Stephen A. Lesko

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.

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.

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

Abstract 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 °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.

Induction of 6-thioguanine-resistant mutants by hyperoxia and γ-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.

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.

Involvement of Radicals in Chemical Carcinogenesis

Chemical carcinogens comprise a large and structurally diverse group of synthetic and naturally occurring compounds. It appears almost axiomatic that such compounds must react with tissue components in order to induce neoplastic transformation. With the exception of the carcinogenic alkylating agents, most chemical carcinogens are not reactive per se and must be converted to reactive forms either chemically or metabolically. Data are emerging to indicate that electrophilic reactants are the ultimate form of most, if not all, chemical carcinogens (Miller, 1970). The proximate forms of a number of chemical carcinogens might be convertible to free radicals, i.e., electrophilic reactants, and suggest a possible role for the free radical in carcinogenesis. The presence of free radicals in tobacco smoke has been demonstrated (Lyons and Spence, 1960; Bluhm et al., 1971) and the increased incidence of lung cancer in cigarette smokers based on epidemiological studies is well known. The chemical and metabolic conversion of a number of chemical carcinogens to free radicals and the interaction of these radicals with DNA will be described in this communication.