Disruption of mitochondrial respiration by melatonin in MCF-7 cells.

Clinical and laboratory studies have provided evidence of oncostatic activity by the pineal neurohormone melatonin. However, these studies have not elucidated its mechanism of action. The following series of MCF-7 breast tumor cell studies conducted in the absence of exogenous steroid hormones provide evidence for a novel mechanism of oncostatic activity by this endogenous hormone. We observed a 40--60% loss of MCF-7 cells after 20-h treatment with 100 nM melatonin, which confirmed and extended previous reports of its oncostatic potency. Interestingly, there were no observed changes in tritiated thymidine uptake, suggesting a lack of effect on cell cycle/nascent DNA synthesis. Further evidence of a cytocidal effect came from morphologic observations of acute cell death and autophagocytosis accompanied by degenerative changes in mitochondria. Studies of mitochondrial function via standard polarography revealed a significant increase in oxygen consumption in melatonin-treated MCF-7 cells. Enzyme-substrate studies of electron transport chain (complex IV) activity in detergent permeabilized cells demonstrated a concomitant 53% increase (p < 0.01) in cytochrome c oxidase activity. Additional studies of succinate dehydrogenase activity (complex II) as determined by reduction of (3-4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide demonstrated a significant increase (p < 0.05) in melatonin-treated cells and further confirmed the accelerated ET activity. Finally, there was a 64% decrease (p < 0.05) in cellular ATP levels in melatonin-treated cells. The G-protein-coupled melatonin receptor antagonist luzindole abrogated the cytotoxic and mitochondrial effects. These studies suggest a receptor-modulated pathway of cytotoxicity in melatonin-treated MCF-7 tumor cells with apparent uncoupling of oxidative phosphorylation.

The neoplastic transformation of SCID cells by radiation.

Severe combined immunodeficiency (SCID) cells are hypersensitive to killing by ionizing radiation because of deregulation of DNA-dependent protein kinase (DNA-PK) and a concomitant deficiency in the repair of DNA double-strand breaks. The effect of this condition on the neoplastic transformation of SCID fibroblasts, designated SCID 3T1, has been investigated. The spontaneous transformation rate was approximately 2 x 10(-5) at early passages and increased up to approximately 7 x l0(-3) at later passages. The radiation survival curves of transformed cells had thresholds and therefore appeared to be qualitatively similar to the survival curves of C3H 10T(1/2) mouse fibroblast cells, but the initial slopes were steeper. In contrast, per unit dose, SCID cells were more sensitive to transformation than 10T(1/2) cells. Eight transformed clones were tested for tumorigenicity, and all produced fibrosarcomas in athymic nude mice. Properties associated with the tumor suppressor Trp53 (formerly known as p53) were examined in three of the clones. In these clones, although Trp53 protein was overexpressed, a lower expression of Cdkn1a (formerly known as p21, Cip1) protein was observed compared to parental cells. The expression of Trp53 and Cdkn1a and the G(1)-phase arrest (one set of data on G(1)-phase delay is included as an example) was not induced by ionizing radiation in these transformed clones; each clone carried a point mutation in Trp53. This suggests that the deficiency in the repair of DNA double-strand breaks increased the tumorigenicity and the genomic instability of transformed SCID cells.

Wortmannin sensitizes mammalian cells to radiation by inhibiting the DNA-dependent protein kinase-mediated rejoining of double-strand breaks.

Wortmannin has been shown to be an efficient radiosensitizer. Since wortmannin is able to inhibit DNA-dependent protein kinase (DNA-PK) and double-strand break (DSB) rejoining, it is believed that its mechanism of radiation sensitization is through the inhibition of DNA-PK-mediated repair of DSBs. However, since wortmannin is not a specific inhibitor, the possibility that other kinases are inhibited and thereby may contribute to radiosensitization cannot be ruled out. Here we present data confirming the radiosensitizing effect of wortmannin on cells of different cell lines. In the same range of wortmannin concentrations, survival after exposure to ionizing radiation correlated well with DSB rejoining and the induction of micronuclei, suggesting that the inhibition of the processing of DSBs is involved in the sensitizing effect. Pretreatment with wortmannin enhanced the radiosensitivity of ataxia telangiectasia (AT) cells, thereby precluding the participation of ATM protein in the radiation sensitization by wortmannin. At the same time, irradiated DNA-PK-deficient cells were not significantly affected by pretreatment with wortmannin. These observations support a likely mechanism; that is, wortmannin sensitizes cells to radiation through inhibition of the DNA-PK-mediated rejoining of DSBs.

Phase transitions in the growth of C3H 10T1/2 cells.

In systems used to express transformation using focus formation as the end point, nontransformed cells generally express a down-regulation of cell growth and division made evident by the formation of a monolayer of cells that completely covers the growth surface. In C3H 10T1/2 cells, down-regulation is thought to be progressively effected principally by cell-to-cell communication via gap junctions. Starting with a sparse population in asynchronous growth--e.g. containing cells in all phases of the growth cycle--as the area density increases, cells are progressively lost from the distribution in the order M phase, G2 phase, S phase and G1 phase, leading to the accumulation of viable cells out of cycle in so-called G0 phase. We have measured the progressive phase transitions as a function of inoculum size and time. The influence of a promoter and an antipromoter was also examined as well as the expression of the cyclin/cyclin-dependent kinase inhibitors p21Waf1/Cip1 and p27Kip1 as the cells grew into confluence. Using cells synchronized in mitosis, we found that with increasing cell density the expression of p27 increased and concomitantly p21 decreased.

Re-investigation of the circumsporozoite protein-based induction of sterile immunity against Plasmodium berghei infection.

Although the circumsporozoite protein (CSP) of the malaria parasite is the most immunologically characterized protein, the goal of using this protein in an effective vaccine has not yet been realized. Monoclonal antibody against the repetitive immunodominant B-epitope of the CSP can protect mice from malaria, but vaccines that induce antibody against this epitope do not consistently induce protection. Toward developing a rationale for a CSP-based effective vaccine, we have re-investigated the ability of anti-CSP repeat antibodies, as induced by different CSP vaccine formulations with several adjuvants, to confer sterile immunity against sporozoite challenge. Using Plasmodium berghei rodent malaria model and several CSP subunit vaccine constructs, we found that a formulation consisting of the P. berghei CSP repetitive epitope, (DPPPPNPN)2 (CS), conjugated to BSA by carbodiimide, formulated in a block copolymer and detoxified lipopolysaccharide (RaLPS) adjuvant, was particularly promising. Mice were immunized and boosted with vaccines that contain varying malarial peptide-carrier ratios of 6:1 (CS6-BSA), 55:1 (CS55-BSA) and 170:1 (CS170-BSA). Following immunization, the animals were challenged with live sporozoites. Two types of effects were observed in vaccinated mice. First, sterile immunity was induced in 100%, 50% and 29% of mice that were immunized with the CS170-BSA, CS55-BSA, and CS6-BSA vaccine conjugates, respectively. The second effect of immunization was observed with the CS170-BSA conjugate vaccine primed mice; a boost in IFA titers followed sporozoite challenge. In addition, we observed that IgG1 isotype titer against the surface of the sporozoite, as measured by IFA, and antibody avidity parallel sterile immunity. These findings reiterate the potential of the CSP as a malaria vaccine candidate antigen, and suggest that the induction of sterile immune responses depends on inducing antibody of the appropriate isotype, avidity and specificity.

Enhanced cytochrome P450 (Cyp1b1) expression, aryl hydrocarbon hydroxylase activity, cytotoxicity, and transformation of C3H 10T1/2 cells by dimethylbenz(a)anthracene in conditioned medium.

The basal and benz(a)anthracene-induced aryl hydrocarbon hydroxylase activities of C3H 10T1/2 mouse embryo fibroblasts have been shown to vary with population growth. We report here that, in the case of dimethylbenz(a)anthracene, cytotoxicity and transformation (neoplastic/morphological transformation and focus formation) increased as a consequence of population growth and, at high cell densities, DNA adduct formation was elevated. Among the factors that may contribute to these changes, we have found that conditioning of the medium with population growth plays a significant role. Cells treated with medium conditioned by several days of cell growth supported increases in the dimethylbenz(a)anthracene induction of mRNA expression of a new mouse cytochrome P450 gene designated Cyp1b1, aryl hydrocarbon hydroxylase activity, cytotoxicity, and the frequency of neoplastic transformation. These results suggest a cause and effect relationship between the enhanced expression of Cyp1b1 due to medium conditioning and the enhanced expression of the cellular endpoints cytotoxicity and transformation.

cDNA cloning, sequence analysis, and induction by aryl hydrocarbons of a murine cytochrome P450 gene, Cyp1b1.

C3H mouse embryo fibroblast cells, designated 10T1/2, can be transformed by physical and chemical agents including polycyclic aromatic hydrocarbons. In a previous report (Shen et al., Proc. Natl. Acad. Sci. USA 90, 11483-11487, 1993), we identified a cytochrome P450 gene induced by polycyclic aromatic hydrocarbons (PAHs) that is different from 1A1 or 1A2, and which we tentatively named P450CMEF. Here, we report the entire cDNA sequence of P450CMEF (5,128 bp) and the amino acid sequence deduced from it (543 residues). A comparison of the latter sequence with known cytochrome P450s indicates that P450CMEF is in a new subfamily of family 1 of the P450 superfamily. Accordingly, the Committee on Standardized Cytochrome P450 Nomenclature designated the gene Cyp1b1. Exposure to various aryl hydrocarbons (2.5 hr) induced Cyp1b1 mRNA in 10T1/2 cells to different degrees: 2,3,7,8-tetrachlorodibenzo-p-dioxin, 7,12-dimethylbenz[a]anthracene, benz[a]anthracene, benzo[a]pyrene, and beta-naphthoflavone were strong inducers; alpha-naphthoflavone and 3-methylcholanthrene, were moderate inducers; and benzo[e]pyrene was a weak inducer.

Identification of a cytochrome P450 gene by reverse transcription--PCR using degenerate primers containing inosine.

A cytochrome P450-like gene, tentatively named P450CMEF, was amplified by a mixed oligonucleotide-primed amplification of cDNA from C3H mouse embryo fibroblast cells, designated 10T1/2, that had been treated with 7,12-dimethylbenz[a]anthracene (DMBA) or benz[a]anthracene (BA). A set of inosine-containing degenerate primers that were targeted to two conserved regions of known cytochrome P450 cDNAs were used. One primer was coded for the well-described and conserved heme-binding region of P450 enzymes, and the second was designed based upon other considerations of homology among P450 molecules. One of the four PCR-amplified cDNA products hybridized to two major RNA bands, 4.2 and 5.3 kb, that were induced by DMBA or BA. The amino acid sequence of the fragment deduced from the base-sequence data indicate that the amplified cDNA has a 50-55% identity with the cytochrome P450 subfamily 1A. The induction of P450CMEF mRNA preceded the induction of aryl hydrocarbon hydroxylase activity after DMBA or BA treatment, suggesting that the product of P450CMEF is involved in the metabolism of these polycyclic aromatic hydrocarbons in 10T1/2 cells. From the partial sequence of the cDNA identified by this procedure, we propose that P450CMEF is a member of the P450 superfamily, possibly in a subfamily of family 1, that is induced in 10T1/2 cells by DMBA and BA. This method should be useful in identifying additional P450 genes and genes in other gene families.

Transformation-sensitive cells in G2/M-phase are not promoted by TPA following 137Cs gamma-rays.

Mouse C3H 10T1/2 cells are most sensitive to radiation-induced neoplastic transformation in the G2/M-phase of the cell cycle. When synchronized 10T1/2 cells were exposed to phorbol 12-myristate 13-acetate (TPA) after irradiation, transformation of cells not in the transformation-sensitive window was enhanced, but transformation of cells already in the transformation-sensitive window was not. Earlier work showed that (a) TPA enhances the frequency of transformation of both high and low dose-rate gamma-irradiated cells by about the same factor, but that (b) TPA enhances the transformation of cells exposed to low dose-rate fission spectrum neutrons appreciably less than cells exposed to high dose-rate fission spectrum neutrons. The latter observation is consistent with the inability of TPA to promote cells in the transformation-sensitive window and with the role of such cells in enhancing transformation by protracted doses of neutrons. The data provide a cellular basis for studying the biochemical/molecular aspects of TPA promotion in vitro.

Neoplastic transformation of C3H mouse embryo cells, 10T1/2: cell-cycle dependence for 50 kV X-rays and UV-B light.

The variation of neoplastic transformation induced by 50 kV X-rays, and by solar-simulating UV-B light, was studied through the cell cycle of C3H mouse embryo cells designated 10T1/2. A mitotic shake-off method was used to harvest mitotic cells. The progression through the cell cycle of initially mitotic cells was followed as a function of time by flow cytometry, DNA labelling for passage through S-phase, and growth curves for cell number. At 2-3 h after shake-off, about 90% of the cells were in early G1-phase and by 15 h 60-70% of cells had reached S-phase. For 2.5 Gy, the transformation frequency per viable cell in M-phase was some five times higher than in S-phase. In contrast, at similar survival levels, UV-B light is less efficient in transforming mitotic cells. For both types of radiation, the frequency of neoplastic transformation per viable cell was roughly inversely proportional to survival.

Cycle sequencing using degenerate primers containing inosines.

A modified protocol for cycle sequencing DNA amplified by polymerase chain reaction (PCR) is described. The method involves two sequential linear PCR amplifications using a small amount of double-stranded DNA as a template and a stringent annealing temperature: 1) alpha-35S-dATP labeling of specific primers initially in degenerate primer mixture and 2) dideoxy-ribonucleotide termination of the extended and alpha-35S-dATP-labeled specific primers. The method does not require end labeling and is useful in sequencing PCR-amplified DNA sequences from highly degenerate primers when the sequences of the regions flanking those to be primed are unknown.

Enhanced sensitivity to neoplastic transformation by 137Cs gamma-rays of cells in the G2-/M-phase age interval.

C3H mouse 10T1/2 cells, exposed to low doses of fission-spectrum neutrons, have an enhanced frequency of neoplastic transformation if protracted exposures are used (Hill et al. 1982, 1984a, 1985). To explain this anomaly, a biophysical model was proposed (Elkind 1991a,b) having the following essential features: (1) a narrow age interval exists in the growth cycle of 10T1/2 cells in which cells have high sensitivities to transformation; (2) in the latter age interval, cells are also sensitive to killing; (3) with increasing dose, cells at ages earlier in the growth cycle are progressively delayed from entering the sensitive age window; and (4) with increasing dose, the transformation sensitivity of cells in the sensitive window is not expressed due to increased killing. Protracted low doses result in elevated frequencies because of less killing, and reductions in delays in cell progression. Therefore, transformation-sensitive cells can progress into the sensitive interval to replace those that have progressed out of it. The unique shape and radiobiological properties of cells in and around mitosis, led to the proposal that the sensitive window is mitosis and possible cells just preceding or just following M phase (Elkind 1991a,b). Because of the likelihood that the properties of the cells in a sensitive window would not be evident only when fission-spectrum neutrons are used, this study was undertaken using 137Cs gamma-rays. We have found that late G2- to M-phase 10T1/2 cells have a maximal sensitivity to neoplastic transformation as well as to killing by 137Cs gamma-rays.

Abrogation of killing and neoplastic transformation of C3H10T1/2 cells due to 7,12-dimethylbenz[a]anthracene.

The combined action of 7,12-dimethylbenz[a]anthracene (DMBA) and alpha-naphthoflavone (alpha NF) on the survival and neoplastic transformation of C3H10T1/2 mouse embryo fibroblasts has been examined and correlated with DNA adduct formation and removal. When a 24 h DMBA treatment of asynchronously growing cells was followed for the next 24 h by a treatment with alpha NF + DMBA, both killing and transformation per viable cell were abrogated to a large extent. In some instances, transformation was completely abrogated--i.e. reduced to control frequencies--even at nontoxic concentrations of DMBA, indicating that changes in survival were not the reason for the reduction in transformation. Even at toxic concentrations of DMBA, post-treatment with alpha-NF + DMBA resulted in 10-fold reductions in transformation frequency. 3-Methylcholanthrene (3MC) also reversed DMBA cytotoxicity but with a dependence on 3MC concentration that was qualitatively different from that for alpha NF. The abrogation of cell killing occurred at lower molar ratios of alpha NF:DMBA than the abrogation of transformation; less than or equimolar concentrations resulted in maximal abrogation of killing, but about equal concentrations were required to abrogate transformation. Although the preceding findings suggest that different mechanisms may be involved in these endpoints, taken together they suggest that second treatments make apparent the repair of lesions due to a first treatment with DMBA alone. To test this hypothesis, the formation and removal of DMBA-DNA adducts were measured. Adducts were not removed when the second treatment was growth medium alone, but enhanced removal was observed when second treatments consisted of DMBA alone or DMBA plus one of several other polycyclic aromatic hydrocarbons (PAHs). Relative to killing and neoplastic transformation, these results suggest DMBA induces a repair process that limits its own effectiveness--a process that can be sustained by other PAHs.

Noninvasive fetal ECG mode fetal heart rate monitoring by adaptive digital filtering.

Beat-to-beat variability (BTBV) of the fetal heart rate (FHR) is considered an indication of the neural integrity and is an important prognostic indicator of fetal well-being. We report the initial evaluation of a recently developed abdominal fetal ECG (AFECG) mode of FHR monitoring using Adaptive Digital Filtering (ADF) to accurately obtain BTBV noninvasively. Five women in labor at term were monitored with the direct fetal scalp electrode (FSE) and simultaneously with the AFECG using ADF. A computer analysis of 3298 seconds (55 minutes) of data provided a one-to-one comparison of the R-R intervals. One analysis of the direct FSE data with a second simultaneous analysis from the same electrode, to serve as control, was compared with the noninvasive AFECG data. The study group has a standard deviation of only 1.50 bpm compared to 0.79 bpm for the control group. The AFECG method agrees with the direct FSE method within 1 bpm for 92.6% of the reported R-R intervals and within 2 bpm for 98.9% of the reported intervals. This new noninvasive AFECG technique with ADF provides a continuous record of instantaneous FHR and BTBV that may be relied upon to provide an accurate continuous clinical record. The reliability of the technique has yet to be determined over a wide range of subjects.

Aryl hydrocarbon hydroxylase activity assayed in whole cell lysates using synchronous fluorescence spectroscopy.

Synchronous fluorescence spectrophotometry has been used to measure induced and constitutive levels of aryl hydrocarbon hydroxylase activity in lysates of C3H 10T1/2 mouse embryo fibroblasts. Without compromising sensitivity, the method was reproducible, eliminated the need to extract metabolites, and made the procedure simpler and less time consuming than other methods. Moreover, since the assay was tailored to directly measure 3-hydroxybenzo(a)pyrene, a metabolite produced by several cytochrome P-450s, it may be more generally applicable than dealkylation assays, which apparently detect only P-450-IA1.

Additivity of cytotoxic damage due to dimethylbenz(a)anthracene and X rays.

7,12-Dimethylbenz(a)anthracene is one of a group of polycyclic aromatic hydrocarbons that are known to be indirectly acting carcinogens. As a product of the incomplete combustion of complex hydrocarbons, dimethylbenzanthracene is present in the environment and may therefore act on living systems in conjunction with ionizing radiation. We have studied the cytotoxic effects of dimethylbenzanthracene by itself, together with other polycyclic aromatic hydrocarbons, and combined with X radiation. Pre- or postirradiation treatment of mouse C3H 10T1/2 cells with dimethylbenzanthracene progressively removes the shoulder of the X-ray survival curve and, consistent with that observation, the survival sparing from dose fractionation is progressively lost. The cotreatment of cells with 3-methylcholanthrene and dimethylbenzanthracene largely abrogates the killing due to the latter compound alone and, accordingly, returns the shoulder to the survival curve. The application of dimethylbenzanthracene, or similar compounds, between X-ray dose fractions, separated by 4 h, is without effect quite likely because of the need for metabolic activation of the compound for effectiveness. Dimethylbenzanthracene is believed to be genotoxic because, after it is activated, it forms bulky adducts with DNA. Hence these results suggest that bulky adducts are a form of DNA damage operationally equivalent to sublethal X-ray damage.