Detection of intact prostate cancer cells in the blood of men with prostate cancer.

OBJECTIVES: To develop a procedure to be used to find, identify, and characterize the living prostate cancer cells in the blood of patients with prostate cancer. METHODS: The procedure is based on a negative selection approach that removes most of the blood cells and collects the remaining prostate cancer cells, which are identified and characterized by fluorescent in situ hybridization with deoxyribonucleic acid probes and by indirect fluorescent immunocytochemical staining. The blood cells are removed via density gradient centrifugation. RESULTS: Using the prostate cancer LNCaP cells as a model, the recovery rate of the added prostate cancer cells to 10 mL of blood was about 85%, with a dilution of 1 LNCaP cell to 10,000 white blood cells or more. Blood samples varying from 9 to 27 mL were collected and analyzed from 8 men aged 54 to 79 years who had varying levels of PSA in serum. In one blood sample, prostate cancer cells were not found; in the seven other samples, the number of prostate cancer cells found per milliliter of blood varied from 1 to 20. Prostate cancer cells were not found in 7.5 to 15-mL blood samples from 3 healthy younger men. The prostate cells were found to be aneuploid for chromosomes 7 and 8, highly suggestive that these cells were cancerous. CONCLUSIONS: Using a negative selection approach, prostate cells can be found in the blood of patients with prostate cancer, as identified by prostate cell-specific probes and antibodies. These cells were found to be aneuploid.

Formation, intracellular distribution and efflux of glutathione-bimane conjugates in drug-sensitive and -resistant MCF-7 cells.

The rate of reaction of monochlorobimane with glutathione (GSH) was measured in native human mammary MCF-7 adenocarcinoma cells (MCF-7wt) and sublines displaying resistance to 4-hydroperoxycyclophosphamide (MCF-7hc) and adriamycin (MCF-7adr) prior to examination by epifluorescence and confocal microscopy. After a 60-min incubation period at 37 degrees C, essentially all GSH was conjugated in the MCF-7wt and MCF-7adr cell lines whereas only 80% of the GSH was conjugated in the MCF-7hc line. All three lines displayed significant export of the conjugate from the cell during this period, with the MCF-7adr line displaying the most rapid efflux with 85% of the conjugate exported within 60 min. Epifluorescence microscopy detected an approximately 20% increase in integrated fluorescence intensity in the nuclear region in all three lines. Confocal microscopy however, indicated that most of the cells examined showed a homogeneous fluorescence distribution. The cells grown in monolayers were found to be thicker in the nuclear region suggesting that the observed increase in fluorescence intensity in the nuclear region in the images from epifluorescence microscopy was probably derived from fluorescence from an out-of-focus plane. Cells depleted of GSH with buthionine sulfoximine followed by treatment with mBCl showed significant fluorescence intensity resulting from nonspecific binding of this probe. These studies illustrate the need for measuring the rate of GSH conjugate export and for determining probe specificity, and emphasizes the need for using confocal techniques for the quantitative evaluation of the distribution of intracellular fluorescence.

The experimental homologous and heterologous separation distance histograms for the centromeres of chromosomes 7, 11, and 17 in interphase human T-lymphocytes.

The simultaneous 3D arrangement of the interphase centromeres of chromosomes 7, 11, and 17 in unstimulated human T-lymphocytes is analyzed. Using triple in situ hybridization in combination with optical sectioning and image processing, the identification of three pairs of centromeres in each nucleus and the assignment of 3D coordinates to each centromere are made. The homologous and heterologous centromere separation distance histograms are determined and compared to the hypothesized histograms for randomly distributed centromeres. The experimental nuclei are truncated spheres in shape with a principal radius of 3.7 +/- 0.3 micron and a truncated hemispherical height of 2.6 micron. None of the separation distance distributions appears to be statistically significantly different from a random model distribution.

Quantitation and mapping of integrated human papillomavirus on human metaphase chromosomes using a fluorescence microscope imaging system.

Integrated human papillomavirus type 16 (HPV-16) DNA was directly visualized on metaphase chromosomes in the two human cervical carcinoma cell lines SiHa and CaSki by fluorescence in situ hybridization with a biotinylated DNA probe (7.9 kb). The fluorescence intensities of hybridization signals from single copies and dispersed clusters of integrated HPV-16 DNA were quantified using a microscope equipped with a cooled-CCD camera that was interfaced to an image processor and host computer. Hybridization signals were localized on chromosomes using separate, registered images of 4',6-diamidino-2-phenylindole (DAPI) or propidium iodide stained metaphase chromosome spreads. In both SiHa and CaSki spreads, a single fluorescein signal was observed on one or both chromatids of chromosome 13, which was identified by simultaneous hybridization with a biotinylated centromere probe specific for chromosomes 13 and 21. Ratios of the distance from 13pter to the HPV-16 signals to the entire chromosome length were approximately 0.63 +/- 0.05 in both SiHa and CaSki cells, indicating the possibility of a common integration domain on chromosome 13. In SiHa cells, no additional signals were observed on other chromosomes. This observation, taken together with literature reports that SiHa cells contain 1 to 2 copies of the HPV-16 genome in this region of chromosome 13, suggests that each fluorescein signal on chromosome 13 represents one equivalent of the HPV-16 genome. The total integrated fluorescence intensity in isolated CaSki metaphase chromosome spreads was approximately two orders of magnitude greater than that of a single copy of HPV-16 DNA in SiHa cells, indicating an increase in HPV-16 copy number.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous in situ detection of beta-interferon mRNA and DNA in the same cell.

We report a quantitative method that combines in situ mRNA hybridization with microfluorometric analysis of DNA content to detect gene expression in single cells of a heteroploid cell population. The model was a human fibrosarcoma HT1080 cell line which consisted of diploid and tetraploid cells that were induced with polyI:polyC for production of beta-interferon. The level of beta-interferon mRNA detected by in situ hybridization was found to be two to three times higher in tetraploid compared to diploid HT1080 cells, and correlated with beta-interferon activity in that a subclone of tetraploid HT1080 cells secreted two- to fivefold more beta-interferon than a subclone of diploid HT1080 cells. Interestingly, beta-interferon-related transcripts were detected during S-phase in uninduced tetraploid HT1080 cells. In addition, beta-interferon induced by polyI:polyC was expressed in all phases of the cell cycle as demonstrated with a human diploid fibroblast, HF926. The unique features offered by the combination of microfluorometry and in situ hybridization provide a valuable tool to investigate specific gene expression related to ploidy or cell-cycle stage in the same individual cell of an unsynchronized population. Since the method allows direct observation of morphology, one can be assured that all quantitative measurements were made on whole cells with intact nuclei.

Production of interferon-beta upon induction with polyinosinic acid:polycytidylic acid during the cell cycle of human fibroblasts.

The production of interferon-beta was examined at various stages of the cell cycle in synchronized and unsynchronized cell populations induced by poly(I):poly(C). Human fibroblasts were synchronized with mitotic detachment and, at different stages of the cell cycle, poly(I):poly(C) was added for induction of interferon-beta. One hour after induction, cell-free medium was collected and assayed for secreted interferon-beta. The cells were then fixed and stained with a DNA-specific fluorochrome, 4',6-diamidino-2-phenylindole (DAPI), for cell cycle analysis by microfluorometry. The data indicated that interferon-beta was produced in every stage examined of the cell cycle. In addition, the level of intracellular interferon-beta was quantitatively measured in single cells of an unsynchronized cell population using a specific antibody. In the same individual cell, DAPI fluorescence intensity was measured for determination of the cell cycle position. The results show that interferon-beta protein can be detected throughout the cell cycle.

Quantitative immunofluorescence assay for cyclobutyldithymidine dimers in individual mammalian cells.

An indirect immunofluorescence procedure was developed for the measurement of cyclobutyl dithymidine dimers in DNA of individual Syrian hamster embryo cells using a specific monoclonal antibody. A fluorescein-labeled secondary antibody and a fluorochrome which binds to DNA were used to measure the photoproduct and total DNA in the same nucleus. Fluorescence intensity was quantitated with a computer-assisted microfluorometric system which was calibrated with a uranyl oxide impregnated glass slide. Similar dose-response curves, i.e. normalized fluorescence intensity plotted as a function of dose of germicidal irradiation, were obtained with two different cell types. Normalized fluorescence intensity per nucleus was related to thymidine dimer content with a competitive enzyme-linked immunosorbent assay using DNA isolated from cells given doses of germicidal irradiation identical to those used in the immunofluorescence assay. Thymidine dimer levels produced by 10 J/m2 of germicidal irradiation (approximately 8 x 10(5)/nucleus) and which allow for 15-30% cell survival can readily be detected. The specific monoclonal antibody was labeled with tritium and used in the immunofluorescence assay to relate the number of antibodies bound to the number of thymidine dimers per cell. The data revealed that approximately 45% of the thymidine dimers in cells exposed to 100 J/m2 of germicidal irradiation and essentially all the T mean value of T in cells receiving 20 J/m2, were being detected in the indirect immunofluorescence assay. This technique can provide a sensitive means for measuring various types of DNA damage in individual cells given that the appropriate probes are available. It can be especially useful for monitoring occupationally or environmentally exposed populations where usually only small samples of cells or tissues are available.

DNA damage induced in mammalian cells by active oxygen species.

The active oxygen species generated by ionizing radiation, hyperoxia, paraquat and hydrogen peroxide induced unscheduled DNA synthesis and DNA synthesis rate inhibition in human fetal lung fibroblast (IMR-90) and transformed cell line derived from Syrian hamster embryo fibroblast (BP6T). The D-glucose and sucrose, which were unable to generate active oxygen species, could induce neither unscheduled DNA synthesis nor DNA synthesis rate inhibition. The indicator of DNA synthesis rate inhibition for active oxygen species was more sensitive than that of unscheduled DNA synthesis. All the enhancing chemicals on active oxygen species aggravated DNA damage, while all the inhibiting chemicals alleviated DNA damage. Results showed that active oxygen species do damage DNA. The active oxygen species mechanism for carcinogenesis, mutation and aging is applicable.

Somatic mutation, DNA damage and cytotoxicity induced by benzo[a]pyrenedione/benzo[a]pyrenediol redox couples in cultured mammalian cells.

BP-3,6-dione was found to be mutagenic, cytotoxic and to induce DNA damage in a transformed line of Syrian hamster fibroblasts at low concentrations, 2 micrograms/ml and less. Inhibition of sulfate and glucuronic acid conjugating enzymes with salicylamide potentiated the above effects of BP-3,6-dione. Diminishing cellular capacity to scavenge superoxide anion radicals also potentiated the mutagenic and cytotoxic action of the dione. The presence of dicumarol, a specific inhibitor of the two-electron reduction of quinones by DT-diaphorase, afforded some protection against cytotoxicity. The results indicate that BP-3,6-dione undergoes two-electron reduction to an unstable hydroquinone, BP-3,6-diol, or one-electron reduction to a semiquinone radical intermediate and that both of these reduced forms undergo rapid univalent oxidation to generate active reduced oxygen species. The data are consistent with the hypothesis that active oxygen species generated by BP-dione/BP-diol redox cycling are responsible, at least in part, for the mutagenic and cytotoxic effects observed with BP-3,6-dione.

Induction of 6-thioguanine-resistant mutants by hyperoxia and gamma-irradiation: effect of compromising cellular antioxidant systems.

Hyperoxia and gamma-irradiation were found to be mutagenic in a transformed Syrian hamster cell line in a dose-dependent manner. The frequency of resistance to 6-thioguanine increased from 10 per 10(6) survivors after 48 h of growth in 70% O2 to 32.6 (highly significant) after 75 h. Increasing the oxygen tension to 95% resulted in a significant mutagenic response in only 44 h. At equitoxic doses, gamma-irradiation was 4 times more mutagenic than 70% O2. After growth in hyperoxia, the cells showed an enhancement of catalase activity, glutathione peroxidase activity and glutathione levels but there was little effect on superoxide dismutase activity. Diethyldithiocarbamate (3 mM, 1.5 h) was mutagenic in normoxia and potentiated the mutagenic activity of both gamma-irradiation and hyperoxia. Cells thus treated showed an 855 reduction in superoxide dismutase activity. When diethyldithiocarbamate was used in conjunction with a direct-acting alkylating agent, the mutagenic response was only additive. Depletion of cellular glutathione with buthionine sulfoximine (0.2 mM) or inhibition of catalase activity with aminotriazole (100 mM) was also effective in potentiating the mutagenic response of gamma-irradiation and hyperoxia. The data demonstrates that endogenously produced activated oxygen species are mutagenic to hamster cells in culture and suggest that aerobic organisms are subject to an unavoidable background risk due to living in an oxygen atmosphere.

Benzo[a]pyrene dione-benzo[a]pyrene diol oxidation-reduction couples; involvement in DNA damage, cellular toxicity, and carcinogenesis.

Three isomeric quinone metabolites of the environmental carcinogen benzo[a]pyrene undergo reversible, univalent oxidation-reduction cycles involving the corresponding benzo[a]pyrene diols and intermediate semiquinone radicals. Under anaerobic conditions, benzo[a]pyrene 1,6-dione, benzo[a]pyrene 3,6-dione, and benzo[a]pyrene 6,12-dione are readily reduced by mild biological agents such as NADH and glutathione. The benzo[a]pyrene diols, in turn, are very rapidly autooxidized to diones when exposed to air. Substantial amounts of hydrogen peroxide are produced during these autooxidations. The benzo[a]pyrene diol/benzo[a]pyrene dione interconversions proceed by one-electron steps; the corresponding semiquinone radicals were detected as intermediates when the reactions were carried out at high pH. Benzo[a]pyrene diones are electron-acceptor substrates for NADH dehydrogenase. Catalytic amounts of these metabolites, together with this respiratory enzyme, function as cyclic oxidation-reduction couples to link NADH and molecular oxygen in the continuous production of hydrogen peroxide. Benzo[a]pyrene diones induce strand scissions when incubated with T7 DNA. The damage is modified by conditions that indicate that reduced oxygen species propagate the reactions responsible for strand scission. Benzo[a]pyrene diones are cytotoxic at low concentrations to cultured hamster cells. The cytotoxic effect can be substantially reduced by depletion of oxygen from the growth medium and the atmosphere in which the cells are incubated. The results support the hypothesis that the biological activity of benzo[a]pyrene diones is due to the regenerative oxidation-reduction cycles involving quinone and hydroquinone forms; activated oxygen species and semiquinone radicals formed during these cycles are most likely responsible for the observed cytotoxic action. The role of activated oxygen species in carcinogenesis is discussed.

Deoxyribonucleic acid-protein and deoxyribonucleic acid interstrand cross-links induced in isolated chromatin by hydrogen peroxide and ferrous ethylenediaminetetraacetate chelates.

DNA-protein and DNA interstrand cross-links were induced in isolated chromatin after treatment with H2O2 and ferrous ethylenediaminetetraacetate (EDTA). Retention of DNA on membrane filters after heating of chromatin in a dissociating solvent indicated the presence of a stable linkage between DNA and protein. Treatment of protein-free DNA with H2O2/Fe2+-EDTA did not result in enhanced filter retention. Incubation of cross-linked chromatin with proteinase K completely eliminated filter retention. Resistance to S1 nuclease after a denaturation-renaturation cycle was used to detect DNA interstrand cross-links. Heating the treated chromatin at 45 degrees C for 16 h and NaBH4 reduction enhanced the extent of interstrand cross-linking. The following data are consistent with, but do not totally prove, the hypothesis that cross-links are induced by hydroxyl radicals generated in Fenton-type reactions: (1) cross-linking was inhibited by hydroxyl radical scavengers; (2) the degree of inhibition of DNA interstrand cross-links correlated very closely with the rate constants of the scavengers for reaction with hydroxyl radicals; (3) cross-linking was eliminated or greatly reduced by catalase; (4) the extent of cross-linking was directly related to the concentration of Fe2+-EDTA. Partial inhibition of cross-linking by superoxide dismutase indicates that superoxide-driven Fenton chemistry is involved. The data indicate that DNA cross-linking may play a role in the manifestation of the biological activity of agents or systems that generate reactive hydroxyl radicals.

Toxicity of metabolic benzo(a)pyrenediones to cultured cells and the dependence upon molecular oxygen.

The three quinone metabolites of carcinogenic benzo(a)pyrene, the isomeric benzo(a)pyrenediones (6, 12; 1,6; 3,6), are toxic to cultured hamster cells at low concentrations. The reduction in cell number, observed after treatment with these metabolites, is the result of both direct cell killing and the inhibition of growth, since DNA synthesis is inhibited very early after treatment with benzo(a)pyrene 1,6-dione when little cell death has occurred. The rate of RNA synthesis was also inhibited by treatment of cells with benzo(a)pyrene 3,6-dione. These actions of the benzo(a)pyrenediones toward hamster cells can be eliminated or substantially reduced by the removal of oxygen from the growth medium and atmosphere in which the cells are incubated. In contrast, anaerobic conditions do not reduce the cytotoxicity observed with the alkylating agent ethyl methanesulfonate. These results support the hypothesis that benzo(a)pyrenediones, and other biologically active quinones, owe their activity to oxidation-reduction cycles involving quinone, hydroquinone, and molecular oxygen; the reactive reduced oxygen radicals and semiquinone radical formed during these cycles may be responsible for the observed cellular injury and inhibition of cellular processes.

Mutagenic action, loss of transforming activity, and inhibition of deoxyribonucleic acid template activity in vitro caused by chemical linkage of carcinogenic polycyclic hydrocarbons to deoxyribonucleic acid.

In a solvent system of 10(-2)m phosphate buffer (pH 6.8)-ethanol (2:1, v/v) and in an iodine-induced reaction, the polycyclic hydrocarbons [(3)H]3,4-benzpyrene and [(3)H]3,4-BP/[(3)H]9, 10-dimethyl-1,2-benzanthracene (DMBA) can be covalently linked to deoxyribonucleic acid (DNA) at room temperature. By stepwise addition of the hydrocarbon and repeating the reaction two to three times after isolating the hydrocarbon DNA adduct, it was possible to introduce as many as one covalently bound hydrocarbon molecule per 100 nucleotide bases. When 3,4-BP and DMBA were linked in this way to biologically active transforming DNA of Bacillus subtilis, they caused (i) reduction of the transforming activity of the DNA accompanied by (ii) significant increases in the frequency of forward mutations. The majority of these hydrocarbon-induced mutations were not able to revert spontaneously. These samples of DNA covalently linked with hydrocarbons showed much lower levels of survival of biological activity when assayed in recipient strains (hcr(-)) which are known to be deficient in the enzymes required for repair of ultraviolet light-induced damage to DNA. 3,4-BP covalently linked to calf thymus DNA at a level of approximately one hydrocarbon molecule per 330 bases was shown to cause up to 80% inhibition of the in vitro transcription of the DNA by highly purified ribonucleic acid polymerase prepared from Micrococcus luteus under the experimental condition of template saturation. The presence of 3,4-BP and DMBA molecules covalently bound to B. subtilis DNA samples was also found to prevent complete denaturation of the bihelical structure of certain DNA molecules and thus appears to effect a cross-link in these DNA molecules.