phenanthroline, a reducing agent, and H2O2 causes DNA base damage as well as strand breakage. We have reported in previous work that a strong chemiluminescence was followed by DNA base damage in this system, which is characteristic of guanine. In the present work, the mechanism of the chemiluminescence was studied. Results show that the luminescence was inhibited by all three classes of reactive oxygen species (*OH, O2-, (1)O2) scavengers to different degrees. Singlet oxygen scavengers showed the most powerful inhibition while the other two classes of scavengers were relatively weaker. The emission intensity in D2O was 3-fold that in H2O. Comparing the effect of scavengers on the luminescence of DNA with that of dGMP, the ratio of inhibition was similar. On the other hand, DNA breakage analysis showed that inhibition by the singlet oxygen scavenger NaN3 of strand breakage was strong and comparable to that of the scavengers of the two oxygen radicals. The results suggest that singlet oxygen may be a major factor for the chemiluminescence of guanine, while DNA strand breakage may be caused by many active species.

Phenanthroline-Cu complex-mediated chemiluminescence of DNA and its potential use in antioxidation evaluation.

The chemiluminescence (CL) concomitant with phen-Cu2+/ascorbate/H2O2-induced DNA damage has been studied. The emission intensity increases linearly with increasing DNA concentration. The emission spectrum has a maximal wavelength at about 410 nm. The luminescence is inhibited by histone in a histone concentration-dependent manner. The CL is characteristic of guanine. Of all common kinds of bases and nucleotides, only guanine or guanine nucleotides can give rise to luminescence. The possibility of using the luminescence as a means of studying antioxidation related to DNA damage is discussed. Several kinds of well-defined antioxidants have been used as testing reagents and show that this method can not only evaluate the antioxidative effect but also distinguish different types of antioxidants.

Photodynamic effects of hypocrellin A on three human malignant cell lines by inducing apoptotic cell death.

Hypocrellin A (HA), a lipid-soluble peryloquinone derivative extracted from the fungus Hypocrella bambusae, has been proved to be effective in treating many diseases. In this study, we have examined the photodynamic effects of HA on three different human malignant epithelioid cell lines, i.e., HIC, MGC-803 and HeLa cells. They all respond to photodynamic therapy (PDT) by committing apoptosis or necrosis evidenced by morphological changes, DNA fragmentation and a decrease of mitochondria dehydrogenase activity. The sensitivity order of the three cell lines to cytotoxicity by HA is HIC > MGC-803 > HeLa. The extent of apoptosis is also dependent on light dose, post-treatment time and cell type. Vitamin E can significantly protect cells against cell death, indicating that reactive oxygen species (ROS) play an important role in apoptotic or necrotic cell death. These results may be to some extent instructive for clinical cancer treatment.

Formation and characteristics of an unusual lambda-DNA species.

An unusual DNA species, termed as DNA species A, has been isolated and purified from thermal-denatured lambda-DNA Hind III by Sephadex G-200 gel filtration. Our studies indicate that DNA species A is resistant to DNase I digestion and has a higher melting point. The new DNA species showed a lower absorbency at 260 nm, and a lower fluorescence quantum yield after interaction with ethidium bromide (EB) than native double-stranded lambda-DNA. CD spectrum of DNA species A consists of a broad positive band centered at 245 nm and a weak negative band at 220 nm. transmission electron microscope (TEM) visualizations showed that their lengths of DNA species A fell mainly in three regions (300-500 nm, 750-1000 nm and 1500 nm) that corresponded to three fluorescence bands in the EB-stained gels. Their apparent width and height were 65-75 nm and 2.2 nm respectively as observed by images of atomic force microscope (AFM).

Effect of natural antioxidant tanshinone II-A on DNA damage by lipid peroxidation in liver cells.

Tanshinone II-A (TSII-A) isolated from the root of Salvia miltorrhiza Bunge, a traditional medicine in China, is a derivative of phenanthrenequinone, which is known to have antioxidant properties. In the present study, effects of TSII-A on DNA damage by lipid peroxidation were investigated using liver cells, labeled with [3H] arachidonic acid, in the presence of FeCl2-DTPA. The results show that the nuclear DNA isolated from treated cells had higher radioactivity compared to controls and the radioactivity increased with longer incubation times. Purified lipid-DNA adducts had a characteristic fluorescent spectra and showed a decrease of hyperchromicity and melting point. TSII-A could inhibit the association of peroxidation products with DNA in liver cells and prevent a decrease in cell viability and in the the activity of O6-methylguanine acceptor protein with increasing incubation time. Compared with other antioxidants, TSII-A had a higher inhibitory ratio, which was similar to vitamin E and butylated hydroxy-toluene (BHT), but markedly stronger than NaN3, mannatol, and superoxide dismutase (SOD). These data suggest that TSII-A represents a new and effective antioxidant that inhibits the association of lipid peroxidation products with DNA. Its protective effect may be through breaking the chain reactions of peroxidation by scavenging lipid free radicals, thereby decreasing their cytotoxicity.

A molecular model of braid-like DNA structure.

The three-dimensional molecular models of DNA triple helices and triple-stranded brain-like structure were built up by molecular architecture, and their structural features and energy decomposition were examined. The results showed: (i) The base triplet is the element forming braid-like and triple helix DNA; (ii) Under specified conditions, DNA could form the triplet-stranded braid-like structure; (iii) DNA stability of the braid-like structure is less than that of the triple helix structure.

Evidence that lipid peroxidation products bind to DNA in liver cells.

Three lines of evidence are presented indicating the association of lipid peroxidation products with DNA in liver cells, labeled with [3H]arachidonic acid, in the presence of Fe(2+)-DTPA: (1) the nuclear DNA isolated from treated cells had higher radioactivity, compared to controls and the radioactivity increased with longer incubation times, (2) lipid-DNA adducts with a characteristic fluorescence spectrum were formed during the incubation with Fe(2+)-DTPA; (3) the association of peroxidation products with DNA could be inhibited by vitamin E and BHT. Compared with control DNA, purified lipid-DNA adducts showed a decrease of hyperchromicity and melting point, and partial resistance to hydrolysis by DNase I. On the other hand, the repair test shows that the lipid-DNA adducts in cells were not repaired by 4 h after removal of Fe(2+)-DTPA. A decrease in cell viability and in the activity of O6-alkylguanine acceptor protein was also observed with increasing incubation time. These data suggest that the lipid-DNA association, an oxidative DNA damage, occurs in cells treated by Fe(2+)-DTPA and could result in cytotoxicity if not repaired.

Oxidative damage to DNA: levels of thymine glycol and thymidine glycol in neoplastic human urines.

We have measured the levels of thymine glycol (TG) and thymidine glycol (dTG) in human urine, using an HPLC method. The results show that all 30 specimens examined (including 10 non-neoplastic and 20 neoplastic human urines) contained significant amounts of TG and dTG, average levels (mean +/- SEM) were 0.376 +/- 0.026 and 0.138 +/- 0.009 nmol/kg body weight/day respectively. The average levels of TG and dTG were 0.435 +/- 0.038 and 0.164 +/- 0.017 nmol/kg body weight/day in 10 healthy human urine specimens and 0.347 +/- 0.035 and 0.125 +/- 0.010 nmol/kg body weight/day in 20 neoplastic human urine specimens respectively. No significant differences were found between female and male as well as between the non-neoplastic and neoplastic human urine specimens. There were also wide interindividual variations, which were not age-dependent.

DNA damage induced by hypocrellin-A photosensitization.

Hypocrellin-A (HC-A) isolated from Hypocrellia bambusae Sacc., is a new and effective photosensitizer. Illumination of sarcoma 180 cells with visible light in the presence of HC-A leads to a decrease in cell viability and 3H-TdR incorporation, causes DNA strand breakage, and results in the selective destruction of guanine moieties in DNA. HC-A photosensitization causes an increase in the theta 260/theta 280 ratio in the circular dichroism spectra of DNA in vitro. Of the four usual 2'-deoxynucleotides illuminated in the presence of HC-A only 2'-deoxyguanylic acid was destroyed.

Levels of O6-methylguanine acceptor protein in extracts of human breast tumor tissues.

We have measured the abilities of extracts of tissues from human breast tumors to demethylate adducts of O6-meG in exogenous DNA by transfer of the methyl group to an acceptor protein. The results have shown that all 21 specimens examined (including 5 non-neoplastic, 11 malignant tumors and 5 benign growth) contained significant amounts of O6-meG acceptor activity, removing on average 221.1 +/- 2.1 (SEM) fmol O6-meG per mg protein or 10.07 +/- 0.98 (SEM) fmol O6-megG per microgram DNA in the extracts. There were also wide interindividual variations, which were not age-dependent, and there were no significant differences between the non-neoplastic and neoplastic tissues obtained from individuals with benign or with malignant disease. It was estimated that the average number of O6-meG acceptor molecules per cell in normal human breast tissues was calculated as 46,000 +/- 7000 (SEM).

Study on enhancement of tumor-killing effect and formation of new fluorophore in tumor cells.

Hyperthermia (42-44 degrees C) and photosensitizing therapy can destroy S180 tumor cells, reduce malignant ascites and prolong the survival times of mice with carcinomas. The highest curative effect was observed when using a combination of the two treatments. Heating to 44 degrees C has a greater destructive effect on tumor cells than has heating to 42 degrees C. The results show that this is due to a synergistic interaction between these two treatments. The fluorescence spectrum of S180 cells was determined before and after treatment, and the indication was that the synergistic effect is probably related to a new fluorescence product; the greater the intensity of the new fluorescence, the more marked the synergy of hyperthermia and photosensitizing therapy. The maximum emission wavelength was 460nm (excitation wavelength 370nm).

Hematoporphyrin derivative photosensitization and cellular fluorescent photoproduct formation.

A new fluorescence band with an emission maximum at 460 nm was observed in S180 tumour cells and human cancer tissues photosensitized by Y-HPD. This fluorescence was the result of a photochemical reaction involving specific proteins and Y-HPD in the cells.

Presence of O6-methylguanine acceptor protein in the tissues of different classes of vertebrates and invertebrates.

We have measured the ability of extracts of tissues from several species of mammals, birds, reptiles, amphibia and fish to demethylate adducts of O6-methylguanine in exogenous DNA by transfer of the methyl group to an acceptor protein. Our study also encompassed tissues from a smaller number of invertebrates, from arthropods, molluscs and annelids. The vertebrate tissues used were liver, brain, spleen and kidney. In the case of the invertebrates we sampled liver, neural tissue, gonads, digestive tract and hepatopancreas. There was no consistent change in the amount of acceptor activity per unit of protein or DNA going from cold-blooded to warm-blooded vertebrates. Liver invariably had the highest amount; this finding was not unexpected since metabolic processes in the liver are high, and good cellular protective mechanism important. Inter-class comparisons within the vertebrates are highly speculative, and hindered by the fact that there is little information on carcinogenesis in animals other than rodents and humans. O6-methylguanine acceptor activity was found in all the invertebrate tissues tested. The amounts were variable, 0.003-0.0051 fmol/micrograms cellular DNA, but the values fell within the range of those found in the tissues of vertebrates.

Levels of O6-methylguanine acceptor protein in tissues of rats and their relationship to carcinogenicity and aging.

N-nitroso compounds react with cellular DNA to produce various damaging adducts, one of the more important being O6-alkylguanine. DNA restoration is accomplished by transfer of the alkyl group to a cysteine residue of an acceptor protein. The levels of acceptor activity were compared in several tissues from well-fed and dietary-restricted inbred SD rats 30-1,194 days of age. Striking and consistent differences were found in the levels of acceptor activity in different tissues from both groups; these levels corresponded to their sensitivity to tumorigenesis by alkylating agents. Acceptor activity levels were highest in the liver and somewhat less in the spleen; there were significantly lower levels in brain and kidney. The random loss with time in the integrity of DNA may cause alterations in cellular function or limit cellular proliferation, thus leading to senescence and death. DNA repair processes may alter the rate of accumulation of damage, thereby affecting potential longevity. There were no significant age-associated changes in the ability of cells from either dietary group to remove DNA adducts and there was no evidence of alterations in the acceptor protein with age that would compromise its functional activity.